Laboratory Animal Medicine, Second Edition (American College of Laboratory Animal Medicine)

Laboratory Animal Medicine 2nd edition AMERICAN COLLEGE OF LABORATORY ANIMAL MEDICINE SERIES Steven H. Weisbroth, Ro...

Author: James G. Fox | Lynn C. Anderson | Franklin M. Loew | Fred W. Quimby

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Laboratory Animal Medicine 2nd edition

AMERICAN COLLEGE OF LABORATORY ANIMAL MEDICINE SERIES

Steven H. Weisbroth, Ronald E. Flatt, and Alan L. Kraus, eds.: The Biology of the Laboratory Rabbit, 1974 Joseph E. Wagner and Patrick J. Manning, eds.: The Biology of the Guinea Pig, 1976 Edwin J. Andrews, Billy C. Ward, and Norman H. Altman, eds.: Spontaneous Animal Models of Human Disease, Volume 1, 1979; Volume 2, 1979 Henry J. Baker, J. Russell Lindsey, and Steven H. Weisbroth, eds.: The Laboratory Rat, Volume 1: Biology and Diseases, 1979; Volume 2: Research Applications, 1980 Henry L. Foster, J. David Small, and James G. Fox, eds.: The Mouse in Biomedical Research, Volume 1: History, Genetics, and Wild Mice, 1981; Volume 2: Diseases, 1982; Volume 3: Normative Biology, Immunology, and Husbandry, 1983; Volume 4: Experimental Biology and Oncology, 1982 James G. Fox, Bennett J. Cohen, and Franklin M. Loew, eds.: Laboratory Animal Medicine, 1984 G. L. Van Hoosier, Jr., and Charles W. McPherson, eds.: Laboratory Hamsters, 1987 Patrick J. Manning, Daniel H. Ringler, and Christian E. Newcomer, eds." The Biology of the Laboratory Rabbit, 2nd Edition, 1994 B. Taylor Bennett, Christian R. Abee, and Roy Henrickson, eds.: Nonhuman Primates in Biomedical Research: Biology and Management, 1995 Dennis E Kohn, Sally K. Wixson, William J. White, and G. John Benson, eds.: Anesthesia and Analgesia in Laboratory Animals, 1997 B. Taylor Bennett, Christian R. Abee, and Roy Henrickson, eds.: Nonhuman Primates in Biomedical Research: Diseases, 1998 James G. Fox, Lynn C. Anderson, Franklin M. Loew, and Fred W. Quimby, eds.: Laboratory Animal Medicine, 2nd edition, 2002

La orator Anima Mec/icine 2nd edition EDITED BY

James G. Fox

Massachusetts Institute of Technology Cambridge, Massachusetts

Lynn C. Anderson Merck Research Laboratories Rahway, New Jersey

Franklin M. Loew Becker College Worcester, Massachusetts

Fred W. Quimby Laboratory Animal Research Center Rockefeller University New York, New York

ACADEMIC PRESS An Imprint of Elsevier

Amsterdam Boston Heidelberg London New York Oxford Paris San Diego San Francisco Singapore Sydney Tokyo

This book is printed on acid-free paper. @ Copyright 2002, 1984, Elsevier, All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permissions may be sought directly from Elsevier's Science and Technology Rights Department in Oxford, UK. Phone: (44) 1865 843830, Fax: (44) 1865 853333, e-mail: permissions@elsevi'er.co.uk. You may also complete your request on-line via the Elsevier homepage: http://www.elsevier.com by selecting "CustomerSupport" and then "ObtainingPermissions". Explicit permission from Academic Press is not required to reproduce a maximum of two figures or tables from an Academic Press Chapter in another scientific or research publication provided that the material has not been credited to another source and that full credit to Academic Press is given. Academic Press An Imprint of Elsevier 525 B Street, Suite 1900, San Diego, California 92101-4495, USA http ://www. academicpres s.com Academic Press 32 Jamestown Road, London NW 1 7BY, UK http://www.academicpress.com Library of Congress Catalog Card Number: 2002100883 ISBN-13:978-0-12-263951-7 ISBN'10:0-12-263951-0 Printed in the United States of America 06 07 MV 9 8 7 6 5 4

Contents

B e n n e t t J. C o h e n

ix

List of C o n t r i b u t o r s

xi

Recombinant DNA Research Guidelines References B i o l o g y and D i s e a s e s of M i c e

Chapter 3

Robert O. Jacoby, James G. Fox, and Muriel Davisson

xv

Preface

I.

List of R e v i e w e r s for C h a p t e r s in This V o l u m e Chapter 1

xvii

L a b o r a t o r y A n i m a l M e d i c i n e : Historical

C ha p t e r 2

35 41 53 113

B i o l o g y and D i s e a s e s of Rats I~

II. III.

Chapter 5

Introduction Biology Diseases References

121 123 134 158

B i o l o g y and D i s e a s e s of H a m s t e r s

F. Claire Hankenson and Gerald L. Van Hoosier, Jr. Syrian Hamster

14 15 15 15 16

Laws, R e g u l a t i o n s , and Policies Affecting

Introduction II. Biology III. Diseases Introduction Biology Diseases Armenian Hamster

193

Chinese Hamster

I. II. III.

Lynn C. Anderson 19 20 29 31

168

168 173 180 190 190 190 191

I~

I. Introduction II. Biology III. Diseases

the U s e of L a b o r a t o r y A n i m a l s I. Introduction II. Animal Welfare III. Importation and Exportation of Animals and Animal Products IV. Hazardous Substances


Dennis F Kohn and Charles B. Clifford

Franklin M. Loew and Bennett J. Cohen I. Introduction II. Origins of Animal Experimentation III. Early Veterinarians in Laboratory Animal Science and Medicine IV. The Organizations of Laboratory Animal Science V. Education and Training in Laboratory Animal Medicine VI. Impact of Laws, Regulations, and Guidelines on Laboratory Animal Medicine VII. Regulation of Animal Research in the United Kingdom and Canada VIII. Commercial and Academic Breeding of Rodents IX. Conclusion References

II. III.

Chapter 4

Perspectives

31 32

European Hamster

I. Introduction II. Biology III. Diseases Djungarian Hamster

193 194 194 194

194 194 195 196

vi

CONTENTS I. Introduction II. Biology III. Diseases References

Chapter 6

196 196 196 197

I. Introduction II. Biology III. Diseases References

203 206 212 241

Chapter 8

248 250 254 257 259 261 263 265 267 268 270 272 275 279 281 284 286 291

309 312 319 327

B i o l o g y and Diseases of Rabbits

C h a p t e r 10

329 331 339 358

M i c r o b i o l o g i c a l Q u a l i t y C o n t r o l for L a b o r a t o r y R o d e n t s and L a g o m o r p h s

William R. Shek and Diane J. Gaertner I. Overview II. Introduction III. Biosecurity

I. II. III. IV. V. VI.

C h a p t e r 13

Introduction Sources of Cats Housing Breeding Colony Management Nutrition and Feeding Infectious Disease Exclusion and Control References

365 366 366

460 462 463 466 474 475 480

B i o l o g y and Diseases of Ferrets

Robert P. Marini, Glen Otto, Susan Erdman, Lori Palley, and James G. Fox I. Introduction II. Biology III. Diseases References C h a p t e r 14

483 485 490 513

B i o l o g y and Diseases of R u m i n a n t s : Sheep, Goats, and Cattle

Margaret L. Delano, Scott A. Mischler, and Wendy J. Underwood

C h a p t e r 15

519 525 537 611

B i o l o g y and Diseases of S w i n e

Kathy E. Laber, Mark T. Whary, Sarah A. Bingel, James A. Goodrich, Alison C. Smith, and M. Michael Swindle I. Introduction II. Biology III. Diseases References

Mark A. Suckow, David W. Brammer, Howard G. Rush, and Clarence E. Chrisp I. Introduction II. Biology III. Diseases References

D o m e s t i c Cats as L a b o r a t o r y A n i m a l s


Christine A. Bellezza, Patrick W. Concannon, William E. Hornbuckle, Lois Roth, and Bud C. Tennant

Chapter 9

395 397 405 454

Brenda Griffin and Henry J. Baker

W o o d c h u c k s as L a b o r a t o r y A n i m a l s


372 387

B i o l o g y and Diseases of D o g s

I. Introduction II. Biology III. Diseases References C h a p t e r 12

B i o l o g y and D i s e a s e s of O t h e r R o d e n t s

Introduction Ground Squirrels or Susliks: Spermophilus Prairie Dogs: Cynomys Pocket Gophers: Geomyidae Kangaroo Rats: Dipodomys Wood Rats or Pack Rats: Neotoma Grasshopper Mice: Onychomys White-Footed Mice or Deer Mice: Peromyscus Rice Rats: Oryzomys Cane Mice: Zygodontomys Cotton Rats: Sigmodon White-Tailed Rats: Mystromys Gerbils and Jirds: Meriones Volesand Meadow Mice: Microtus Multimammate Rats: Mastomys Degus or Trumpet-Tailed Rats: Octodon Chinchillas: Chinchilla References

Microbiological (Health) Surveillance References

Robert C. Dysko, Jean A. Nemzek, Stephen L Levin, George J. DeMarco, and Maria R. Moalli

Thomas M. Donnelly and Fred W. Quimby I. II. III. IV. V. VI. VII. VIII. IX. X. XI. XII. XIII. XIV. XV. XVI. XVII.

C h a p t e r 11

B i o l o g y and Diseases of G u i n e a Pigs

John E. Harkness, Kathleen A. Murray, and Joseph E. Wagner

Chapter 7

IV.

C h a p t e r 16

615 618 629 665

Nonhuman Primates

Bruce J. Bernacky, Susan V. Gibson, Michale E. Keeling, and Christian R. Abee I. II. III. IV. V. VI.

Introduction Taxonomy Biology Principles of Colony Management Medical Management Diseases References

676 677 680 715 724 730 777

vii

CONTENTS

Chapter 17 Biology and Diseases of Amphibians Dorcas P. O'Rourke and Terry Wayne Schultz I. II. III.


793 801 814 823

IV. V. VI. VII.

Dogs Swine Small Ruminants Nonhuman Primates References

973 979 986 990 997

Chapter 23 Techniques of Experimentation Robert J. Adams

Chapter 18 Biology and Diseases of Reptiles Dorcas P. O'Rourke and Juergen Schumacher I. II. III.


827 837 848 857

Chapter 19 Biology and Management of the Zebrafish Ke.ith M. Astrofsky, Robert A. Bullis, and Charles G. SagerstriJm I~

II. III. IV. V. VI. VII.

Introduction Experimental Model in Biomedical Research Health Management in the Aquatic Animal Facility Environmental Factors Important to Health Nutrition and Feeding Acquisition Infectious Diseases References

862 863 866 866 874 875 875 882

Chapter 20 Biology and Health of Laboratory Fishes Introduction Facility Design Management and Husbandry Medical Protocols Zoonotic Considerations Diseases References

886 886 893 894 898 899 907

Introduction Identification Methods Blood Collection and Intravenous Injection Vascular Cannulation Intraperitoneal Injection Subcutaneous and Intramuscular Injection Digestive System Urinary System Techniques Respiratory System Techniques Reproductive System Cardiovascular Techniques Endocrine System Techniques Orthopedic Procedures for Laboratory Animals Neurosurgical Techniques Tumor Transplantation Imaging Techniques Radiotelemetry References

1006 1006 1008 1013 1015 1015 1015 1019 1021 1023 1027 1028 1029 1029 1032 1033 1034 1034

Chapter 24 Control of Biohazards Associated with the Use of Experimental Animals Thomas E. Hamm, Jr. I.

Michael K. Stoskopf I. II. III. IV. V. VI.

I. II. III. IV. V. VI. VII. VIII. IX. X. XI. XII. XIII. XIV. XV. XVI. XVII.

II. III. IV. V. VI.

Introduction Risk Assessment Managing Facilities in Which Biohazards Are Used Other Considerations Responsibility for Reviewing and Approving Protocols Involving Biohazards Summary References

1047 1048 1048 1053 1056 1056 1056

Chapter 25 Selected Zoonoses Chapter 21 Design and Management of Animal Facilities

James G. Fox, Christian E. Newcomer, and Harry Rozmiarek I. Introduction

Jack R. Hessler and Steven L. Leary I. Introduction II. Facility Planning and Design III. Equipment IV. Commissioning and Validation V. Management VI. Conclusions References

909 910 931 946 946 947 948

Chapter 22 Preanesthesia, Anesthesia, Analgesia, and Euthanasia

Introduction Rodents Rabbits

Viral Diseases Rickettsial Diseases Chlamydial Infections Bacterial Diseases Fungal Diseases Protozoal Diseases Helminth Infections Arthropod Infestations References

1060 1060 1074 1076 1077 1088 1089 1093 1098 1098

Chapter 26 Xenozoonoses: The Risk of Infection

M. Michael Swindle, George A. Vogler, Linda K. Fulton, Robert P. Marini, and Sulli Popilskis I. II. III.

II. III. IV. V. VI. VII. VIII. IX.

after Xenotransplantation Marian G. Michaels 956 956 966

I. II. III.

Introduction Lessons from Allotransplantation Xenotransplantation References

1107 1107 1109 1113

viii

CONTENTS

C h a p t e r 27

III. History of Animal Use in Biomedical Research References

Genetic Monitoring

John J. Sharp, Evelyn E. Sargent, and Peter A. Schweitzer I. II. III. IV. v. vI.

C h a p t e r 28

Introduction The Need for Genetically Defined Animals Sources and Monitoring of Genetic Variability Colony Management Monitoring Methods Summary References

1117 1118 1119 1121 1122 1126 1127

C h a p t e r 31

I~ Introduction II. Choice of Mouse Strains for Trafisgenic Programs III. Production of Transgenic Mice: Animal Requirements IV. Management of the Transgenic Mouse Colony References

1129 1130

C h a p t e r 32

C h a p t e r 29

Henry J. Baker and J. Russell Lindsey I. II. III. IV. v. vI.

1131 1134 1140

I. II. III. IV. v. vI.

Research

Neil S. Lipman and Scott E. Perkins

C h a p t e r 30

1143 1143 1147 1165

A n i m a l M o d e l s in B i o m e d i c a l R e s e a r c h

Fred W. Quimby What Is an Animal Model? II. The Nature of Research I~

1185 1200

Introduction The Research Process Research Training Research Resources Support for Research Summary References

1228 1229 1231 1234 1235 1237 1237

Laboratory Animal Behavior

Kathryn A. L. Bayne, Bonnie V. Beaver, Joy A. Mench, and David B. Morton

Factors T h a t M a y I n f l u e n c e A n i m a l

I. Introduction II. Intrinsic Considerations III. Extrinsic Considerations References

R e s e a r c h in L a b o r a t o r y A n i m a l and Comparative Medicine

T r a n s g e n i c and K n o c k o u t M i c e

Glenn M. Monastersky and James G. Geistfeld

1206 1214

Index

Introduction Rodents Rabbits Laboratory Dogs and Cats Nonhuman Primates Farm Animals References

1240 1241 1245 1246 1248 1252 1256 1265

Bennett We would like to dedicate this book to our friend and colleague Bennett J. Cohen, D.V.M., Ph.D., who died on August 23, 1990, at the age of 65. Ben was a valued coeditor of the first edition of "Laboratory Animal Medicine." We would like to honor him with our heartfelt appreciation of his leadership, wisdom, and friendship throughout our individual careers. After graduating from Cornell University Veterinary School in 1949, Ben obtained a Ph.D. in physiology from Northwestern Medical School in 1953. He then spent several years on the faculty and was director of the vivarium at the University of California Medical School in Los Angeles. He was then recruited to the University of Michigan, where he founded the Unit for Laboratory Animal Medicine (ULAM) at the University of Michigan, and was its director for 23 years. He obtained the rank of professor of laboratory animal medicine in 1968. Dr. Cohen was a pioneer and visionary in the field of laboratory animal science for more than 40 years. His career of caring for animals used in medical research began at Northwestern University in 1949. A year later, he and veterinary colleagues in the Chicago area founded the Animal Care Panel, which later became the American Association for Laboratory Animal Science (AALAS). Ben served as the association's first secretary, as a member of the board of trustees, and later as president. Three years later Ben and a few colleagues saw the need to establish standards of training and experience for veterinarians

J.

Cohen

engaged in laboratory animal medicine. They persuaded the American Veterinary Medical Association to accept the veterinary specialty of laboratory animal medicine and establish a specialty certification board. This became the American College of Laboratory Animal Medicine (ACLAM). Today there are more than 600 board-certified veterinarians in the United States. In 1963, Dr. Cohen chaired the National Academy of Sciences committee that wrote the first edition of the document that later became "The Guide for Care and Use of Laboratory Animals." Since then, more than 400,000 copies have been distributed, and it has been accepted as a primary reference on laboratory animal care and use. The National Institutes of Health (NIH) now requires that awardee institutions comply with the provisions of the guide. Dr. Cohen was the recipient of all of the major national and international awards in laboratory animal science. In 1966, he received the Griffin Award from the American Association for Laboratory Animal Science. This, the association's highest award, was presented for "outstanding accomplishments in the improvement of care and quality of laboratory animals." In 1980, he received the Charles River Prize, the highest award of the American Veterinary Medical Association. The inscription reads, "You have been a moving force in laboratory animal science and a major figure in the founding of national

/x

X

organizations that have brought strength, cohesion, and credibility to the field." In 1990, the governing board of the International Council for Laboratory Animal Science (ICLAS) presented Dr. Cohen with the council's highest award, the Muhlbock Award, for his work in establishing high standards of laboratory animal care and use worldwide. In addition to his activity in national and international organizations, Dr. Cohen was an active clinician and scientist. He published more than 70 articles in peer-reviewed scientific journals and served on many NIH study sections and advisory boards. Dr. Cohen also established a national reputation in the field of gerontology. He originated health standards for aging animals and undertook long-term studies of rodent diseases of aging. At the University of Michigan he established the Core Facility for Aged Rodents (CFAR) in the Institute of Gerontology and the Gerontology Research and Training Center. The CFAR provides aged rodents for study by scientists campuswide.

BENNETT J. C O H E N

An additional and lasting legacy of Ben's impact on laboratory animal medicine is the stellar record he achieved in training future generations of specialists in the field. He trained 36 postdoctoral veterinary fellows from 1959 to 1985. Dr. Cohen was originally awarded the NIH training grant while at UCLA in 1960 and transferred the training grant to Ann Arbor when he relocated to the University of Michigan in 1962. This grant has been funded since its inception and is recognized internationally for its record of excellence. Dr. Cohen was a friend and an inspiration to his colleagues worldwide. He loved his work, his science, and his colleagues. He was truly a humanitarian and a lasting example--for those of us engaged in pursuits of veterinary medicine--of the values of professionalism and integrity. James G. Fox Lynn C. Anderson Franklin M. Loew Fred W. Quimby

List of Contributors

Numbers in parentheses indicate the pages on which the authors' contributions begin.

Christian R. Abee (675), Department of Comparative Medicine, University of South Alabama, College of Medicine, Mobile, Alabama 36688 Robert J. Adams (1001), Division of Comparative Medicine, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 Lynn C. Anderson (19), Department of Comparative Medicine, Merck Research Laboratories, Rahway, New Jersey 07065 Keith M. Astrofsky (861), Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Henry J. Baker (459, 1227), College of Veterinary Medicine, Scott-Ritchey Research Center, Auburn University, Auburn, Alabama 36849 Kathryn A. L. Bayne (1239), Association for Assessment and Accreditation of Laboratory Animal Care International, Rockville, Maryland 20852 Bonnie u Beaver (1239), Department of Small Animal Medicine and Surgery, College of Veterinary Medicine, Texas A&M University, College Station, Texas 77843 Christine A. Bellezza (309), Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853 Bruce J. Bernacky (675), Department of Veterinary Sciences, M. D. Anderson Cancer Center, Bastrop, Texas 78602 Sarah A. Bingel (615), Department of Comparative Medicine, Medical University of South Carolina, Charleston, South Carolina 29425 David W. Brammer (329), Parke-Davis Pharmaceutical Research, Ann Arbor, Michigan 48105

Robert A. Bullis (861), Center for Applied Aquaculture, The Oceanic Institute, Waimanalo, Hawaii 96795 Clarence E. Chrisp 1 (329), Unit for Laboratory Animal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109 Charles B. Clifford (121), Charles River Laboratories, Inc., Wilmington, Massachusetts 01887 Bennett J. Cohen 2 (1), Unit for Laboratory Animal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109 Patrick W. Coneannon (309), Department of Biomedical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853 Muriel Davisson (35), Jackson Laboratories, Bar Harbor, Maine 04609 Margaret L. Delano (519), Animal Care, University of Massachusetts, Amherst, Massachusetts 01003 George J. DeMarco (395), Unit for Laboratory Animal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109 Thomas M. Donnelly 3 (247), The Kenneth S. Warren Institute, Ossining, New York 10562 Robert C. Dysko (395), Unit for Laboratory Animal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109 Susan Erdlnan (483), Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

1Deceased February 8, 2000. 2Deceased August 23, 1990. 3present address: Post Graduate Foundation in Veterinary Science, University of Sydney, Australia.

xi

xii James G. Fox (35,483, 1059), Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Linda K. Fulton (955), Godley-Snell Research Center, Clemson University, Clemson, South Carolina 29634 Diane J. Gaertner (365), Institute for Animal Studies, Albert Einstein College of Medicine, Bronx, New York 10461 James G. Geistfeld (1129), Taconic, Inc., Germantown, New York 12526 Susan V. Gibson (675), Department of Comparative Medicine, College of Medicine, University of South Alabama, Mobile, Alabama 36688 James A. Goodrich (615), Department of Comparative Medicine, Medical University of South Carolina, Charleston, South Carolina 29425 Brenda Griffin (459), College of Veterinary Medicine, ScottRitchey Research Center, Auburn University, Auburn, Alabama 36849 Thomas E. Hamm, Jr. (1047), 105 Martinique Place, Cary, North Carolina 27511 F. Claire l-lankenson (167), Unit for Laboratory Animal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109 John E. l-larkness (203), College of Veterinary Medicine, Office of Research, Mississippi State University, Mississippi State, Mississippi 39762 Jack R. l-lessler (909), Washington University School of Medicine, St. Louis, Missouri, 63110 William E. l-lornbuckle (309), Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853 Robert O. Jacoby (35), Section of Comparative Medicine, Yale University School of Medicine, New Haven, Connecticut 06520 Michale E. Keeling (675), Department of Veterinary Sciences, M. D. Anderson Cancer Center, Bastrop, Texas 78602 Dennis F. Kohn (121 ), Institute of Comparative Medicine, College of Physicians and Surgeons, Columbia University, New York, New York 10032 Kathy E. Laber (615), Department of Comparative Medicine, Medical University of South Carolina, Charleston, South Carolina 29425 Steven L. Leary (909), Washington University School of Medicine, St. Louis, Missouri 63110 Stephen I. Levin (395), Unit for Laboratory Animal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109 J. Russell Lindsey (1227), Department of Comparative Medicine, University of Alabama Schools of Medicine and Dentistry, Birmingham, Alabama 35294 Neil S. Lipman (1143), Research Animal Resource Center, Memorial Sloan-Kettering Cancer Center, Weill Medical College of Cornell University, New York, New York 10021

LIST OF CONTRIBUTORS Franklin M. Loew (1), Becker College, Worcester, Massachusetts 01609 Robert P. Marini (483, 955), Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139 Joy A. Mench (1239), Department of Animal Science, University of California, Davis, California 95616 Marian G. Michaels (1107), Division of Allergy, Immunology, and Infectious Disease, The Children's Hospital of Pittsburgh, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 11521 Scott A. Mischler (519), Animal Care and Management, University of Vermont, Burlington, Vermont 05405 Maria R. Moalli (395), Department of Surgery and Unit for Laboratory Animal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109 Glenn M. Monastersky (1129), Hoffman-LaRoche, Nutley, New Jersey 07110 David B. Morton (1239), Department of Biomedical Ethics, The Medical School Edgbaston, University of Birmingham, Birmingham, B 15 211 United Kingdom Kathleen A. Murray (203), Technical Operations, Charles River Laboratories, Wilmington, Massachusetts 01887 Jean A. Nemzek (395), Department of Pathology and Unit for Laboratory Animal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109 Christian E. Newcomer 4 (1059), Division of Laboratory Animal Medicine, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599 Dorcas P. O'Rourke (793, 827), Department of Comparative Medicine, College of Veterinary Medicine, University of Tennessee, Knoxville, Tennessee 37901 Glen Otto (483), Department of Comparative Medicine, Stanford University, Stanford, California 94305 Lori Palley (483), Astra Research Boston, Cambridge, Massachusetts 02139 Scott E. Perkins (1143), Division of Laboratory Animal Medicine, Tufts University, School of Veterinary Medicine, Boston, Massachusetts 02111 Sulli Popilskis (955), Institute of Comparative Medicine, Columbia University, New York, New York 10032 Fred W. Quimby (247, 1185), Laboratory Animal Research Center, Rockefeller University, New York, New York 10021 Lois Roth (309), Department of Pathology, MSPCA Angell Memorial, Boston, Massachusetts 02130 Harry Rozmiarek (1059), Laboratory Animal Resources, University of Pennsylvania, Philadelphia, Pennsylvania 19104 Howard G. Rush (329), Unit for Laboratory Animal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109 4presentaddress:VeterinaryResourcesProgram,NationalInstitutesof Health, Bethesda, Maryland20892.

LIST OF CONTRIBUTORS

Charles G. Sagerstriim (861), Department of Biochemistry and Molecular Pharmacology, University of Massachusetts Medical Center, Worcester, Massaschusetts 01655 Evelyn E. Sargent (1117), Genetic Quality Assurance, Jackson Laboratories, Bar Harbor, Maine 04609 Terry Wayne Schultz (793), Department of Comparative Medicine, College of Veterinary Medicine, University of Tennessee, Knoxville, Tennessee 37901 Juergen Schumacher (827), Department of Comparative Medicine, College of Veterinary Medicine, University of Tennessee, Knoxville, Tennessee 37901 Peter A. Schweitzer (1117), Genetic Quality Assurance, Jackson Laboratories, Bar Harbor, Maine 04609 John J. Sharp (1117), SAIC-Frederick, Frederick, Maryland 21702 William R. Shek (365), Charles River Laboratories, Wilmington, Massachusetts, 01887 Alison C. Smith (615), Department of Comparative Medicine, Medical University of South Carolina, Charleston, South Carolina 29425 Michael K. Stoskopf (885), Environmental Medicine Consortium, College of Veterinary Medicine, North Carolina State University, Raleigh, North Carolina 27606

xiii

Mark A. Suckow (329), Friemann Life Science Center, University of Notre Dame, Notre Dame, Indiana 46556 M. Michael Swindle (615, 955), Department of Comparative Medicine, Medical University of South Carolina, Charleston, South Carolina 29425 Bud C. Tennant (309), Department of Clinical Sciences, College of Veterinary Medicine, Cornell University, Ithaca, New York 14853 Wendy J. Underwood (519), Animal Science and Veterinary Services, Eli Lilly and Company, Greenfield, Indiana 46140 Gerald L. Van Hoosier, Jr. (167), Department of Comparative Medicine, University of Washington School of Medicine, Seattle, Washington 98195 George A. Vogler (955), Department of Comparative Medicine, St. Louis University, School of Medicine, St. Louis, Missouri 63104 Joseph E. Wagner (203), College of Veterinary Medicine, University of Missouri, Columbia, Missouri 65211 Mark T. Whary (615), Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

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Preface

The American College of Laboratory Animal Medicine (ACLAM) was founded in 1957 to encourage education, training, and research in laboratory animal medicine and to recognize veterinary medical specialists in the field by certification and other means. Continuing education has been an important activity in ACLAM from its inception. The second edition of this teaching text, "Laboratory Animal Medicine," reflects the College's continuing effort to foster education. It is, in part, an updated distillation for teaching purposes of a series of volumes on laboratory animals developed by ACLAM over the past two decades: "The Biology of the Laboratory Rabbit" published in 1974, with a second edition in 1994, "The Biology of the Guinea Pig" in 1976, and a two-volume work "Biology of the Laboratory Rat" in 1979 and 1980, followed by the publication "Laboratory Hamsters" in 1987. Also, in 1979 the college published a two-volume text on "Spontaneous Animal Models of Human Disease." In 1981-1983, four volumes of "The Mouse in Biomedical Research" were published. Most recently, a twovolume treatise on "Nonhuman Primates in Biomedical Research" was published in 1995 and 1998, and, finally, a text "Anesthesia and Analgesia in Laboratory Animals" in 1997. Most major advances in biology and medicine in one way or another have depended on the study of animals. During the past generation, the health, genetic integrity, and environmental surroundings of the animals have been recognized as important factors to be taken into account in planning animal studies. The ultimate responsibility for insuring the validity of scientific resuits, together with humane and scientifically appropriate animal care, resides with two categories of scientists: veterinarians responsible for the acquisition, care, nutrition, anesthesia, and other aspects of humane animal use and scientific investigators who use animals as subjects of study. This book therefore is intended for students of veterinary medicine and others in the fields of biology and medicine who utilize animals in biomed-

ical research. The editors and contributors hope it will prove useful in introducing students and scientists embarking on their careers to important concepts related to animals in research. The contents of this second edition have been greatly expanded and are presented in thirty-two chapters that provide information on the diseases and biology of the major species of laboratory animals used in biomedical research. The history of laboratory animal medicine, legislation affecting laboratory animals, experimental methods and techniques, design and management of animal facilities, zoonoses, biohazards, animal models, and genetic monitoring are also covered. Reflective of the ever increasing use of genetically engineered mice, new chapters include rodent and lagomorph surveillance and quality assurance, and transgenic and knockout mice. Also, added are chapters dealing with the emerging interest in fish biology and the use of xenotransplantation. The editors acknowledge the contributors' outstanding efforts to follow the guidelines on content and accept sole responsibility for any significant omissions. As with all volumes of the ACLAM series texts, the contributors and editors of this book have donated publication royalties to the American College of Laboratory Animal Medicine to foster continuing education in laboratory animal science. It could not have been completed without the support and resources of the editors' parent institutions. A special thanks also is extended to the reviewers of each chapter whose excellent and thoughtful suggestions helped the authors and editors present the material in a meaningful and concise manner. We acknowledge and thank Lucille Wilhelm for her excellent secretarial assistance. The assistance of the staff of Academic Press also is greatly appreciated and acknowledged. James G. Fox Lynn C. Anderson Franklin M. Loew Fred W. Quimby XI2


List of Reviewers for Chapters in This Volume

Astrofsky, Keith Balk, Melvin Bayne, Kathryn A. Bell, Judith A. Bennett, B. Taylor Bowser, Paul Clemons, Donna Collins, Bobby R. Cullen, John DeTolla, Louis J. Erdman, Susan Faith, Robert E. Forsythe, Diane B. Geistfeld, James Herbst, Lawrence H. Huerkamp, Michael Hurley, Julie Hurley, Richard Jaax, Gerald P. James, Mary Lou Klein, Hilton Lipman, Neil S. Lohmiller, Jeffrey J. Marini, Robert P. Morrissey, J. Motzel, Sherri Noga, Edward J. Patterson, Mary Percy, Dean H. Perkins, Scott Rand, Michael S. Ringler, Daniel H. Saperstein, George Scipioni, Roberta Sive, Hazel L. Smith, Abigail Spaulding, Glen Straw, Barbara Webster, William S. White, William J.

Massachusetts Institute of Technology, Cambridge, Massachusetts Charles River Laboratories, Inc., Wilmington, Massachusetts AAALAC International, Bethesda, Maryland University of Guelph, Ontario University of Illinois, Chicago, Illinois Cornell University, Ithaca, New York Covance Laboratories, Inc., Madison, Wisconsin University of Florida, Gainesville, Florida North Carolina State University, Raleigh, North Carolina University of Maryland, Baltimore, Maryland Massachusetts Institute of Technology, Cambridge, Massachusetts Baylor College of Medicine, Houston, Texas National Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina Taconic, Inc., Germantown, New York Albert Einstein College of Medicine, New York, New York Emory University, Atlanta, Georgia Consultant, Boston, Massachusetts Children's Hospital, Boston, Massachusetts Kansas State University, Manhattan, Kansas Consultant, Regulatory Compliance Merck Research Laboratories, West Point, Pennsylvania Memorial Sloan-Kettering Cancer Center, New York, New York Taconic, Inc., Germantown, New York Massachusetts Institute of Technology, Cambridge, Massachusetts The Animal Medical Center, New York, New York Merck Research Laboratories, West Point, Pennsylvania North Carolina State University, Raleigh, North Carolina Massachusetts Institute of Technology, Cambridge, Massachusetts University of Guelph, Ontario Memorial Sloan-Kettering Cancer Center, New York, New York University of Arizona, Tucson, Arizona University of Michigan, Ann Arbor, Michigan Tufts University School of Veterinary Medicine, North Grafton, Massachusetts Marshall Farms, North Rose, New York Whitehead Institute, Cambridge, Massachusetts The Jackson Laboratory, Bar Harbor, Maine Tufts University School of Veterinary Medicine, North Grafton, Massachusetts Michigan State University, East Lansing, Michigan University of Massachusetts Medical School, Worcester, Massachusetts Charles River Laboratories, Inc., Wilmington, Massachusetts xvii


Chapter 1 Laboratory Animal Medicine: Historical Perspectives Franklin M. Loew and Bennett J. Cohen*

I. II. III. IV.

V. VI. VII. VIII. IX.

I.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Originsof Animal Experimentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . EarlyVeterinarians in Laboratory Animal Science and Medicine . . . . . . . The Organizations of Laboratory Animal Science . . . . . . . . . . . . . . . . . . . A. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. The National Society for Medical Research . . . . . . . . . . . . . . . . . . . . C. The American Association for Laboratory Animal Science . . . . . . . . D. The Institute of Laboratory Animal Resources . . . . . . . . . . . . . . . . . . E. The American College of Laboratory Animal Medicine . . . . . . . . . . . Educationand Training in Laboratory Animal Medicine . . . . . . . . . . . . . . Impactof Laws, Regulations, and Guidelines on Laboratory Animal Medicine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Regulationof Animal Research in the United Kingdom and Canada . . . . . Commercialand Academic Breeding of Rodents . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

INTRODUCTION

Five key terms identify the fields or activities that relate to the care and use of animals in research, education, and testing. Animal experimentation refers to the scientific study of animals, usually in a laboratory, for the purpose of gaining new biological knowledge or solving specific medical, veterinary medical, dental, or biological problems. Most commonly, such experimentation is carried out by or under the direction of persons holding research or professional degrees. Laboratory ani*Deceased. LABORATORY ANIMAL MEDICINE, 2nd edition

1

2 3 7 7 8 10 12 13 13 14 15 15 15 16

mal care is the application of veterinary medicine and animal science to the acquisition of laboratory animals and to their management, nutrition, breeding, and diseases. The term also relates to the care that is provided to animals as an aid in managing injury and pain. Laboratory animal care usually is provided in scientific institutions under veterinary supervision or guidance. Laboratory animal medicine is recognized by the American Veterinary Medical Association as the specialty field within veterinary medicine that is concerned with the diagnosis, treatment, and prevention of diseases in animals used as subjects in biomedical activities. Laboratory animal medicine also encompasses the methods of minimizing and preventing pain or discomfort in research animals and the identification of Copyright 2002, Elsevier Science (USA). All fights reserved. ISBN 0-12-263951-0

2

FRANKLIN M. L O E W AND BENNETT J. COHEN

complicating factors in animal research. Comparative medicine is "the study of the nature, cause and cure of abnormal structure and function in people, animals and plants for the eventual application to and benefit of all living things" (Bustad et al., 1976). Laboratory animal science is the body of scientific and technical information, knowledge, and skills that bears on both laboratory animal care and laboratory animal medicine and that is roughly analogous to "animal science" in the agricultural sector. Laboratory animal medicine has grown rapidly because of its inherent scientific importance and because good science and the public interest require the best possible care for laboratory animals. In this chapter, we trace briefly the historical evolution of laboratory animal medicine and consider its relationship to other areas of biology and medicine.

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ORIGINS OF ANIMAL EXPERIMENTATION

The earliest references to animal experimentation are to be found in the writings of Greek philosopher-physicians of the fourth and third centuries BC. Aristotle (384-322 BC), characterized as the founder of biology, was the first to make dissections that revealed internal differences among animals (Wood, 1931). Erasistratus (304-250 BC) probably was the first to perform experiments on living animals, as we understand them today. He established in pigs that the trachea was an air tube and the lungs were pneumatic organs (Fisher, 1881). Later, Galen (AD 130-200) performed anatomical dissections of pigs, monkeys, and many other species (Cohen and Drabkin, 1948; Cohen, 1959a). Galen justified experimentation as a long, arduous path to the truth, believing that uncontrolled assertion that was not based on experimentation could not lead to scientific progress. Dogma replaced experimentation in the dark centuries following Galen's lifetime. Whereas anatomical dissection of dead animals and people had been among the earliest types of experimentation, in medieval times this practice was prohibited by ecclesiastical authorities who wanted to prevent acquisition of knowledge about the natural world that could

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be considered blasphemous. Not until the 1500s was there a reawakening of interest in science. Andreas Vesalius (15141564), the founder of modern anatomy, used dogs and pigs in public anatomical demonstrations (Saunders and O'Malley, 1950) (Fig. 1). This "vivisection" led to great leaps in the understanding of anatomy's correspondence with physiology. In 1628, Sir William Harvey published his great work on the movement of the heart and blood in animals (Singer, 1957). By the early 1700s, Stephen Hales, an English clergyman, reported the first measurement of blood pressure, using as his subject a horse "fourteen hands high, and about fourteen years of age, [and with] a fistula on her withers" (Hoff et al., 1965) (Fig. 2).

3

1. LABORATORY A N I M A L MEDICINE: H I S T O R I C A L P E R S P E C T I V E S

Fig. 3. Claude Bernard, often referred to as the founder of experimental medicine, developed and described highly sophisticated methods of animal research in his laboratory in Paris. (Photograph from Garrison, 1929.)

During the 1800s, France became a primary center of experimental biology and medicine. Scientists, such as Franqois Magendie (1783-1855) and Claude Bernard (1813-1878) (Fig. 3) in experimental physiology and Louis Pasteur (1822-1895) in microbiology, contributed enormously to the validation of the scientific method, which included the use of animals. Bernard (1865) commented: ... it is proper to choose certain animals which offer favorable anatomical arrangements or special susceptibility to certain influences. For each kind of investigation we shall be careful to point out the proper choice of animals. This is so important that the solution of a physiological or pathological problem often depends solely on the appropriate choice of the animal for the experiment so as to make the result clear and searching.

Pasteur studied infectious diseases in a variety of animals, such as silkworms ("pebrine"), dogs (rabies), and sheep (anthrax). "Pebrine" (pepper) was an economically important disease of silkworms in France when silk was a major fabric; Pasteur and others demonstrated the parasite that caused the disease (Duclaux, 1920). As pathogenic organisms were identified that could be related to specific human diseases, their animal disease counterparts also were studied. Pasteur and others perceived that the study of animal diseases benefited animals and enhanced understanding of human diseases and pathology. The extraordinary power of the experimental approach, including experiments on animals, led to what has been called the Golden Age of scientific

medicine. Despite advances in physiological and bacteriological understanding, however, criticisms of the use of animals in science began, particularly in England (Loew, 1982). The first Society for the Prevention of Cruelty to Animals (SPCA) was established in England, followed in the 1860s by an American SPCA in New York, a Philadelphia SPCA, and a Massachusetts SPCA. Objections to the use of animals in science were part of the concerns of these societies, particularly because Darwin's findings on evolution made "differences" between animals and humans less sure in many persons' minds (Loew, 1982). Most American and Canadian scientists, physicians, and veterinarians soon applied emerging scientific concepts in their research. D. E. Salmon, recipient of the first D.V.M. degree awarded in the United States (by Cornell University in 1879), studied bacterial diseases, and the genus Salmonella, a ubiquitous human and animal pathogen, was named for him. Cooper Curtice (Fig. 4), Theobald Smith, and others first demonstrated the role of arthropod victors in disease transmission in their studies of bovine Texas fever (Schwabe, 1978). The first paper published at the then fledgling Johns Hopkins Hospital and School of Medicine was by the physician William H. Welch, for whom Clostridium welchii was named, and was entitled "Preliminary Report of Investigations concerning the Causation of Hog Cholera" (Welch, 1889). Thus, it became evident that the study of the naturally occurring diseases of animals could illuminate principles applicable to both animals and mankind and lead to improved understanding of biology in general. John Call Dalton, M.D. (1825-1889), an American physiologist, spent a year in Bernard's laboratory in Paris, about 1850. He was highly impressed with Bernard's instructional methods, which included demonstrations in living animals of important physiological principles. Subsequently, Dr. Dalton included such demonstrations in his teaching at the College of Physicians and Surgeons in New York City (Mitchell, 1895), the forerunner of the "animal labs" in which generations of students in biology and medicine were once trained. When Alexis Carrel received the Nobel Prize in 1912, the citation stated in part: " . . . you have.., proved once again that the development of an applied science of surgery follows the lessons learned from animal experimentation" (Malinin, 1979). Thus, starting in ancient times and continuing to the present day, animal experimentation has been one of the fundamental approaches of the scientific method in biological and medical research and education.

llI.

EARLY VETERINARIANS IN LABORATORY ANIMAL SCIENCE AND MEDICINE

On September 15, 1915, Dr. Simon D. Brimhall (1863-1941; V.M.D., University of Pennsylvania, 1889) (Fig. 5) joined the staff of the Mayo Clinic in Rochester, Minnesota, the first

4

FRANKLIN M. LOEW AND BENNETT J. COHEN

Fig. 4. Dr. CooperCurtice, examiningticks on a cow dead of Texas fever. Curtice contributedimportantlyto the demonstrationthat arthropodscan act as carriers of mammaliandiseases. (Courtesyof The Nation's Business.)

veterinarian to fill a position in laboratory animal medicine at an American medical research institution (Cohen, 1959b; Physicians of the Mayo Clinic and the Mayo Foundation, 1937). No such field was recognized at the time, of course; but Dr. Brimhall's activities--management of the animal facilities, development of animal breeding colonies, investigation of laboratory animal diseases (Brimhall and Mann, 1917; Brimhall and Hardenbergh, 1922), and participation in collaborative and independent research (Brimhall et al., 1919-1920)mwere the prototype of the present role of "laboratory animal veterinarians" in scientific institutions throughout the world. The decision to employ a veterinarian at the Mayo Clinic in 1915 appears to have resulted from a unique juxtaposition of institutional needs and personalities. Although the Mayo Clinic was already world renowned, organized research was in only a rudimentary stage of development. About 1910, an unsuccessful effort was made to convert an old barn, belonging to the chief of surgical pathology, Dr. Louis B. Wilson, for animal experimentation (Braasch, 1969). Then, in 1914, with Dr. William J. Mayo's active encouragement, the Division of Experimental Surgery and Pathology was created, the first real research laboratory at the clinic. Dr. Frank C. Mann, a young medical scientist from Indiana, was invited to head the division, with the primary assignment of developing a first-class animal research laboratory. Dr. Brimhall's employment followed within a year and was accompanied by the planning and ultimate construction of new animal facilities (Figs. 6 and 7). Christopher Graham, M.D., then head of the Division of Medicine, greatly influenced

the decision to employ Dr. Brimhall. Perhaps the fact that Dr. Graham also was a veterinarian (V.M.D., University of Pennsylvania, 1892) provided insights into the contributions that veterinary medicine could make to experimental surgery and pathology. Certainly, the concept of mutual support among the professions was not at that time widely held; there was, in fact, relatively little interprofessional communication between medicine and veterinary medicine then. Dr. Brimhall retired in 1922 and was succeeded by Dr. John G. Hardenbergh (1892-1963; V.M.D., University of Pennsylvania, 1916). During his 5-year tenure at the Mayo Clinic, Dr. Hardenbergh was an active clinical investigator (Hardenbergh, 19261927) as well as animal facility manager. In a stout defense of animal experimentation, he also demonstrated the communication skills in the public arena that were to serve him well later in his career (1941-1958) as executive secretary of the American Veterinary Medical Association (Hardenbergh, 1923). Dr. Carl F. Schlotthauer (1893-1959; D.V.M., St. Joseph Veterinary College, 1923), who had joined the Mayo Clinic staff in 1924 as assistant in veterinary medicine, succeeded Dr. Hardenbergh in 1927. By this time, the Mayo Foundation was functioning as the graduate medical education and research arm of the Mayo Clinic and had become formally affiliated with the University of Minnesota. Dr. Schlotthauer ultimately became head of the Section of Veterinary Medicine at the Mayo Foundation (1952) and professor of veterinary medicine at the University of Minnesota Graduate School (1945). Thus, Dr. Schlotthauer was the first veterinarian to attain a full professorship

1. LABORATORY ANIMAL MEDICINE: HISTORICAL PERSPECTIVES

5

the Mayo Foundation between 1915 and 1950, Dr. Brimhall, Dr. Hardenbergh, and Dr. Schlotthauer were the ones most closely associated with activities that today are identified with laboratory animal medicine. It is noteworthy that the Mayo Clinic/Foundation has maintained a program in animal medicine continuously for more than 85 years, having initiated it long before most medical research institutions were prepared even to consider the possible value of adding veterinarians to their professional staff (P. E. Zollman, personal communication, 1982). Dr. Karl E Meyer (1884-1974; D.V.M., University of Zurich, 1924; M.D. [honorary], College of Medical Evangelists, 1936) was an internationally known epidemiologist, bacteriologist, and pathologist. Dr. Meyer was intensely interested in matters related to laboratory animals for most of his professional life. He was the author of an early review of laboratory animal diseases (Meyer, 1928), one of the first publications of its kind in the United States. Dr. Meyer was a unique personality--vigorous, dynamic, active--a world traveler on missions related to international health; a scientist who engendered in his students respect, admiration, love, and fear in varying proportions. Together with his longtime associate Bernice Eddy (Ph.D.), a bacteriologist, Dr. Meyer developed a model animal facility at the George Williams Hooper Foundation at the University of California, San Francisco, during a 30-year tenure as director (1924-1954). Dr. Meyer often was away from the laboratory, and it fell to Dr. Eddy to supervise the animal facility, which she did with great skill and dedication. Dr. Meyer foresaw the need for and was an early advocate of the participation of veterinarians in the operation of institutional laboratory animal colonies Fig. 5. SimonD. Brimhall, V.M.D., the first veterinarian in laboratoryani- (Meyer, 1958). He figured importantly in the planning that led mal medicine at an Americanmedicalresearch institution, workedat the Mayo the University of California to create the position of "statewide Clinic from 1915to 1922. (Courtesyof Universityof Minnesota Press and Dr. veterinarian" in 1953, which subsequently was superseded by Paul E. Zollman.) the appointment of veterinarians at each of the university's major campuses. Among his many honors, Dr. Meyer received the for laboratory animal medicine-related academic activities. Charles A. Griffin Award of AALAS in 1959. Dr. Charles A. Griffin (1889-1955; D.V.M., Cornell UniverDr. Schlotthauer vigorously opposed antivivisectionist attacks on animal research. He was a leader in the statewide campaign sity, 1913) was a bacteriologist at the New York State Board of that led to adoption of the Minnesota "pound law" in 1950, i.e., Health, Division of Laboratories, Albany, New York, from 1919 a law authorizing the requisitioning for research and education to 1954. Dr. Griffin pioneered the concept of the development by approved scientific institutions of impounded, but unclaimed of "disease-free" animal colonies long before gnotobiotic techdogs and cats. Dr. Schlotthauer believed that open and honest nology had evolved (Brewer, 1980). In the 1940s, Dr. Griffin communication between medical scientists and humane society utilized progeny testing to establish a rabbit colony free of pasworkers could lead to better public understanding and support teurellosis. Additionally, he showed that Salmonella spp. could of animal research. Consequently, he was also active in humane be transmitted in meat meal (Griffin, 1952). This led feed mansociety activities, serving for many years on the board of direc- ufacturers to improve the processing of laboratory animal diets tors of the Minnesota Society for the Prevention of Cruelty to so as to eliminate Salmonella contamination. The Charles A. Animals. Dr. Schlotthauer also was an important figure in the Griffin Award of AALAS was established and named in Dr. early years of the American Association for Laboratory Animal Griffin's honor. He received the award posthumously in 1955, Science (AALAS). He was a founding member of its board of the first recipient of this prestigious award. The Griffin Laboradirectors and presented a paper on animal procurement at its tory at the New York State Board of Health central facility in first meeting, in 1950 (Schlotthauer, 1950). Albany, New York, also is named in his honor. Although other veterinarians also held appointments at Dr. Nathan R. Brewer (D.V.M., Michigan State University,

6


Fig. 6.

Dog breeding facility, Institute of Experimental Medicine, Mayo Clinic, constructed in the mid- 1920s. (Courtesy of Dr. Paul E. Zollman.)

Fig. 7. Interior of guinea pig breeding house, Institute of Experimental Medicine, Mayo Clinic, constructed in the early 1920s. (Courtesy of Dr. Paul E. Zollman.)


7

overcOme, and Dr. Brewer became supervisor of the Central Animal Quarters. Laboratory animal medicine began its modern evolution in the following years. Dr. Brewer's role was seminalmas a founder of the American Association for Laboratory Animal Science, as first president of AALAS (1950-1955), and as a "father figure" for the then youthful group of veterinarians that had been employed by other medical schools and medical research institutions in the Chicago area between 1945 and 1949. Dr. Brewer received the AALAS Griffin Award in 1962. Although retired since 1969, he remains active and attends local and national AALAS meetings. To honor his contributions to the field, AALAS instituted an annual award in 1994, the Nathan Brewer Scientific Achievement Award. Other personalities that played important roles in the early history of laboratory animal science and medicine have been characterized and their roles assessed by Brewer (1980).

IV.

THE O R G A N I Z A T I O N S OF L A B O R A T O R Y A N I M A L SCIENCE

A.

Fig. 8. Dr. NathanR. Brewer,directorof the CentralAnimalQuarters at the Universityof Chicago (1945-1969) and firstpresident of the AmericanAssociation for Laboratory Animal Science. Photograph taken in the late 1940s. (Courtesy of Dr. N. R. Brewer.)

1937; Ph.D., University of Chicago, 1936) headed the laboratory animal facilities at the University of Chicago from 1945 until his retirement in 1969 (Fig. 8). Dr. Brewer's interest in laboratory animals originated in the mid-1920s, when he started veterinary school, and continued during his graduate student years in the Department of Physiology at the University of Chicago. About 1935, Professors Anton J. Carlson (Ingle, 1979) and A. B. Luckhardt first approached Dr. Brewer about managing the University of Chicago animal facilities. They saw merit in the concept of a veterinarian, well grounded in the scientific method, as animal facility manager. They believed this arrangement would contribute to public confidence in the care and treatment of animals in research and would help to turn aside antivivisection activists. However, many investigators at the university feared that a veterinarian would dictate the conditions of care and use of animals, and they opposed the creation of this position. It was not until 1945 that this opposition was

Background

Organizations are important in scientific life as a means of implementing the content and activities of the fields they represent. Present-day students of laboratory animal science are confronted with a confusing array of organizational acronyms: AAALAC International, AALAS, ACLAM, ASLAP, CALAS, ILAR, ICLAS, NABR, and so on. It is instructive to examine why organizations such as these came into being and to evaluate their impact on laboratory animal science. Consider the milieu for research in biology and medicine in the United States about 1945. A new national policy was just being initiated to provide increased federal support of science. The use of laboratory animals began to expand rapidly as the funding of medical and biological research increased, and a host of problems as well as challenges accompanied this development. The base of knowledge about the care and diseases of laboratory animals was small. Published information was scattered and sparse. Few veterinarians were devoting themselves te "laboratory animal care," which was not yet recognized as a special field. In many institutions, animal facilities and administrative arrangements for operating them were poor. Institutions were ill prepared to accommodate increasingly large animal colonies. Simultaneously, medical scientists were under increasingly vigorous attack from antivivisectionists whose objective was to stop or limit animal research. It became essential for scientists both to confront their persistent critics and to face up to the problems they knew existed. The Chicago area was a hotbed of antivivisection activity in

8


the Medical School, were notified hastily. They intercepted Mrs. McLaughlin and the dog at the entrance of the Medical School. At this point, Mrs. McLaughlin made a citizen's arrest of Dr. Ivy and Dean Miller, and the protagonists proceeded to the Chicago Avenue police station. The dog was returned to the Medical School, and the arrests subsequently were nullified. However, the incident was given wide publicity in the media, especially in the Chicago Herald-Examiner, reflecting the antivivisection sentiments of publisher William Randolph Hearst and Mr. Hearst's close friends, Mrs. McLaughlin and actress Marion Davies. This incident illustrates the flavor of the relationships between animal research scientists and their critics in the mid- and late 1940s. Without realizing it, Mrs. McLaughlin had alerted the scientific community to the significant and determined opposition it faced. An organized response was a clear necessity.

B.

Fig. 9.

Cover page of antivivisection brochure, late 1940s.

1945 (Fig. 9). The National Antivivisection Society, based in Chicago, was distributing its literature widely and working for legislative abolition of animal research in Illinois and elsewhere. Orphans of the Storm, a humane society with a strong antivivisection outlook, was headed by its founder, Irene Castle McLaughlin, a famous dancer of the World War I era. Mrs. McLaughlin had been appointed to the Animal Advisory Committee of the Arvey Ordinance. The ordinance permitted the medical schools in Chicago to obtain unclaimed dogs and cats from the public pound. On one occasion, during an inspection of the animal facilities at Northwestern University Medical School, Mrs. McLaughlin deliberately removed a dog from its cage because she felt that the animal was not receiving adequate treatment. She planned to take the dog to her shelter in Winnetka. Dr. Andrew C. Ivy, then professor and chairman of the Department of Physiology, and Dr. J. Roscoe Miller, then dean of

The National Society for Medical Research

The National Society for Medical Research (NSMR) was created in 1946 by the Association of American Medical Colleges (AAMC) and about 100 supporting groups (Grafton, 1980). AAMC had become concerned that progress in medical science could be jeopardized if antivivisectionists were successful in their numerous campaigns to prohibit or restrict animal experimentation. It was deemed essential to establish a separate organization to counter these antiscience activities and, especially, to promote better public understanding of the needs and accomplishments of animal experimentation. Public support of animal research depended upon such understanding. NSMR headquarters were established in Chicago, and Dr. Anton J. Carlson was elected the organization's first president (Fig. 10). From its inception, NSMR contributed importantly to campaigns conducted at the state, city, and county levels to win public support for the use of public pounds as a source of unclaimed dogs and cats for research (Fig. 11). Antivivisection efforts to restrict or prohibit animal experimentation were fought successfully in several states. NSMR also developed much educational material about animal research and distributed it throughout the country. In the late 1940s, NSMR provided legal counsel to several Chicago-area research scientists who had been attacked by the Hearst newspapers. Dr. Nathan Brewer was among this group. Libel suits were filed and dragged on for several years, until shortly before William Randolph Hearst's death in 1951. The suits were concluded in favor of the scientists, but without significant monetary settlement. The Hearst publications agreed to stop publishing statements tending to damage the reputations of scientists involved in animal research. The suits and Mr. Hearst's death brought to an end the extremist approach of the Hearst publications to the vivisection-antivivisection issue. In 1952, a cause celebre developed within the American Physiological Society (APS) that also involved other constitu-

9

1. LABORATORY ANIMAL MEDICINE: H I S T O R I C A L P E R S P E C T I V E S We are determining the amount of abuse that life will endure in unanaesthetized animals m i n the name of science. We are producing frustration ulcers in experimental animals under shocking conditions--in the name of science. We are observing animals for weeks, months, or even years under infamous conditions m i n the name of science. Yet it is the National Society for Medical Research and its New York satellite that are providing the means to these ends. And how is it being accomplished? By undermining one of the finest organizations of our country. THE AMERICAN H U M A N E SOCIETY.

With the aid of the halo supplied by the faith of the American people in medical science, the NSMR converts sanctuaries of mercy into animal pounds at the beck and call of experimental laboratories regardless of how the animals are to be used. What a travesty of humanity! This may well prove to be the blackest in the history of medical science.

Fig. 10. Dr. Anton J. Carlson, professor of physiology at the University of Chicago and first president of the National Society for Medical Research. (Courtesy of the University of Chicago Archives and Dr. N. R.. Brewer.)

ent societies of the Federation of American Societies for Experimental Biology (FASEB) and the NSMR. Robert Gesell, M.D., professor and chairman of the Department of Physiology at the University of Michigan from 1923 to 1954 (Fig. 12), made the following statement at the APS business meeting on April 15: The National Society for Medical Research would have us believe that there is an important issue in vivisection versus antivivisection. To a physiologist there can be no issue on vivisection per se. The real and urgent issue is humanity versus inhumanity in the use of experimental animals. But the NSMR attaches a stigma of antivivisection to any semblance of humanity. Antivivisection is their indispensable bogie which must be kept before the public at any cost. It is their only avenue towards unlimited procurement of animals for unlimited and uncontrollable experimentation. The NSMR has had but one idea since its organization, namely, to provide an inexhaustible number of animals to an ever growing crowd of career scientists with but little biological background and scant interest in the future of man. Consider what we are doing in the name of science, and the issue will be clear. We are drowning and suffocating unanaesthetized animalsmin the name of science.

Dr. Gesell had supported the formation of NSMR but subsequently took issue with Dr. Carlson on NSMR involvement in pound legislation. Dr. Gesell also was dissatisfied with what he perceived to be a lack of interest by NSMR in promulgating more detailed humane criteria for the care and use of animals than existed at that time. He knew of the formation of the American Association for Laboratory Animal Science in 1950 and of the assistance that NSMR provided to AALAS in its formative years. This did not soften his view that NSMR was not constructively dealing with the issue of humane use of animals in research. Dr. Gesell asked that his statement be made part of the minutes. Vigorous discussion followed. Dr. Ralph Gerard, APS president, explained that it was not APS policy to include all statements by APS members in the minutes. Finally, a motion by Dr. Maurice Visscher, then professor and chairman of the Department of Physiology at the University of Minnesota (later, president of NSMR), was adopted, to be included in the minutes, "that Dr. Gesell had made a statement concerning animal experimentation which criticized physiologists and the NSMR and that the statement had been challenged" (APS minutes, April 15, 1952). At a second business meeting, on April 17, 1952, APS adopted the following formal response: The American Physiological Society reaffirms its sincere belief that the moral justification for humane animal experimentation, for the purpose of furthering biological and medical knowledge, in the interest of both human and animal welfare, is completely established. The American Physiological Society rejects the sweeping allegations made by Dr. Gesell in a recent business meeting. The American Physiological Society rejects unequivocally the inference that its members are insensitive to the moral responsibilities which they have in protecting the welfare of man and animals. The American Physiological Society expresses the hope that in the future all of its members will act in unison in promoting conditions facilitating humane animal experimentation.

Despite efforts by Dr. Gesell to prevent and suppress use of his statement by antivivisection groups, it was distributed

10

Fig. 11.


Insidecover and page 1 of NSMR Bulletin, May-June 1949,reporting a national opinion poll on favorable public attitudes toward animal research.

widely by these groups in their campaigns for legislative restriction of animal research. After all, it reflected the views of a respected American physiologist. The APS response was not similarly distributed by these groups. Dr. Carlson prepared a lengthy and thoughtful rebuttal of the Gesell statement for members of FASEB, but it too had only a limited distribution (A. J. Carlson, letter to FASEB members, September 17, 1952). After Dr. Gesell's death in 1954, his daughter, Christine Stevens, a founder and the president since 1950 of the Animal Welfare Institute, continued to espouse her father's views and her own strong opinion that too many scientists were insufficiently concerned about humane treatment of animals in research. These views have included critical commentary about NSMR and AALAS (Stevens, 1963, 1976, 1977). The Gesell-APS-NSMR controversy highlighted issues that, to this day, underlie the difficult relations between the scientific community and the animal welfare movement. Perhaps a positive result has been that the controversy also contributed to the climate of opinion that led additional numbers of medical research institutions to employ veterinarians to care for research animals. Ultimately, the controversy raised questions that influenced and should continue to influence all those having a constructive concern for both science and animal welfare. What, if any, are the appropriate limits on scientific freedom in

animal research? Who is best qualified to make judgments about the propriety of animal studies? Can "humaneness" be legislated? Is there not a moral imperative to conduct animal studies in the interest of human and animal welfare? How best can refinement of animal studies, reduction in the numbers of animals used, and replacement of animals, where appropriate, best be incorporated into the design of experiments (Russell and Burch, 1959)? In the 1980s, the National Society for Medical Research merged with the Association for Biomedical Research (ABR), which had been organized in 1979, to become the National Association for Biomedical Research (NABR), with Dr. Edward C. Melby as its first president. NABR works with scientists and elected officials on behalf of biomedical research. The Scientists' Center for Animal Welfare (SCAW) was formed about the same time to contribute scientific perspectives to laboratory animal welfare.

CO The A m e r i c a n Association for L a b o r a t o r y A n i m a l Science

By 1949, veterinarians were managing the laboratory animal facilities at five Chicago-area institutions: the University of


Fig. 12. Dr. Robert Gesell, professor and chairman, 1923-1954, Department of Physiology,Universityof Michigan. Dr. Gesell's statement at the APS business meetingin 1952became a cause celebre. Photographtaken in the late 1930s. (Courtesy of the Bentley Historical Library, Universityof Michigan.)

Chicago (Nathan R. Brewer), the University of Illinois (Elihu Bond), Northwestern University (Bennett J. Cohen), the Argonne National Laboratory (Robert J. Flynn), and the Hektoen Institute for Medical Research of Cook County Hospital (Robert J. Schroeder). The veterinarians sought one another out to exchange information and experience on the day-to-day problems they were encountering. The group met at least monthly, starting during the summer of 1949. Among the subjects reviewed at these meetings were husbandry and diseases of laboratory animals, the need to develop basic standards of animal care, and the need to counter the strident antivivisection attacks on medical science in the Chicago area. The Chicago veterinarians knew that few other veterinarians elsewhere in the country were engaged in the activity they had begun to identify as "laboratory animal care." For example, in reviewing the proceedings of a symposium on animal colony maintenance, held under the sponsorship of the New York Academy of Sciences in 1944 (Farris et al., 1945), they noted that not a single veterinarian had presented a paper. Their perception was that the problems of laboratory animal care merited organized attention, and they wondered whether others felt the same way. Special meetings were arranged when colleagues from other institutions visited Chicago. Among these colleagues were C. E Schlotthauer, D.V.M., Mayo Clinic; Charles A. Slanetz, Ph.D., director of the Central Animal Facility at the College of Physicians and Surgeons, Columbia University; Harry Herrlein, Rockland Farms, New City, New York (then a major commer-

11

cial rodent and rabbit breeding facility); C. N. W. Cumming, Carworth Farms, New City, New York (also a major rodent breeding facility at the time); and W. T. S. Thorp, D.V.M., then chief of the Laboratory Aids Branch, National Institutes of Health. These meetings were exciting, interesting, and rewarding to the participants. They demonstrated that interest in laboratory animal problems extended well beyond the Chicago area and included individuals who had a broad range of scientific, professional, and technical backgrounds. In a letter signed by the five Chicago veterinarians and sent in May 1950 to individuals in the United States and Canada thought to have an interest in the care of laboratory animals, the development of a national organization was proposed "to be open to all individuals interested in animal care work on an institutional scale" (Flynn, 1980). The response was overwhelmingly favorable, and the first meeting was convened in Chicago on November 28, 1950, with an attendance of 75. The founding members named the organization the Animal Care Panel (ACP), reflecting their broad concern with the c a r e of laboratory animals (Fig. 13). "Panel" was used in the name to emphasize the organization's purpose as a forum for the exchange of information on all aspects of animal care. Dr. Brewer was elected the first president, a post he held until 1955. During the

Fig. 13. Coverof early descriptive brochure about the Animal Care Panel, now the American Associationfor LaboratoryAnimal Science.

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early meetings of the ACP, its programs were dominated by papers on animal colony management, design of facilities and equipment, and descriptions of common diseases. This reflected the relatively underdeveloped "state of the art" with respect to the technology of animal care. ACP meetings became more sophisticated with each passing year. By the sixth meeting, in 1955, original research was being presented (Flynn, 1980). Shortly thereafter, the annual Proceedings of the Animal Care Panel was transformed into the scientific journal Laboratory Animal Care and subsequently renamed Laboratory Animal Science. The ACP grew rapidly in its institutional and individual membership and was characterized by the unique diversity of scientific,, professional, and technical backgrounds of its members. By 1960, the ACP was able to employ a full-time executive secretary, Joseph J. Garvey. He had served earlier as assistant executive secretary of NSMR and, in this position, had assisted with ACP administration, reflecting the support and encouragement the ACP received from NSMR in its formative years. From its inception, the ACP also worked to enhance the stature and training of laboratory animal technicians. This activity is exemplified in the career and contributions of George Collins (1917-1974), who served successively as supervisor of the animal facilities at the Argonne National Laboratory, Rockefeller University, and the AMA Education and Research Foundation (Brewer, 1980). He was a founding member of AALAS and, in 1963, received the AALAS Animal Technician Award. In 1967, he edited the first edition of the AALAS "Manual for Animal Laboratory Technicians," a landmark in its time (Collins, 1967). Development of standards was another early activity of the ACP. Indeed, the first edition of the "Guide for Laboratory Animal Facilities and Care" (Cohen, 1963), now known as the "Guide for the Care and Use of Laboratory Animals" (Moreland, 1978) was prepared under ACP auspices. The guide has become the basic standard regarding the use and care of animals in American research institutions. In 1967, the name of the Animal Care Panel was changed to the American Association for Laboratory Animal Science. Today, AALAS has more than 10,000 individual and institutional members and more than 48 local branches. Its annual meeting and scientific journalsmComparative Medicine (formerly Laboratory Animal Science) and a relatively new journal, Contemporary Topics in Laboratory Animal Sciencemare the principal means of scientific exchange in the field. AALAS recently published its 50-year history (McPherson and Mattingly, 1999).

D.

The Institute of Laboratory Animal Resources

Many problems of supply, standardization, and procurement of animal resources accompanied the rapid growth of medical and biological research after World War II. Concerns surfaced about these problems within the National Academy of Sciences

(NAS). These concerns developed independent of those that led to the formation of AALAS. The NAS is a private organization with a federal charter. Since 1863, it has been a principal advisor to the federal government on matters related to science and science policy (Seitz, 1967). Election to membership in NAS or its Institute of Medicine is among the highest honors a scientist can receive. It is a prestigious organization, and therefore NAS advisory groups, all of which serve without compensation, have a standing and authority they might not otherwise have. In the early 1950s, organized efforts to improve and standardize animal supply and quality had barely been initiated. Scientific standards for laboratory animal production, genetics, breeding, husbandry, and transportation did not exist. There were no good mechanisms to facilitate information exchange about laboratory animals internationally. Education and training in laboratory animal science were in an undeveloped state, and no guidelines for such training existed. Problems such as these led Dr. Paul Weiss, then chairman of the Division of Biology and Agriculture of the National Research Council (the NAS advisory arm), to appoint a Committee on Animal Resources in 1952. Dr. Weiss appointed Dr. Clarence Cook Little, the eminent geneticist and founder of the Jackson Laboratory (Bar Harbor, Maine), to be chairman. The Committee on Animal Resources recommended establishment of an Institute of Animal Resources (IAR). IAR commenced full-time operation in July 1953 (Hill, 1980). In 1956, it was renamed Institute of Laboratory Animal Resources. It was again renamed, to Institute for Laboratory Animal Research (ILAR), in the late 1990s. Historically, the ILAR office has been headed by an executive secretary (now named the director), with oversight from an advisory council and executive committee that is appointed in accordance with NAS-NRC procedures. Dr. Orson Eaton, a geneticist from the Bureau of Animal Industry, was the first executive secretary. He was succeeded by the vigorous and energetic Berton F. Hill, who also had a background in genetics. During Hill's tenure (1955-1965), ILAR became established as the major standards-development organization within laboratory animal science. In 1965, Hill was succeeded by Dr. Robert H. Yager, former director of the animal facilities at the Walter Reed Army Institute of Research. Dr. Yager was one of the "founding fathers" of ILAR, having served on the Committee on Animal Resources in 1952. Among many important ILAR accomplishments during Dr. Yager's tenure were the development of the first guidelines for education and training in laboratory animal medicine (Clarkson, 1967); the publication of an important national survey of animal facilities in the United States (Trum, 1970), following up on the first such survey, during Hill's tenure (Thorp, 1964); and enlargement of United States participation in international laboratory animal activities through support of the International Council on Laboratory Animal Science (ICLAS), known then as the Intentional Committee on Laboratory Animals (ICLA). During the formative years of ILAR and AALAS, there were


obvious areas of overlap. Both organizations had been involved in standards development, both were holding scientific meetings, and in many other areas their interests coincided. In 1962, the executive committees agreed on a division of responsibility that solidified ILAR's role in standards development (Garvey and Hill, 1963). This proved to be an important agreement because it enabled ILAR and AALAS to concentrate on the things each could do best. It also was important because of the position that ILAR standards subsequently achieved under the umbrella of the National Academy of Sciences.

13

sections, divisions, or departments of laboratory animal medicine or comparative medicine. With this development, laboratory animal medicine began to establish its separate identity. The committee then proceeded to seek recognition of specialization in the field by the AVMA. Late in 1956, the committee disbanded in favor of the American Board of Laboratory Animal Medicine and the new specialty was born.

V.

E D U C A T I O N AND TRAINING IN LABORATORY

ANIMAL MEDICINE E.

The American College of Laboratory Animal Medicine

Formal recognition of veterinary medical specialty fields by the American Veterinary Medical Association (AVMA) began in 1951 with the establishment of the American Board of Veterinary Public Health and the American College of Veterinary Pathologists (Grafton, 1974). In 1957, laboratory animal medicine was accorded the same recognition, when the American Board of Laboratory Animal Medicine (ABLAM) was incorporated under the laws of the state of Illinois, with 18 "Charter Fellows." In August 1961, the name was changed to American College of Laboratory Animal Medicine (ACLAM), and the designation "Fellow" was discontinued in favor of "Diplomat," a term used by other specialties. ACLAM was established to encourage education, training, and research in laboratory animal medicine, to establish standards of training and experience for qualification of specialists, and to certify specialists by examination. These objectives, which today are well understood and accepted, were but a vague concept in the early 1950s. On June 23, 1952, thirty-four veterinarians assembled in a meeting room at the Ambassador Hotel in Atlantic City, during the AVMA meeting, to consider the role of veterinarians in laboratory animal care. There was a lively discussion about this rapidly developing field, with special emphasis on defining activities that veterinarians were uniquely qualified to pursue ("News reports," 1952). Those in attendance noted that medical schools were employing an increasing number of veterinarians and that further growth seemed likely. They felt that more specific definition of this newly developing field was needed. The group organized as the Committee on the Medical Care of Laboratory Animals, with Dr. Nathan R. Brewer as chairman, Dr. Mark Morris as vice chairman, and Dr. W. T. S. Thorp as secretary. The decision was made to organize programs of special interest to laboratory animal veterinarians at future AVMA meetings. During the ensuing 4 years, the term "laboratory animal medicine" came into use to differentiate the activities of veterinarians from other professional or technical people working in the broad area of laboratory animal science. Additionally, within this period, a number of laboratory animal veterinarians were able to establish academic units in their institutions (Clarkson, 1961 a), some of which were identified as

Veterinarians entering "laboratory animal care" in the 1940s and early 1950s had to be largely self-trained. They relied on their basic education in veterinary medicine and on what they could learn from one another at AALAS and AVMA meetings (Clarkson, 1980). There were no post-D.V.M, training programs. The establishment of ACLAM in 1957, with its strong commitment to fostering education and training, stimulated more specific discussion of training needs in this new field. At this same time, NIH-supported training programs were being initiated in basic medical science and clinical fields in leading scientific institutions throughout the country. As mentioned in Section IV, E, by the late 1950s "laboratory animal medicine" was being conducted as an academic program in a few medical schools (Clarkson, 1961a). In such settings it became possible to consider establishing postdoctoral training. The animal resources program in the Division of Research Resources (DRR) at NIH had not yet matured, and the Animal Resources Branch (ARB) did not have training authority. However, with great insight about the underlying significance of laboratory animal medicine, the Physiology Training Committee, within the Division of General Medical Sciences (later to become the National Institute of General Medical Sciences [NIGMS]), decided to accept applications to establish a few research training programs in this new field. During the time this matter was under consideration, Dr. Howard Jenerick and Dr. J. H. U. Brown, both physiologists, served as secretaries of the committee. The committee chairmen were Dr. T. C. Ruch, professor and chairman of the Department of Physiology at the University of Washington, and Dr. Wallace O. Fenn, professor and chairman of the Department of Physiology at the University of Rochester. The committee's decision to sponsor such training was of paramount importance, because for the first time, training in laboratory animal medicine was placed on a par with other areas of research training in the medical and biological sciences. In the ensuing years, the specialists that now compose the academic core of present-day laboratory animal medicine were trained in such programs. The first training program was established in January 1960 at the Bowman Gray Medical School, directed by then assistant professor of laboratory animal medicine, Thomas B. Clarkson.

14

In July 1960, a second program was started at UCLA Medical School, directed by Bennett J. Cohen, then assistant professor of physiology and director of the vivarium. The program moved with Dr. Cohen to the University of Michigan in 1962. Later, programs were established at other medical schools and universities: Tulane University (1963, Dr. K. E Burns), Stanford University (1965, Dr. O. A. Soave), University of Florida (1965, Dr. A. E Moreland), Johns Hopkins University (1968, Dr. E. C. Melby), and University of Missouri (1968, Dr. C. C. Middleton). Edgewood Arsenal, Maryland, and Brooks Air Force Base, Texas, became the sites of training programs for military veterinarians. With strong encouragement from Dr. Jules S. Cass, chief veterinary medical officer at the Veterans Administration, a program was established in the mid-1960s at the Hines Veterans Administration Medical Center in the Chicago area. This program was guided initially by Dr. Robert E Locke. The "core of knowledge" comprising laboratory animal medicine was not well defined at the time these early programs were started. The curricula of the training programs simply reflected the outlook of the directors and the settings in which they were conducted. Some were formal graduate programs leading to a master of science degree. Others stressed residency training analogous to that of residency programs in the medical specialties. Thus, there were research-oriented programs and others that focused more on the clinical or managerial aspects of laboratory animal medicine (Clarkson, 1961b). By 1964, the need for better definition of the field had become apparent. An ILARsponsored workshop held in that year pointed to the need for educational guidelines to be used by all training programs (Clarkson, 1965). The first such guidelines subsequently were published (Clarkson, 1967). A formal process is now in place by ACLAM that reviews and approves training programs in laboratory animal medicine. In the mid-1960s, ARB received training authority, and the training grants in laboratory animal medicine were transferred there. Other aspects of the NIH extramural animal resources program also grew significantly, as, for example, the laboratory animal science program. Overall, the impact of these programs on laboratory animal medicine has been enormously beneficial. Some of the early training programs have been terminated, but most have continued, and several new programs have been started. These programs emphasize research training in comparative medicine and continue to be funded by NIH. Furthermore, a number of institutions have initiated residency programs independent of NIH sponsorship. Post-D.V.M. training is recognized today as necessary for a career in academic laboratory animal medicine. The American Society of Laboratory Animal Practitioners (ASLAP) was founded in 1967 to promote dissemination of knowledge about laboratory animal medicine, to foster research, and to serve as a spokesman for veterinarians in laboratory animal practice. ASLAP, together with ACLAM, has played an important role in encouraging continuing education


programs in the field. Continuing education has become an important adjunct to the formal training programs in laboratory animal medicine, and such activities now are a regular component of the scientific program at AVMA and AALAS meetings.

VI. I M P A C T OF LAWS, R E G U L A T I O N S , A N D G U I D E L I N E S ON L A B O R A T O R Y A N I M A L M E D I C I N E

Prior to 1966, no federal law existed in the United States specifically regulating the acquisition or care of research animals. Pressure for federal legislation mounted steadily in the late 1950s and early 1960s. Animal welfare organizations, such as the Humane Society of the United States, the Society for Animal Protective Legislation, and the Animal Welfare Institute, argued for legislation to curb alleged "pet stealing" and abuse of animals in laboratories. They used the media effectively to generate public interest in their causes (e.g., cover headline of Life, February 4, 1966: "Concentration Camps for Lost and Stolen Pets: Your Dog Is in Cruel Danger"). Organizations of the scientific community, such as AALAS, NSMR, and FASEB, argued against the proposed legislation. Their position was that the best way to foster the humane use and care of animals was to provide better support of research and training, provide funds to upgrade animal facilities, and strengthen selfregulation through mechanisms such as the newly organized (1965) American Association for Accreditation of Laboratory Animal Care (AAALAC) and institutional committees to assess the adequacy of animal care and use programs (Galton, 1967). In the mid-1990s, AAALAC assumed a more international role and renamed itself the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC International). After holding hearings on a series of bills in the House of Representatives and the Senate dealing with the regulation of animal research, Congress passed the Laboratory Animal Welfare Act in 1966. The principal purposes were to regulate commercial traffic in dogs, cats, monkeys, rabbits, guinea pigs, and hamsters and to establish standards for their housing and transportation and for "adequate veterinary care." The act, administered by the U.S. Department of Agriculture (USDA), established a legal requirement for scientific institutions to provide appropriate care for research animals by or under the direction of a veterinarian. Since its initial passage, the act has been broadened and its name changed to Animal Welfare Act (see Chapter 2). According to the USDA Animal Welfare Report of Fiscal Year 2000, the number of registered research facilities totals 1,231. Thus, the act has contributed to the betterment of animal care through its requirement for participation of veterinarians in institutional animal medicine programs. The National Institutes of Health has long recognized that


good research requires animals that are healthy and well cared for. In 1963, NIH published the first edition of the "Guide for Laboratory Animal Facilities and Care" (Cohen, 1963), as developed by the Standards Committee of the Animal Care Panel. Revised several times since 1963 by ILAR's Committee on Revision of the Guide, it is now the "Guide for the Care and Use of Laboratory Animals." Since 1963, NIH and other granting agencies have required scientific institutions to provide assurance of compliance with the standards in the guide as a condition for receiving funds for research. The guide also is used as the basis for accreditation by AAALAC. One of the basic requirements established in the guide is for the provision of adequate veterinary medical care, a concept also expressed in the regulations of the Department of Agriculture. In 1978, the Food and Drug Administration (FDA) promulgated regulations for the conduct of animal experiments relating to new or existing pharmaceutical medicinal substances, food additives, or other chemicals. These regulations, known as the Good Laboratory Practice (GLP) regulations, also specify the need for adequate diagnosis, treatment, and control of diseases in animals used in such studies. Thus, the standards of AAALAC, NIH, USDA, and FDA all include specific references to veterinary medical participation in the care of laboratory animals. In fact, these standards provide the basis for implementation of the legal requirement that research animals receive "adequate veterinary care."

VII.

REGULATION OF ANIMAL RESEARCH IN THE UNITED K I N G D O M AND C A N A D A

Until 1986, the use of animals in the United Kingdom was governed by the Cruelty to Animals Act of 1876 (French, 1975) (see Chapter 2). The Animals (Scientific Procedures) Act 1986 is now the governing legislation in the U.K. Major debates about "vivisection" occurred in Parliament in the late 1860s and early 1870s. Finally, the act was passed with the active support of leading scientists of that time. Antivivisectionists had been working for a law that would have prohibited animal research or regulated it more strictly than called for in the act. The act requires the licensing of scientists using animals. There also is provision for certificates to be issued to scientists, depending upon the species used and the nature of the experimentation. The legal regulatory relationship is between the government and the scientist, not the government and the institution, as it is in the United States. Veterinarians, as such, do not have legal standing in the implementation of the act, although within recent years veterinarians have been added to the Home Office inspectorate. In the private sector, the British Laboratory Animals Veterinary Association was organized in the early 1970s and is affiliated with the British Veterinary Association. The

15

specialty of laboratory animal medicine does not have standing comparable to that accorded ACLAM by the AVMA in the United States. Nevertheless, laboratory animal medicine clearly is an emerging field in Great Britain, and excellent animal medicine programs are evolving under the leadership of British laboratory animal veterinarians. The use of animals in Canada is not specifically regulated by federal law. However, in 1968, the Canadian Council on Animal Care (CCAC) was established by the major agencies that fund animal research (see Chapter 2). Dr. Harry Rowsell has played an instrumental role in the founding of CCAC and in its operation over the years. The CCAC assesses animal care in Canadian research laboratories based on the standards in the "Guide to the Care and Use of Experimental Animals" (CCAC, 1980). This program has been a major factor in the elevation of animal care standards and the employment of veterinarians in Canadian research laboratories. In addition, some of the provinces have laws that apply to the requisition or use of animals in research in those provinces (e.g., Ontario and Alberta).

VIII.

C O M M E R C I A L AND ACADEMIC BREEDING OF RODENTS

The development of gnotobiology in the 1950s represented a major conceptual and technological advance in the commercial breeding of healthier rodents for research (Foster, 1958). This advance had been preceded by laboratory research (Trexler and Reynolds, 1957; Reyniers, 1957) and years of attempts to breed animals that would lead to the most unambiguous results in research as possible. With the introduction of genetically engineered mice, academic and industrial animal resource programs have increasingly been engaged in breeding and characterizing numerous lines of mice with unique genetic makeup (see Chapter 3).

IX.

CONCLUSION

A complete history of the individuals and organizations that have influenced the development of laboratory animal science and medicine would require a separate volume. Some of these topics are the history of laboratory animal science internationally; the history of the major commercial and institutional animal colonies and of the important genetic stocks and strains of laboratory animals; the evolution of animal technology, including the field of gnotobiology; the contributions of animal technicians to laboratory animal science; the origins of the NIH extramural and intramural laboratory animal science programs;

16


and reviews, in historical perspective, of the major diseases of laboratory animals. Some of these topics are dealt with elsewhere (Cohen, 1979; Foster, 1980; Lindsey, 1979; McPherson, 1980; McPherson and Mattingly, 1999). Others must await documentation of the historical record. Laboratory animal science and medicine are fields of expanding horizons that provide challenging opportunities for satisfying professional careers. The following chapters clearly document the progress that has occurred while pointing to many challenges that lie ahead. It remains for each generation of laboratory animal scientists to build on the base of knowledge established by its predecessors and so determine its own future.

REFERENCES

Bernard, C. (1865). "An Introduction to the Study of Experimental Medicine" (trans. H. C. Greene, p. 123. Reprinted by Dover, New York, 1957). Braasch, W. E (1969). "Early Days of the Mayo Clinic," p. 141. Thomas, Springfield, Illinois. Brewer, N. R. (1980). Personalities in the early history of laboratory animal science and medicine. Lab. Anim. Sci. 30(4), Part 2, 741-758. Brimhall, S. D., and Hardenbergh, J. G. (1922). A study of so-called kennel lameness: Preliminary report. J. Am. Vet. Med. Assoc. 61, 145-154. Brimhall, S. D., and Mann, E C. (1917). Pathologic conditions noted in laboratory animals. J. Am. Vet. Med. Assoc. 52, 195-204. Brimhall, S. D., Mann, E C., and Foster, J. P. (1919-1920). The relation of the common bile duct to the pancreatic duct in common domestic and laboratory animals. J. Lab. Clin. Med. 5, 203-206. Bustad, L. K., Gorham, J. R., Hagreberg, G. A., and Padgett, G. A. (1976). Comparative medicine: Progress and prospects. J. Am. Vet. Med. Assoc. 169, 90-105. Canadian Council on Animal Care (CCAC) (1980). "Guide to the Care and Use of Experimental Animals," Vol. 1, 112. CCAC, Ottawa, Ontario. Clarkson, T. B. (1961a). Laboratory animal medicine and the medical schools. J. Med. Educ. 36, 1329-1330. Clarkson, T. B. (1961b). Graduate and professional training in laboratory animal medicine. Fed. Proc., Fed. Am. Soc. Exp. Biol. 20, 915-916. Clarkson, T. B. (1965). Laboratory animals. 4. Graduate education in laboratory animal medicine. Proceedings of a workshop. NAS-NRC, Publ. 1284, 33. Committee on Professional Education, Institute of Laboratory Animal Re. sources. Clarkson, T. B. (1967). A guide to postdoctoral training in laboratory animal medicine. NAS-NRC, Publ. 1483, 9. Committee on Professional Education, Institute of Laboratory Animal Resources. Clarkson, T. B. (1980). Evolution and history of training and academic programs in laboratory animal medicine. Lab. Anim. Sci. 30(4), Part 2, 790792. Cohen, B. J. (1959a). The early history of animal experimentation and animal care. 1. Antiquity. Lab. Anim. Sci. 9, 39-45. Cohen, B. J. (1959b). The evolution of laboratory animal medicine in the United States. J. Am. Vet. Med. Assoc. 135, 161-164. Cohen, B. J. (1963). "Guide for Laboratory Animal Facilities and Care." Anim. Facilities Stand. Comm., Anim. Care Panel, Public Health Serv. Publ. 1024, p. 33. U.S. Dept. of Health, Education and Welfare, Washington, D.C. Cohen, B. J. (1979). ILAR News 22(2), 26. Cohen, M. R., and Drabkin, I. E. (1948). "Sourcebook in Greek Science," p. 479. McGraw-Hill, New York.

Collins, G. R. (1967). "Manual for Laboratory Animal Technicians." Animal Technician Training Committee, American Association for Laboratory Animal Science Publ. 67-3. Joliet, Illinois. Duclaux, E. (1920). "Pasteur--The History of a Mind" (trans. E. E Smith and E Hedges), p. 363. Saunders, Philadelphia. Farris, E. J., Carnochan, E G., Cumming, C. N. W., Farber, S., Hartman, C. G., Hutt, E B., Loosli, J. K., Mills, C. A., and Ratcliffe, H. L. (1945). Animal colony maintenance. Ann. N.Y. Acad. Sci., 46, 1-126. Fisher, G. J. (1881). Historical and bibliographical notes. 12. Herophilus and Erasistratus. The Medical School of Alexandria, Bc 320-250. Ann. Anat. Surg. 4, 28-67. Flynn, R. J. (1980). The founding and early history of the American Association for Laboratory Animal Science. Lab. Anim. Sci. 30(4), Part 2, 765779. Foster, H. L, (1958). Large scale production of rats free of commonly occurring pathogens and parasites. Proc. Anim. Care Panel 8, 92-99. Foster, H. L. (1980). The history of commercial production of laboratory rodents. Lab. Anim. Sci. 30(4), Part 2, 793-798. French, R. D. (1975). "Antivivisection and Medical Science in Victorian Society," p. 425. Princeton Univ. Press, Princeton, New Jersey. Galton, L. (1967). Pain is cruel, but disease is cruel too. N.Y. Times, Sec. 6 (Magazine), February 26, p. 30. Garrison, F. H. (1929). "An Introduction to the History of Medicine," 4th ed. Saunders, Philadelphia. Garvey, J. J., and Hill, B. E (1963). Cooperation for progress. Lab. Anim. Care 13, 179-180. Grafton, T. S. (1974). The veterinary profession: A review of its progress in the United States and some indications for the future. Vet. Rec. 94, 441-443. Grafton, T. S. (1980). The founding and early history of the National Society for Medical Research. Lab. Anim. Sci. 30(4), Part 2, 759-764. Griffin, C. A. (1952). A study of prepared feeds in relation to Salmonella infection in laboratory animals. J. Am. Vet. Med. Assoc. 121, 197-200. Hales, S. (1740). "Statical Essays.'' Innys and Manby, London. Hardenbergh, J. G. (1923). The value of animal experimentation to veterinary medicine. J. Am. Vet. Med. Assoc. 62, 731-735. Hardenbergh, J. G. (1926-1927). Epidemic lymphadenitis with formation of abscess in guinea pigs due to infection with hemolytic streptococcus. J. Lab. Clin. Med. 12, 119-129. Hill, B. E (1980). The founding and early history of the Institute of Laboratory Animal Resources. Lab. Anim. Sci. 30(4), 780-785. Hoff, H. E., Geddes, L. A., and McCrady, J. D. (1965). The contributions of the horse to knowledge of the heart and circulation. Conn. Med. 29, 795-800. Ingle, D. J. (1979). Anton J. Carlson: A biographical sketch. Perspect. Biol. Med. 29, Part 2, 114-136. Lindsey, J. R. (1979). Origin of the laboratory rat. In "The Laboratory Rat" (H. G. Baker, J. R. Lindsey, and S. H. Weisbroth, eds.), Chap. 1, pp. 2-36. Academic Press, New York. Loew, E M. (1982). Animal experimentation. Bull. Hist. Med. 56, 123-126. McPherson, C. W. (1980). The origins of laboratory animal science at the National Institutes of Health. Lab. Anim. Sci. 30(4), Part 2, 786-789. McPherson, C. W., and Mattingly, S. E (1999). "Fifty Years of Laboratory Animal Science." AALAS, 9190 Crestwyn Hills Dr., Memphis TN 38125. Malinin, T. I. (1979). "Surgery and Life," p. 242. Harcourt Brace Jovanovich, New York. Meyer, K. E (1928). Communicable diseases of laboratory animals. In "The Newer Knowledge of Bacteriology and Immunity" (E. O. Jordan and I. S. Falk, eds.), pp. 607-638. Univ. of Chicago Press, Chicago. Meyer, K. E (1958). Introductory address. Lab. Anim. Sci. 8, 1-5. Mitchell, S. W. (1895). Memoir of John Call Dalton, 1825-1889. Biographical memoirs. Natl. Acad. Sci. 3, 177. Moreland, A. M. (1978). "Guide for the Care and Use of Laboratory Animals." National Academy of Sciences, National Research Council, Institute of Laboratory Animal Resources, Washington, D.C.

1. LABORATORY ANIMAL MEDICINE: HISTORICAL PERSPECTIVES News reports (1952). J. Am. Vet. Med. Assoc. 121, 257. Physicians of the Mayo Clinic and the Mayo Foundation (1937). Univ. of Minnesota Press, Minneapolis (p. 184). Reyniers, J. A. (1957). The control of contamination in colonies of laboratory animals by use of germfree techniques. Proc. Anim. Care Panel 7, 9-29. Russell, W. M. S., and Burch, R. L. (1959). "The Principles of Humane Experimental Technique," p. 238. Methuen, London. Saunders, J. B. de C. M., and O'Malley, C. D. (1950). "The Illustrations from the Works of Andreas Vesalius of Brussels," p. 128. World Publ. Co., New York. Schlotthauer, C. F. (1950). Procurement of animals. Lab. Anim. Sci. 1, 20-25. Schwabe, C. W. (1978). "Cattle, Priests, and Progress in Medicine," p. 277. Univ. of Minnesota Press, Minneapolis. Seitz, E (1967). The National Academy of Sciences. J. Wash. Acad. Sci. 57, 38-41. Singer, C. (1957). "A Short History of Anatomy and Physiology from the Greeks to Harvey," p. 209. Dover, New York. Stevens, C. (1963). Letter to the editor. Perspect. Biol. Med. 7(1) (Autumn), 129-131. Stevens, C. (1976). Humane considerations for animal-models. In "Animal

17 Models of Thrombosis and Hemorrhagic Diseases," DHEW Publ. (NIH) 76-982, pp. 151-158. U.S. Dept. of Health and Human Services, National Institutes of Health, Bethesda, Maryland. Stevens, C. (1977). Humane perspectives. In "The Future of Animals, Cells, Models, and Systems in Research, Development, Education, and Testing," pp. 16-24. National Academy of Sciences, Washington, D.C. Thorp, W. T. S. (1964). "ILAR Committee on the Animal Facilities: Survey Animal Facilities in Medical Research," Final Report and Tabular Appendix, p. 157. National Academy of Sciences, National Research Council, Institute of Laboratory Animal Resources, Washington, D.C. Trexler, P. C., and Reynolds, L. I. (1957). Flexible film apparatus for the rearing and use of germfree animals. Appl. Microbiol. 5, 406-412. Trum, B. E (1970). ILAR Committee on Laboratory Animal Facilities and Resources Survey. Laboratory animal facilities and resources supporting biomedical research. Lab. Anim. Care 20, 795-869. Welch, W. H. (1889). Preliminary report of investigations concerning the causation of hog cholera. Johns Hopkins Hosp. Bull. 1(1), 9-10. Wood, C. A., ed. (1931). "An Introduction to the Literature of Vertebrate Zoology," Chap. 4. Oxford Univ. Press, London.


Chapter 2 Laws, Regulations, and Policies Affecting the Use of Laboratory Animals Lynn C. Anderson

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Animal Welfare . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. U.S. Animal Welfare Act . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. PHS Animal Welfare Policy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. FDA Good Laboratory Practices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Interagency Cooperation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. EPA Good Laboratory Practices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E Professional and Scientific Associations . . . . . . . . . . . . . . . . . . . . . . . G. International Laws and Regulations . . . . . . . . . . . . . . . . . . . . . . . . . . III. Importation and Exportation of Animals and Animal Products . . . . . . . . . A. U.S. Department of Agriculture . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. U.S. Department of Health and Human Services . . . . . . . . . . . . . . . . C. U.S. Department of the Interior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Environmental Protection Agency . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Hazardous Substances . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Biohazards . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Chemical Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Radioactive Materials and Radiation-Emitting Equipment . . . . . . . . . V. Recombinant DNA Research Guidelines . . . . . . . . . . . . . . . . . . . . . . . . . . Appendix: Websites Pertaining to Laboratory Animals . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I.

19 20 20 25 26 27 27 28 28 29 29 30 30 30 31 31 31 31 31 32 32

INTRODUCTION

reliable data f r o m l a b o r a t o r y ani mal s has also h e l p e d assure the h u m a n e and ethical use of these animals. G o o d h u s b a n d r y practices, veterinary care, animal facility m a n a g e m e n t , and labora-

The use of animals in research, testing, and e d u c a t i o n is sub-

tory t e c h n i q u e s are all n e c e s s a r y to help assure the quality of scientific results.

ject to a m y r i a d of laws, regulations, policies, and standards. The public's interest in the t r e a t m e n t of l a b o r a t o r y ani mal s and l o b b y i n g by a n i m a l welfare and antivivisection o r g a n i z a t i o n s

ulations, and policies in the U n i t e d States that pertain to labora-

led to the p a s s a g e of m a n y of these laws and r e g u l a t i o n s during the s e c o n d half of the t w e n t i e t h century. H o w e v e r , the n e e d for

tory animal care and use. In m a n y instances, these g o v e r n i n g principles overlap, and, in s o m e cases, they p r o v i d e conflicting

LABORATORY ANIMAL MEDICINE, 2nd edition

This chapter will p r o v i d e an o v e r v i e w of the federal laws, reg-

Copyright 2002, Elsevier Science (USA). All rights reserved. ISBN 0-12-263951-0

20

LYNN C. ANDERSON

minimal requirements. In addition, every state, the District of Columbia, and many cities and towns have enacted laws or ordinances pertaining to animal cruelty or the release of pound animals to research facilities. The Appendix at the end of this chapter lists websites appropriate for laboratory animal guidelines.

II.

A.

ANIMAL WELFARE

U.S. Animal Welfare Act

The first federal legislation in the United States to protect animals was the 28-Hour Law enacted in 1873. It required that farm animals be provided food, water, and rest at least once every 28 hours during transit. Animals used for research were first protected by federal legislation in 1966, with the passage of the Laboratory Animal Welfare Act (Pub. L. 89-544). To help address public concerns over "pet nabbing," this law required licensing of dealers (individuals or corporations) that bought or sold dogs or cats for research and registration of research facilities that used dogs or cats. It also mandated minimum animal care standards for dogs and cats before and after they were used for research. The standards did not apply while the animals were being used for an experimental purpose. The law authorized the U.S. Department of Agriculture (USDA) to develop and enforce these regulations. The USDA subsequently established standards for nonhuman primates, rabbits, guinea pigs, and hamsters, in addition to those for dogs and cats. Research facilities that used dogs or cats were required to observe the USDA-specified standards for all of these species. However, facilities that did not use dogs or cats were not required to comply with the regulations for the other species. The Laboratory Animal Welfare Act was amended in 1970 and renamed the Animal Welfare Act (Pub. L. 91-579). The scope of protection was broadened to include animals used for teaching, exhibitions, and the wholesale pet industry. "Animals" included dogs, cats, nonhuman primates, rabbits, guinea pigs, and hamsters and, with certain exceptions, any other warm-blooded animal designated by the Secretary of Agriculture. Institutions (except primary and secondary schools) that used these species in research, tests, or experiments were required to register as a research facility. For the first time, zoos were required to be licensed. Agricultural exhibitors and retail pet stores were specifically exempted. The definition of "dealer" was revised to include any person who bought or sold any dog or other animal designated by the USDA for use in research, teaching, or exhibition or as a pet at the wholesale level. The 1970 amendments also expanded the minimal animal care standards. These standards applied to animals during the course of research, not only before and after experimental use.

The act did not allow the Secretary of Agriculture to establish rules, regulations, or orders with regard to the design or performance of the research. However, it required that every research facility submit an annual report that provided the number of regulated species it used and assurance that it met professionally acceptable standards for the care, treatment, and use of animals, including the appropriate use of anesthetic, analgesic, and tranquilizing drugs. In 1976, the Animal Welfare Act was amended again (Pub. L. 94-279) to include regulation of common carriers and intermediate handlers and to establish transportation standards for animals. Standards were established for shipping conditions and for the containers in which animals were shipped. The amendments also prohibited interstate promotion or shipment of animals for animal fighting ventures. The Food Security Act of 1985 (Pub. L. 99-198) included provisions to amend the Animal Welfare Act, referred to as "The Improved Standards for Laboratory Animal Act." These amendments were based on the following congressional findings: 1) the use of animals is instrumental in certain research and education for advancing knowledge of cures and treatment for diseases and injuries which afflictboth humans and animals; 2) methods of testing that do not use animals are being and continue to be developed which are faster, less expensive, and more accurate than traditional animal experiments for some purposes and further opportunities exist for the development of these methods of testing; 3) measures which eliminate or minimizethe unnecessaryduplication of experiments on animalscan result in more productiveuse of federal funds; and 4) measureswhich help meet the public concernfor laboratory animalcare and treatmentare important in assuring that research will continue to progress. Until 1985, the Animal Welfare Act requirements and USDA regulations were essentially limited to animal care, housing, and transportation standards. The new amendments included specific requirements for research facilities that were related to the experimental use of animals. The law clearly states, however, that nothing in the act should be construed as authorizing the Secretary of Agriculture to promulgate rules, regulations, or orders with regard to the design or performance of research protocols. It also mandates that the USDA may not interrupt the conduct of research during inspections. The Pet Theft Act of 1990 was the fourth amendment to the Animal Welfare Act. It was incorporated in the 1990 Farm Bill and referred to as the "Protection of Pets" legislation. This amendment established a 5-day holding period for dogs and cats held at pounds and shelters (both private and public) or research facilities. This period was designed to allow pet owners and prospective owners the opportunity to claim or adopt animals before they are sold or used for research.

1. USDA Regulations The Animal Welfare Act authorizes the USDA to develop regulations based on the act. Within the USDA, the Animal Wel-

2. LAWS, REGULATIONS, AND POLICIES AFFECTING THE USE OF LABORATORY ANIMALS

fare Act is administered through the Animal and Plant Health Inspection Service (APHIS). All rules must be developed in consultation and cooperation with other federal departments and agencies and reviewed and approved by the Office of Management and Budget. The USDA is required to publish any new regulations or changes in existing regulations in the Federal Register and allow a 60-day period during which the public may comment. The final rule on the regulations is published in the Federal Register, along with an effective implementation date. The complete set of USDA regulations and standards are published in the Code of Regulations (CFR), Title 9, Animals and Animal Products, Subchapter A, Animal Welfare. Part 1 defines the terms used, Part 2 provides the regulations, Part 3 specifies the standards, and Part 4 includes the rules of practice governing proceedings under the Animal Welfare Act. In addition, the USDA has issued the "Animal Care Policy Manual" to further clarify the intent of the Animal Welfare Act. The principle components of the animal welfare regulations that pertain to research facilities are provided in Part 2, Subparts C and D, which are summarized below. a. Regulated Species. The regulated species include any live or dead dog, cat, nonhuman primate, guinea pig, hamster, rabbit, aquatic mammal, or any other warm-blooded animal that is being used or is intended for use in research, teaching, testing, experimentation, or exhibition or as a pet. Birds, rats of the genus Rattus, and mice of the genus Mus bred for use in research, teaching, or testing, and horses and farm animals intended for u s e a s food or fiber or used in studies to improve production and quality of food and fiber, are specifically excluded. b.

Licensing.

Any person operating or desiring to operate as a dealer, broker, exhibitor, or operator of an auction sale must be licensed by the USDA and pay an annual fee. A dealer is any person who, for compensation or profit of more than $500 per year, buys, sells or negotiates the purchase of, delivers for transportation, or transports a regulated animal for research, teaching, testing, experimentation, or exhibition or for use as a pet or a dog for hunting, security, or breeding purposes. Retail pet stores are exempt unless they sell to a research facility, exhibitor, or wholesale dealer. Dogs and cats acquired by a dealer or exhibitor must be held for 5 full days, not including the day of acquisition, after acquiring the animal. If the animal was acquired from a contract animal pound or shelter, the animal must be held for at least 10 full days. If the animal is then sold to another dealer, the subsequent dealer is required to hold the animal for a minimum of 24 hours. Research facilities that obtain dogs and cats from sources other than dealers, exhibitors, and exempt persons must

21

also hold the animals for 5 full days, not including the day of acquisition or time in transit, before the animals are used by the facility. c. Registration. Research facilities, intermediate handlers, and common carriers of regulated species must register with the USDA every 3 years; any revisions to the initial registration must be provided at the time of reregistration. Research facilities are defined as any institution, organization, or person that uses live animals in research, testing, or experiments; that purchases or transports live animals; or that receives federal funds for research, tests, or experiments. The Secretary of Agriculture may exempt facilities from registration if they do not use cats, dogs, or a substantial number of other regulated species. d.

IACUC Responsibilities.

To help assure humane experimental animal use, the 1985 amendments required every animal research facility to establish an Institutional Animal Committee, subsequently designated by the USDA as an Institutional Animal Care and Use Committee (IACUC). Congress mandated that the committee include at least three members appointed by the chief executive officer of the research facility. The members must possess sufficient ability to assess animal care, treatment, and practices in experimental research as determined by the needs of the research facility and shall represent society's concerns regarding the welfare of animal subjects. At a minimum, the IACUC must include one member who is a doctor of veterinary medicine and one member who is not affiliated in any way with the research facility other than as a member of the IACUC. The nonaffiliated member cannot be a member of the immediate family of a person who is affiliated with the facility and will provide representation for general community interests in the proper care and treatment of animals. In instances where the IACUC consists of more than three members, not more than three members can be from the same administrative unit of the facility. The IACUC is responsible for making recommendations to the research facility's administrative representative, designated as the Institutional Official, regarding any aspect of the research facility's animal program, facilities, or personnel training. It is required to review, at least once every 6 months, the research facility's program for humane care and use of animals, based on USDA regulations. The IACUC must also conduct an inspection of all animal study areas and animal facilities at least once every 6 months. Exceptions to the study area inspection requirement may be made by the Secretary of Agriculture if animals are studied in their natural environment or the study area is difficult to access. After each program review and inspection, the committee must file reports of its evaluations with the Institutional Official

22

of the research facility. This report must be signed by a quorum, or majority, of the committee members and must include any minority views expressed by committee members. The report must identify any violation of USDA standards, including any deficiencies in animal care or treatment and any deviations in research practices from originally IACUC-approved proposals. Significant deficiencies, defined as those that threaten animal health or safety, must be distinguished from minor deficiencies. A specific plan and reasonable opportunity for correcting problems must also be provided in the report. If, however, corrections are not implemented, the committee, in consultation with the Institutional Official, must notify the USDA and the funding federal agency of the deficiencies or deviations. The report must be maintained on file at the research facility for a minimum of 3 years and be made available during inspections by the USDA or any federal funding agency. Federal research facilities are required to have committees with the same composition and responsibilities, except that they are to report deficiencies or deviations to the head of the federal agency conducting the research. The committee is also charged with reviewing, and, if warranted, investigating concerns involving the care and use of research animals. These concerns may be raised by members of the public or laboratory or research personnel who report issues of noncompliance. The committee must also establish a mechanism for addressing such concerns. In addition, the IACUC is responsible for reviewing and approving all proposed activities or significant changes in activities related to the care and use of animals. It is authorized to require modifications in or withhold approval for these activities; it may also suspend an activity if it determines that the activity is not being conducted in accordance with the IACUC-approved procedures. Any suspended activities must be reported to the USDA. As part of its review, the IACUC must assure that the activities are in accordance with the regulations unless acceptable justification for a departure is presented in writing. A proposal to conduct an activity or to make changes in an ongoing activity involving animals must provide the following information: (1) the species and approximate number of animals to be used; (2) a rationale for involving animals and for the appropriateness of the species and numbers of animals to be used; (3) a complete description of the proposed use of the animals; (4) a description of procedures designed to assure that discomfort and pain to animals will be limited to that which is unavoidable for the conduct of scientifically valuable research, including provision for the use of pharmacologic agents to minimize animal discomfort and pain; and (5) a description of any euthanasia method to be used. Research protocols submitted to the IACUC must also provide assurance that animal discomfort, distress, or pain will be avoided or minimized. A written, narrative description of the methods and sources used to determine that alternatives are not

LYNN C. ANDERSON

available is required for procedures that might cause more than momentary or slight pain or distress. The investigator must also provide written assurance that the activities do not unnecessarily duplicate previous experiments. For potentially painful procedures, a veterinarian must be consuited. Sedatives, analgesics, or anesthetics must be provided, unless withholding them is scientifically justified in writing and approved by the IACUC. In such instances, the pain-relieving agents may be withheld only for the period of time necessary to meet research objectives. During its review, the committee must also be assured that paralytics will not be used without anesthesia. Animals that would otherwise experience severe or chronic pain or distress that cannot be relieved must be euthanatized during or after the procedure. For all research protocols, the method of euthanasia must produce rapid unconsciousness and subsequent death without evidence of pain or distress. Survival surgical procedures must be performed using aseptic technique and sterile instruments; members of the surgical team must wear gloves and masks. Appropriate preoperative and postoperative care must be provided. Major survival surgery on nonrodents may be conducted only in facilities intended for that purpose and must be maintained under aseptic conditions. An animal may not be used in more than one major operative procedure from which it is allowed to recover, unless it is scientifically justified in writing, required as a routine veterinary procedure, or required to protect the health or well-being of the animal. In other special circumstances, requests for exemptions may be made to the administrator of the USDA's Animal and Plant Health Inspection Service. The committee must also be assured that the animals' living conditions will be appropriate for their species. The housing, feeding, and nonmedical care of the animals must be directed by a veterinarian or other scientist trained and experienced in the proper care, handling, and use of the species. The IACUC must also be assured that personnel maintaining or studying animals are appropriately qualified and trained. To help protect trade secrets, the law stipulates that the research facility is not required to disclose trade secrets or commercial or financial information publicly or to the IACUC. It is unlawful for any member of an IACUC to release any confidential information of the research facility, including trade secrets, processes, operations, style of work, or apparatus. Furthermore, the law protects the identity, confidential statistical data, and amount or source of any income, profits, losses, or expenditures of the research facility. Committee members may not use or attempt to use or reveal to any other person any information that is entitled to protection as confidential information. Failure to comply with these requirements could result in a member's being removed from the committee, fined, and imprisoned. Any individual or research facility injured in its business or property by reason of a violation of the confidentiality rules may recover all actual and consequential damages.


e.

Personnel Qualifications.

The 1985 amendments also, for the first time, required research facilities to ensure that all scientists, research technicians, animal technicians, and other personnel involved with animal care, treatment, and use are qualified to perform their duties. This responsibility requires each institution to provide training and instruction on the humane methods of animal maintenance and experimentation, including the basic needs and the proper handling and care of each species, preprocedural and postprocedural care of animals, and methods of aseptic surgery. Personnel must also be instructed about research or testing methods that minimize or eliminate the use of animals or limit animal pain or distress and the utilization of information services that would help them search for alternatives. In addition, they must be informed about the methods whereby deficiencies in animal care and treatment should be reported.

f.

Information Services.

To support the required training, the 1985 amendments mandated the Secretary of Agriculture to establish information services at the National Agriculture Library (NAL) to provide (1) information pertinent to employee training; (2) methods that could prevent unintended duplication of animal experimentation as determined by the needs of the research facility; (3) improved methods of animal experimentation that could reduce or replace animal use and minimize pain and distress, such as anesthetic and analgesic procedures. The Animal Welfare Information Center, NAL, meets these requirements.

g.

Attending Veterinarian.

Each research facility is required to have an attending veterinarian with training or experience in laboratory animal science and medicine who has direct or delegated program responsibility for activities involving animals at the research facility. Parttime or consulting veterinarians must provide a written program of veterinary care and regularly scheduled visits to the research facility. The veterinarian is authorized to ensure the provision of adequate veterinary care and to oversee the adequacy of animal care and use. Adequate veterinary care includes the availability of appropriate facilities, personnel, equipment, and services. It also includes the use of appropriate methods to prevent, control, diagnose, and treat diseases and injuries and the provision of emergency veterinary medical care. The veterinarian must ensure that all animals are observed at least once daily to assess their health and well-being. The veterinarian is also responsible for providing guidance to investigators and other personnel regarding the handling, immobilization, anesthesia, analgesia, tranquilization, and euthanasia of animals. Adequate preprocedural and postprocedural care must be provided in ac-

23

cordance with current established veterinary medical and nursing practices. h.

Records.

The USDA requires records to be maintained for each IACUC meeting; each proposed activity involving animals, including any significant changes; the status of IACUC approval for each activity or change; and semiannual IACUC reports and recommendations. Every research facility must also maintain records concerning any dog or cat purchased, owned, held, transported, euthanatized, or sold. These records must document the animal's source and date of acquisition, USDA-designated unique identification tag or tattoo number, species or breed, sex, date of birth or approximate age, and any distinguishing physical characteristics. The transportation, selling, or other disposition of a dog or cat must also be documented, including the name and address of the carrier (if transported) and of the new owner (if sold or donated). With the exception of the source and date of acquisition, these records must accompany any shipment of dogs or cats. A health certificate signed by a licensed veterinarian must accompany all shipments of dogs, cats, and nonhuman primates. Records that relate directly to activities approved by the IACUC must be maintained for the duration of the activity and for an additional 3 years after completion of the activity. A copy of all other records and reports must be maintained for 3 years and shall be available for inspection and copying by authorized APHIS or federal funding agency representatives. Each research facility must also submit an annual report to the USDA on or before December 1 of each calendar year to provide information relevant to the immediately preceding fiscal year (October 1-September 30). The report must assure (1) that professionally acceptable standards governing the care, treatment, and use of animals were followed; (2) that each principal investigator has considered alternatives to painful procedures; and (3) that the facility is adhering to the USDA standards and regulations, unless the IACUC has approved exceptions specified and explained by the principal investigator. A summary of any exceptions, including a brief explanation and the species and number of animals affected, must be attached to the annual report. In addition, the report must state the location of all facilities where animals were housed or used in actual research, testing, teaching, or experimentation or were held for these purposes. The common names and the numbers of animals used must be reported in one of three categories: (1) activities involving no pain, distress, or use of pain-relieving drugs; (2) experiments, teaching, research, surgery, or tests where appropriate anesthetic, analgesic, or tranquilizing drugs were used; and (3) painful activities where the use of painrelieving agents would have adversely affected the procedures, results, or interpretation of the activity. An explanation of the

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LYNN C. ANDERSON

animal procedure(s) conducted in the third categorymust be attached to the annual report. In addition, the number of animals being bred, conditioned, or held for use, but not yet used, must be listed. The USDA compiles the information contained in the reports from all registered research facilities and submits an annual summary to Congress.

2.

U S D A Standards

Dealers, exhibitors, and research facilities are required to meet minimal housing, operating, animal health, husbandry, and transportation standards. These include feeding, watering, sanitation, lighting, ventilation, shelter from extremes of weather and temperatures, adequate veterinary care, and separation by species where the Secretary of Agriculture finds it necessary for the humane handling, care, or treatment of animals. Specific standards, including minimal space requirements, are provided for dogs, cats, guinea pigs, hamsters, rabbits, nonhuman primates, and marine mammals. The specifications are similar for all species, except marine mammals, and are more detailed for dogs, cats, and nonhuman primates. General standards are also provided for other warm-blooded species, including farm animals used for biomedical research purposes or for testing and production of biologicals for humans or nonagricultural or nonproduction animals. It is beyond the scope of this chapter to provide detailed information regarding these standards. However, several of the most notable aspects, as required by the 1985 amendments to the Animal Welfare Act, are summarized below.

a.

Canine Opportunity for Exercise.

Dogs and cats must be housed in compatible groups. Dealers, exhibitors, and research facilities must develop, document, and follow an appropriate plan, approved by the attending veterinarian, to provide dogs over 12 weeks of age with the opportunity for exercise. This rule does not apply to individually housed dogs provided with at least twice the minimum floor space required and dogs that are group-housed in floor space that meets the minimum space standards for each dog. Bitches with litters and incompatible, aggressive, or vicious dogs are also exempt. The attending veterinarian may also exempt dogs from this program if participation would adversely affect the dog's health or well-being. Such exemptions made by the attending veterinarian must be documented and reviewed at least every 30 days by the veterinarian, unless the condition is permanent. The IACUC may also approve exemptions if the principal investigator determines that it is inappropriate for certain dogs to exercise or be group-housed. This exception must be reviewed annually by the IACUC. Records of these exemptions must be maintained and made available to the USDA or federal funding agency

upon request. If a dog is housed without sensory contact with another dog, it must be provided with positive contact with humans at least daily. b.

Psychological Well-being of Nonhuman Primates.

Dealers, exhibitors, and research facilities must also develop, document, and follow an appropriate plan for environmental enhancement adequate to promote the psychological well-being of nonhuman primates. The plan must be in accordance with currently accepted professional standards as cited in appropriate professional journals or reference guides and as directed by the attending veterinarian. At a minimum, the plan must address the social needs of nonhuman primate species known to exist in social groups in nature. Individual animals that are vicious, overaggressive, or debilitated should be individually housed. Nonhuman primates that are suspected of having a contagious disease must be isolated from healthy animals in the colony as determined by the attending veterinarian. Group-housed nonhuman primates must be compatible, as directed by the attending veterinarian. Individually housed nonhuman primates must be able to see and hear members of their own or compatible species unless the attending veterinarian determines that this arrangement would endanger their health or well-being. Primary enclosures must be enriched by providing means of expressing noninjurious species-specific behavior. Environmental enrichment devices may include perches, swings, mirrors, manipulanda, and foraging or task-oriented food items. Interaction with personnel is recommended, provided it is consistent with safety precautions. Special attention is required for infant and young juvenile nonhuman primates, those that exhibit signs of psychological distress, those entered in IACUC-approved research protocols that require restricted activity, and individually housed nonhuman primates without sensory contact with nonhuman primates of their own or compatible species. Great apes weighing more than 110 lb must be provided additional opportunities to express species-typical behavior. If a nonhuman primate must be maintained in a restraint device for an IACUC-approved protocol, such restraint must be for the minimum period possible. If the protocol requires more than 12 hours of continuous restraint, the nonhuman primate must be provided the daily opportunity for at least 1 continuous hour of unrestrained activity, unless the IACUC approves an exception. Such an exception must be reviewed at least annually. The attending veterinarian may also exempt an individual nonhuman primate from participation in the environmental enhancement plan in consideration of its well-being. However, such an exemption must be documented and reviewed by the attending veterinarian every 30 days. All exemptions must be available for review by the USDA and federal funding agencies upon request and reported in the annual report to the USDA.

2. LAWS,REGULATIONS,AND POLICIES AFFECTING THE USE OF LABORATORYANIMALS 3.

USDA Enforcement

The USDA is also charged with enforcement of the regulations. The Animal Care section of the USDA's Animal and Plant Health Inspection Service (APHIS) is responsible for assuring compliance of transportation, sale, and handling of animals used in laboratory research. The law requires the USDA to inspect each research facility at least once each year and, in the case of deficiencies or deviations from the standards promulgated under the act, to conduct follow-up inspections as necessary until all deficiencies or deviations are corrected. APHIS inspections and investigations may also be conducted as the result of alleged violations of the Animal Welfare Act, in response to public or internal complaints. Each research facility is required to permit APHIS officials to enter its place of business; to examine and make copies of the required records; to inspect the facilities, property, and animals; and to document, by taking photographs and other means, conditions and areas of noncompliance. Animals may be confiscated or euthanatized by the authority of an APHIS official, if the animal is suffering as a result of the research facility's failure to comply with any provision of the regulations or standards. The APHIS official must, however, give the facility the opportunity to correct the condition and provide adequate care to the animal. If deficiencies remain uncorrected, the USDA may take legal action, including the use of fines and/or suspension or revocation of registration or licenses.

B.

PHS Animal Welfare Policy

The Health Research Extension Act of 1985 (Pub. L. 99-158), Section 495, Animals in Research, mandated the Secretary of Health and Human Services, acting through the director of the National Institutes of Health, to establish guidelines for the proper care and treatment of animals used in biomedical and behavioral research. Any institution receiving support through the U.S. Public Health Service (PHS) for animal research, training, biological testing, or animal-related activities, must provide extensive written assurance of their compliance with PHS Policy on Humane Care and Use of Laboratory Animals (PHS Policy). The policy applies to all PHS-conducted or supported activities involving animals regardless of where they are conducted. Most of the federally funded animal research in the United States is channeled through the PHS, including the National Institutes of Health (NIH); Alcohol, Drug Abuse and Mental Health Administration (ADAMHA); Centers for Disease Control and Prevention (CDC); Food and Drug Administration (FDA), Health Resources and Services Administration (HRSA); and Office of the Assistant Secretary for Health. The Office of Laboratory Animal Welfare (OLAW), NIH, is responsible for the implementation, interpretation, and evaluation of compliance with the PHS Policy and for education of in-

25

stitutions and investigators receiving PHS support. No activity involving animals may be conducted or supported by the PHS unless the institution conducting the activity has an approved written Animal Welfare Assurance on file with OLAW. OLAW may approve or disapprove the assurance or may negotiate a satisfactory assurance with the institution. A new assurance must be submitted at least once every 5 years. The assurance must fully describe the institution's program for the care and use of animals in PHS-conducted or PHS-supported activities. OLAW is also responsible for conducting site visits to selected institutions and for evaluating allegations of noncompliance with PHS Policy. If significant problems are identified and not corrected within a reasonable period of time, the director of the NIH may suspend or revoke funding to an individual investigator or institution. The PHS Policy implements the "U.S. Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training," developed by the Interagency Research Animal Committee (Table I). The policy stipulates that institutions must have an Animal Welfare Assurance approved by OLAW and an Institutional Animal Care and Use Committee (IACUC) that is responsible for reviewing proposed projects, evaluating the animal care and use program, and inspecting facilities. The IACUC must maintain records of its activities and must report at least annually to OLAW. The policy also includes the information required for PHS applications or proposals that involve animal use. In addition to requiring compliance with the Animal Welfare Act, the PHS Policy requires institutions to use the "Guide for the Care and Use of Laboratory Animals" (seventh edition, 1996; or subsequent editions) as the basis for developing and implementing an institutional program for activities involving animals. The guide, written under the auspices of the Institute for Laboratory Animal Research of the National Academy of Sciences, applies to all live vertebrate animals, including traditional laboratory animals, farm animals, wildlife, and aquatic animals used in research, teaching, or testing. It emphasizes performance standards, which are less prescriptive and more flexible than engineering standards. The guide also encourages the application of professional judgment when addressing unique circumstances. Recommendations in the guide are based on published data, scientific principles, expert opinion, and experience with methods and practices that are consistent with high-quality, technically and scientifically appropriate, humane animal care and use. The guide provides recommendations for occupational health and safety programs. Numerous relevant references are provided. The PHS Policy also requires that euthanasia of animals be conducted in accordance with the "Report of the American Veterinary Medical Association (AVMA) Panel on Euthanasia." In addition, the guide recognizes accreditation by the Association for the Assessment and Accreditation of Laboratory Animal Care (AAALAC) International. Institutions that

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LYNN C. ANDERSON Table I U.S. GovernmentPrinciples for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training

I. The transportation, care and use of animals should be in accordance with the Animal Welfare Act (US. Code, Vol. 7, Secs. 2131 et seq.) and other applicable Federal laws, guidelines and policies II. Procedures involving animals should be designed and performed with due consideration of their relevance to human or animal health, the advancement of knowledge or the good of society III. The animals selected for a procedure should be of an appropriate species and quality and the minimum number required to obtain valid resuits. Methods such as mathematical models, computer simulation, and in vitro biological systems should be considered IV. Properuse of animals, including the avoidance or minimization of discomfort, distress, and pain when consistent with sound scientific practices, is imperative. Unless the contrary is established, investigators should consider that procedures that cause pain or distress in human beings may cause pain or distress in other animals V. Procedures with animals that may cause more than momentary or slight pain or distress should be performed with appropriate sedation, analgesia, or anesthesia. Surgical or other painful procedures should not be performed on unanesthetized animals VI. Animals that would otherwise suffer severe or chronic pain or distress that cannot be relieved should be painlessly killed at the end of the procedure, or, if appropriate, during the procedure VII. The living conditions of animals should be appropriate for their species and contribute to their health and comfort. Normally the housing, feeding, and care of all animals used for biomedical purposes must be directed by a veterinarian or other scientist trained and experiencedin the proper care, handling, and use of the species being maintained or studied. In any case, veterinary care shall be provided as indicated VIII. Investigators and other personnel shall be appropriately qualified and experienced for conducting procedures on living animals. Adequate arrangements shall be made for their inservice training, including the proper and humane care and use of laboratory animals IX. Where exceptions are required in relation to the provisions of these Principles, the decisions should not rest with the investigators directly concerned but should be made, with due regard to Principle II, by an appropriate review group such as the institutional animal research committee. Such exceptions should not be made solely for the purposes of teaching or demonstration are accredited by AAALAC are assigned "Category 1" assurance status and are not required to submit their most recent semiannual report to OLAW with the assurance statement. Those that are not accredited are awarded "Category 2" assurance status and are required to submit their semiannual report to OLAW. Unlike the Animal Welfare Act, the PHS Policy requires that the IACUC consist of at least five (not three) members, including a doctor of veterinary medicine, a practicing scientist with experience in animal research, an individual whose primary concerns are in a nonscientific area, and an individual who is not affiliated with the institution in any way other than as a member of the IACUC. One person may meet more than one of these four requirements, provided there is a minimum of five IACUC members. The IACUC is responsible for reviewing all proposed research projects or proposed significant changes in ongoing re-

search projects in a manner similar to that required by the USDA animal welfare regulations. Whereas the USDA regulations require annual review of ongoing activities, PHS requires the IACUC to conduct a complete review of ongoing activities at least once every 3 years. R e c o r d s - - i n c l u d i n g the minutes of all IACUC meetings; records of applications, proposals, and proposed significant changes in the care and use of animals and their respective IACUC evaluation; IACUC semiannual program reports and recommendations; and records of accrediting body d e t e r m i n a t i o n s - - m u s t be maintained for at least 3 years. Records related to IACUC-approved activities must be held for 3 years beyond the completion of the activity. All records must be accessible for review and copying by an authorized OLAW or other PHS representative. The PHS also requires the IACUC to report to OLAW, through the Institutional Official, at least once every 12 months. This annual report must include any changes in the institution's accreditation status, program for animal care and use, or IACUC membership, as well as the dates of the IACUC's semiannual evaluations of the institution's program and facilities.

C.

FDA Good Laboratory Practices

The federal Food, Drug, and Cosmetic Act requires the Food and Drug Administration (FDA), under the Department of Health and Human Services, to ensure proper procedures for the care and use of laboratory animals, as implemented by the Good Laboratory Practice (GLP) regulations (21 CFR, Part 58) that became effective June 1979 and were amended in 1987. The regulations establish basic standards for conducting and reporting nonclinical safety testing and are intended to assure the quality and integrity of safety data submitted to the FDA in support of an application for a research or marketing permit. Such permits are required for human and animal drugs, human biological products, medical devices, diagnostic products, food and color additives, and electronic medical products. Basic research studies, clinical or field trials in animals, and human subject trials are not covered by the GLP regulations. Institutions seeking FDA approval of their products must establish written protocols and standard operating procedures (SOPs); provide adequate facilities, equipment, and animal care; properly identify test substances; and accurately record observations and report results for preclinical studies. The FDA relies heavily on documented adherence to the written protocols and SOPs in judging the acceptability of safety data submitted in support of marketing or clinical research permits. Every study conducted under GLP regulations must have a study director, who is ultimately responsible for the implementation of the protocol and conduct of the study. Each institution must also have a quality assurance unit that monitors the conduct of studies to assure that the protocol is being followed and the records are properly maintained.


To help assure compliance with the GLP regulations, the FDA conducts periodic, routine surveillance inspections and data audits of public, private, and government nonclinical laboratories that may be performing tests on GLP-regulated products. They may also conduct directed inspections to verify the reliability, integrity, and compliance of important or critical safety studies being reviewed in support of pending applications for product research or premarketing approval. In addition, the FDA may conduct inspections to investigate potential noncompliance issues brought to the FDA by whistle-blowers, the news media, industry complaints, FDA reviewers, other government contacts, or other sources. Inspections of commercial laboratories are conducted without prior notification. Initial inspections of university and government laboratories are initiated only after the facility has been informed in a letter from the Bioresearch Monitoring Program coordinator, Division of Compliance Policy, Office of Enforcement, FDA, of the intent to inspect. The inspections include a review of the institution's organization and personnel, quality assurance unit, facilities, equipment, testing facility operations, reagents and solutions, test and control articles, protocols and conduct of nonclinical studies, records, and reports. In addition, the animal care program is evaluated to determine if the animal care and housing is adequate to preclude stress and uncontrolled influences that could alter the response of the test system to the test article. The inspection includes the animal housing room(s) and SOPs for the environment, housing, feeding, handling, and care of laboratory animals. Newly received animals must be appropriately isolated, identified, and evaluated for health status. Animals of different species, or animals of the same species on different projects, must be separated. Daily logs of animal health observations are randomly reviewed and treatment of animals must be authorized and documented. Cages, racks, and accessory equipment must be cleaned and sanitized, and appropriate bedding must be used. Feed and water samples must be collected at appropriate sources and analyzed periodically, and the analytical documentation must be retained. The pest control program is also reviewed. Copies of the IACUC's standard operating procedures and meeting minutes are reviewed to verify committee operation. A data audit is also conducted to compare the protocol and amendments, raw data, records, and specimens against the final safety assessment report. This audit is intended to substantiate that protocol requirements were met and that findings were fully and accurately reported. The study methods described in the final report are compared against the protocol and SOPs to confirm that the GLP requirements were met. In addition to reviewing the procedures and methods for animal housing, identification, health observations, and treatment, the audit includes review of the handling of dead or moribund animals and necropsy, histopathology, and pathology procedures. The audit also includes a detailed review of study records and raw data. These data may include animal weight records, food consump-

27

tion records, and clinical pathology analyses and ophthalmologic examinations. Inspection reports are classified according to the findings and whether or not objectionable conditions or practices were found during the inspection. If regulatory and/or administrative actions are recommended, the FDA may hold an informal conference, conduct a reinspection, or issue a warning letter. It may also reject a nonclinical study or studies, disqualify the institution, withhold or revoke a marketing permit, or terminate a permit for preclinical studies.

D.

Interagency Cooperation

As part of the Animal Welfare Act, Congress required the Secretary of Agriculture to consult and cooperate with other federal departments and agencies concerned with the welfare of animals used in research. Specifically, the Secretary of Agriculture must consult with the Secretary of Health and Human Services prior to the issuance of regulations. In 1995, authorized representatives of the USDA, NIH, and FDA signed a Memorandum of Agreement concerning Laboratory Animal Welfare. The cooperating agencies made the following agreements based on mutual concern and interest regarding animal welfare: (1) to share registries, inventories, and listings of organizations that fall under their respective authority; (2) to share significant adverse findings regarding animal care and use and the actions taken by the agency in response to those findings; (3) to share evidence of serious noncompliance with required standards or policies for the care and use of laboratory animals; (4) to inform successive evaluation teams and to avoid redundant evaluations of the same entities; (5) to consult and coordinate with each other on regulatory or policy proposals and significant policy interpretations; and (6) to provide each other with resource persons for scientific and educational seminars, speeches, and workshops related to laboratory animal welfare.

E.

EPA Good Laboratory Practices

The Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), enacted in 1947, authorizes the administrator of the Environmental Protection Agency (EPA) to register and control the use of pesticides. To register a new pesticide, the EPA conducts a premarket review of its potential health and environmental effects. Animal tests must be conducted, according to the EPA's Good Laboratory Practices, which differ in some respects from the FDA's Good Laboratory Practices. According to the final rules published in 1983, the Toxic Substances Control Act (TSCA) also requires the use of GLP standards for conducting chemical studies required by the TSCA.

LYNN C. ANDERSON

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F.

Professional and Scientific Associations

Many scientific and professional associations have adopted position statements regarding the care and use of laboratory animals. Several also provide resources that have made a significant impact on the generally accepted practices for animal facilities. 1.

Institute for Laboratory Animal Research

The Institute for Laboratory Animal Research (ILAR) is a division of the Commission on Life Sciences, one of eight major units with the National Research Council (NRC). The NRC is the working arm of the National Academy of Sciences, a private, nongovernmental, nonprofit organization chartered by Congress in 1863 to "investigate, examine, experiment, and report upon any subject of science or a r t . . , whenever called upon by a federal agency, a group internal to the NRC, or Congress." ILAR is governed by a 15-member council of experts in laboratory animal medicine, zoology, genetics, medicine, ethics, and related biomedical sciences. The council provides direction for ILAR's programs. Many of ILAR's reports provide a framework for governmental and institutional animal welfare policies. The most widely distributed publication from ILAR is the "Guide for the Care and Use of Laboratory Animals," which is recognized by the Public Health Service and AAALAC International as the standard reference on laboratory animal care and use programs. ILAR also published two other standard references that are used to establish and maintain optimal animal care and use programs: "Occupational Health and Safety in the Care and Use of Research Animals" and "The Psychological Well-being of Nonhuman Primates." In addition, ILAR publishes the Laboratory Animal Management Series, which provides specific recommendations for many laboratory animal species. The quarterly ILAR Journal publishes contemporary, authoritative articles relevant to laboratory animal medicine and management. ILAR also maintains a large database of commercial and investigatorheld unique animal models and an international registry of laboratory registration codes on behalf of the International Committee on Standardized Genetic Nomenclature for Mice. 2.

AAALAC International

The Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC) is a nonprofit association that conducts voluntary peer review and accreditation of animal care and use programs. It is a communication-intensive program that stresses application of performance standards and professional judgment, rather than inspection and enforcement of engineering standards. AAALAC uses the "Guide for the Care and Use of Laboratory Animals" as its primary reference for program review and evaluation, but it also considers

the laws and regulations for the country in which the institution is located, and it uses other widely accepted standards and references for animal care and use, occupational health, and biosafety. The use of performance standards allows AAALAC to evaluate each program independently and to provide guidance appropriate to individual situations. This process helps institutions to maintain optimal standards for animal care and use. AAALAC accreditation is recognized by the Public Health Service. The peer review is conducted by a member of the AAALAC Council on Accreditation and at least one ad hoc consultant. Members of the council include distinguished pharmacologists, toxicologists, primatologists, animal scientists, aquatic and poultry specialists, physicians, administrators, and veterinarians. AAALAC is governed by its member organizations, including the American Association for the Advancement of Science (AAAS), the Federation of American Societies for Experimental Biology (FASEB), the American Medical Association, Society of Toxicology, and more than 40 other professional scientific organizations. More than 600 academic, government, and industrial research institutions and hospitals throughout the United States, Canada, Europe, South America, Africa, and Asia are AAALAC accredited. 3.

Federation of Animal Science Societies

To help assure the ethical and humane treatment of farm animals used in agricultural research or teaching, the agricultural community published the "Guide for the Care and Use of Agricultural Animals in Agricultural Research and Testing" ("Ag Guide"). The first edition, published in 1988, was revised in 1999 by the Federation of Animal Science Societies. This document is intended to supplement applicable federal, state, and local laws, regulations, and policies and the "Guide for the Care and Use of Laboratory Animals." It is directed to the care and use of any warm-blooded vertebrate animal that is used to improve understanding of the animal's use in production agriculture and that may require a simulated or actual production agricultural setting consistent with consideration of the well-being of the animal. It provides standards for range or pasture production in naturalistic settings and for various degrees of confinement in relatively intensive and certain less extensive production systems. It includes guidelines for institutional policies, husbandry, veterinary care, and facility construction and maintenance. The "Ag Guide" is used by AAALAC International for relevant program assessment and accreditation purposes.

G.

International Laws and Regulations

It is not the intent of this chapter to provide detailed information on the various international laws and standards governing the care and use of laboratory animals. However, every civilized


Table II CIOMS International Guiding Principles for Biomedical Research I. The advancementof biological knowledge and the developmentof improved means for the protection of the health and well-being both of man and animals require recourse to experimentationon intact live animals of a wide variety of species II. Methods such as mathematical models, computer simulation, and in vitro biological systems should be used wherever appropriate III. Animal experiments should be undertaken only after due consideration of their relevance for human or animal health and the advancementof biological knowledge IV. The animals selected for an experiment should be of an appropriate species and quality, and only the minimum number required to obtain scientifically valid results V. Investigatorsand other personnel should never fail to treat animals as sentient, and should regard their proper care and use and the avoidance or minimization of discomfort, distress, or pain as ethical imperatives VI. Investigatorsshould assume that procedures that would cause pain in human beings cause pain in other vertebrate species, although more needs to be known about the perception of pain in animals VII. Procedureswith animals that may cause more than momentary or minimal pain or distress should be performed with appropriate sedation, analgesia, or anesthesia in accordance with accepted veterinary practice. Surgical or other painful procedures should not be performed on unanesthetized animals paralyzed by chemical agents VIII. Where waivers are required in relation to the provisions of article VII, the decisions should not rest solely with the investigators directly concerned but should be made with due regard to the provisions of articles IV, V, and VI, by a suitably constituted review body. Such waivers should not be made solely for the purpose of teaching or demonstration IX. At the end of, or, when appropriate, during an experiment, animals that would otherwise suffer severe or chronic pain, distress, discomfort, or disablement that cannot be relieved should be painlessly killed. X. The best possible living conditions should be maintained for animals kept for biomedical purposes. Normally the care of animals should be under the supervision of veterinarians having experience in laboratory animal science. In any case, veterinary care should be available as required XI. It is the responsibility of the director of an institute or department using animals to ensure that investigators and personnel have appropriate qualifications or experience for conducting procedures on animals. Adequate opportunities shall be provided for in-service training, including the proper and humane concern for the animals under their care

country in the world has developed and implemented regulatory requirements for the humane and ethical treatment of research animals. Because the biomedical research community has become more global, there is increased interest in the harmonization of international standards for the care and use of laboratory animals. The member states of the European Union, in keeping with Directive 86/609, have agreed to adopt common provisions to protect animals used in experimental and other scientific procedures that may cause pain, suffering; distress, or lasting harm. This directive emphasizes the importance of seeking and en-

29

couraging the use of alternative measures with the aim of reducing the number of animals used in research. The Council for International Organizations of Medical Sciences (CIOMS) is an international, nongovernmental organization composed of medical, biomedical, and research organizations. In 1985, CIOMS developed a broad set of guidelines, the "International Guiding Principles for Biomedical Research Involving Animals" (Table II). Many countries have used these guidelines to form the basis for their regulatory requirements.

Ill.

I M P O R T A T I O N AND EXPORTATION OF ANIMALS AND A N I M A L P R O D U C T S

A.

U.S. Department of Agriculture

The U.S. Department of Agriculture (USDA), APHIS, Veterinary Services (VS), I m p o r t - E x p o r t Products Staff regulates the importation of all animals and animal-derived materials that could represent a disease risk to United States livestock. It also regulates the import and transport of infectious organisms and vectors of disease agents. This category includes not only animal products and by-products but also biological materials that contain or have been in contact with certain organisms and animal materials (including cell cultures and recombinant products). All imported materials must enter the United States through USDA-designated ports of entry. The regulations are set forth in the Code of Federal Regulations (CFR), Title 9, Chapter 1. The individual designated to receive imported material, and who will be responsible for the material, must apply for a USDA permit by submitting a complete VS application form and applicable fee. Importation of cell lines and cell culture products, such as monoclonal antibodies and recombinant proteins, requires an additional form. The information provided must be sufficient for the VS to evaluate disease risk and should include details regarding product processing, production, and nutrient factors. To protect the health of United States livestock and poultry, the USDA requires permits for importation of swine, ruminants, other hoof stock, poultry, and other birds. The USDA imposes few restrictions on the entry of small laboratory mammals. If rats, mice, guinea pigs, and hamsters have not been inoculated with or exposed to infectious agents that affect livestock or poultry, such as bovine and other transmissible spongiform encephalopathy agents, importation of these species is not restricted. However, permits are required for transgenic animals that carry receptors that enable those rodents to develop productive infection with human pathogens. At a minimum, each animal shipment should be accompanied by a health certificate endorsed by a veterinarian or person responsible for the health

LYNN C. ANDERSON

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of laboratory animals at the facility of origin. The certificate must indicate that the animals are (1) clinically healthy, (2) have not been exposed to or inoculated with any infectious disease agents, and (3) have not originated from a facility where work with viruses affecting livestock or poultry is conducted. This certificate should be affixed to the outside of the animal transportation unit so that it is available to the United States officials at the port of arrival. A 30-day quarantine is required for poultry, ruminants, or swine, and a 60-day quarantine for horses from African horsesickness-infested regions. The importer is responsible for arranging quarantine facilities subject to the approval of the VS. The USDA, APHIS, Plant Protection and Quarantine (PPQ) service regulates the importation of plants and other vegetable matter. Feed provided to an animal during transit, such as potatoes or carrots, may be regulated. The importer must consult with the PPQ Permit Office to determine entry requirements. If it cannot be allowed entry, the prohibited vegetable matter must be removed from the cage at the point of entry by a PPQ officer.

B.

U.S. Department of Health and Human Services

Under the direction of the U.S. Department of Health and Human Services, the U.S. Public Health Service, through the Centers for Disease Control and Prevention (CDC), Office of Health and Safety, regulates the importation of nonhuman primates. Only institutions or individuals registered with the CDC may import nonhuman primates or receive them within a 31-day period of their arrival in the United States. Importers are registered for a 2-year period and must comply with CDC record-keeping and reporting requirements. The PHS is also responsible for protecting humans from zoonotic diseases and therefore regulates the importation of other animals that may be infectious to humans. Imported dogs and cats must be free of evidence of rabies, turtles must be free of salmonella, and psittacine birds must not be capable of causing psittacosis. In addition, the PHS regulates the importation or subsequent distribution of any etiologic agent or any arthropod or animal host or vector of human disease. PHS permits must be obtained for importation and distribution of these materials.

C.

U.S. Department of the Interior

The Convention on International Trade in Endangered Species of Wild Fauna and Flora (CITES), established in 1973 and amended in 1979, helps protect wild flora and fauna from extinction by requiring government permits for international trade in threatened wildlife and wildlife products. In the United

States, CITES is enforced by the Department of the Interior, U.S. Fish and Wildlife Service (FWS). It applies to all of the designated vertebrate and invertebrate animal or plant species, whether alive or dead, and any recognizable part of a designated animal. Protection is provided for species in two main categories: (1) those that are most endangered and (2) other species at serious risk. The most endangered species are listed in Appendix I of the CITES agreement. Appendix II includes species that are not currently threatened with extinction but may become so unless trade is subject to strict regulation. Appendix III includes all species that any country identifies as being subject to regulation within its jurisdiction for the purpose of preventing or restricting exploitation and for which the cooperation of other countries is needed. Importation or exportation of these species requires appropriate documents. The FWS is also responsible for enforcement of the Endangered Species Act, which protects threatened and endangered animal and plant species and their habitats from extinction. Protected species include birds, insects, fish, reptiles, mammals, crustaceans, flowers, grasses, and trees. The law prohibits any action, administrative or real, that results in the "taking" of a listed species or adversely affects the habitat of a listed species. It prohibits import, export, and interstate and foreign commerce of listed species. A permit issued by the Federal Wildlife Permit Office is required to use these species for scientific research. The Lacey Act was enacted in 1900 and amended several times, including substantial amendments in 1981. It authorizes the FWS to regulate the importation, exportation, transportation, sale, receipt, acquisition, or purchase of fish, wildlife, or plants that may be injurious to humans or to the interests of agriculture, horticulture, forestry, or United States wildlife resources. The Lacey Act also provides for effective enforcement of state, federal, American Indian tribal, and foreign conservation laws. In addition, the Lacey Act requires that live wildlife be transported into the United States under humane and healthful conditions and that all containers or packages containing wildlife be appropriately labeled when transported in interstate or foreign commerce.

D.

Environmental Protection Agency

The Environmental Protection Agency, which is responsible for enforcing the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), considers the potential adverse impact of pesticides on endangered species and their habitats before licensing the use of pesticides. Under FIFRA, the EPA can issue emergency suspension of certain pesticides to cancel or restrict their use if an endangered species will be adversely affected. FIFRA also provides federal control of pesticide distribution and sale and requires registration of users (farmers, utility companies, and others) and certification of applicators.

2. LAWS,REGULATIONS,AND POLICIES AFFECTING THE USE OF LABORATORYANIMALS IV.

HAZARDOUS SUBSTANCES

A.

Biohazards

The Occupational Safety and Health Act, enacted in 1970, is administered and enforced by the Department of Labor, Occupational Safety and Health Administration (OSHA). The intent is to provide workers with protection against illnesses or injury resulting from unsafe or unsanitary working conditions. The act established the National Institute for Occupational Safety and Health (NIOSH) within the Centers for Disease Control and Prevention. NIOSH plans, directs, and coordinates national programs to develop and establish recommended occupational safety and health standards and to conduct research, training, and related activities to assure safe and healthful working conditions. Chapter 24, "Control of Biohazards Associated with the Use of Experimental Animals," provides a comprehensive overview of and references for the programs, facilities, and practices necessary to help assure employee protection against biohazards. These include animal allergies, zoonoses, recombinant DNA, and infectious experimental agents.

B.

Chemical Agents

Chapter 21 also addresses the potential hazards of and precautions for working with chemical agents used in experiments. The Toxic Substances Control Act (TSCA), enacted in 1976, authorizes the Environmental Protection Agency to require testing of chemical substances entering the environment and to regulate them as necessary. Chemicals used exclusively in pesticides, food, food additives, drugs, and cosmetics are exempt from the TSCA but are regulated by other legislation. The Drug Enforcement Administration of the Department of Justice is responsible for enforcing the Drug Enforcement Act (Pub. L. 93-205). This law requires appropriate security and record management of controlled substances that are considered to be potentially addictive or habituating for human and animal use.

C. Radioactive Materials and Radiation-Emitting Equipment The Atomic Energy Act, enacted in 1954, authorizes the Nuclear Regulatory Commission (NRC) to help assure that the civilian use of radioactive materials is conducted in a manner consistent with public health and safety, environmental quality, national security, and antitrust laws. In 1974, the NRC became an independent regulatory agency under the provision of the Energy Reorganization Act. The NRC licenses individuals and

31

institutions that use radioactive material and regulates the procurement, use, storage, and disposal of these materials. The facilities, instruments, and equipment used for handling and storing radioactive materials must also meet NRC requirements. Personnel must be provided training in the safe handling and use of ionizing radiation. The Radiation Control for Health and Safety Act, enacted in 1968, authorizes the Secretary of Health and Human Services, through the Food and Drug Administration, to regulate the use of products that produce radiation, such as medical diagnostic imaging equipment, irradiators, and electron microscopes.

V.

R E C O M B I N A N T DNA R E S E A R C H GUIDELINES

The Department of Health and Human Services, National Institutes of Health, periodically publishes "Guidelines for Research Involving Recombinant DNA Molecules" in the Federal Register. Recombinant DNA molecules are defined as either (1) molecules that are constructed outside of living cells by joining natural or synthetic DNA segments to DNA molecules that can replicate inside a living cell or (2) DNA molecules that result from the replication of these altered molecules. These guidelines apply to any recombinant DNA research project conducted at or sponsored by an institution that receives any of its support from the NIH, including intramural studies and those conducted abroad. Experiments involving recombinant DNA have been divided into four classes. The level of institutional review and containment required depends on the classification. Special care is required in the evaluation of containment levels for experiments that are likely to enhance the pathogenicity or extend the host range of viral vectors. Each institution must establish and implement policies that provide for the safe conduct of recombinant DNA research and that ensure compliance with the NIH guidelines. The institution must also appoint an Institutional Biosafety Committee (IBC) of no fewer than five members who have experience and expertise in recombinant DNA technology, biosafety, and physical containment. At least two of these members shall not be otherwise affiliated with the institution and shall represent the general interests of the surrounding community with respect to public health or the environment. The IBC acts on behalf of the institution to review all applications, proposals, and activities involving the use of recombinant DNA to assure compliance with the NIH guidelines. These responsibilities include reviewing the containment level required and assessment of the facilities, procedures and practices (including emergency spill plans) and the training and expertise of personnel conducting the research. The institution must also appoint a Biosafety Officer (BSO) if it engages in research at the Biosafety Level 3 (BSL3)or Biosafety Level 4 (BSL4) containment level.

32

LYNN C. ANDERSON Appendix: Websites Pertaining to Laboratory Animals

Resource

Homepage address

AAALAC International Animal Welfare Information Center, National Agriculture Library CITES Code of Federal Regulations Environmental Protection Agency Guidelines for Research Involving Recombinant DNA Molecules Institutional Animal Care and Use Committees Institute for Laboratory Animal Research Office of Laboratory Animal Welfare Public Health Service Policy on Humane Care and Use of Laboratory Animals USDA import permits USDA regulations and standards

http://www.aaalac.org http://www.nal.usda.gov/awic / http://international.fws.gov/cites/cites.html http://www.access.gpo.gov/nara/cfr http://www.epa.gov http ://www4.nih. gov/oba/rac/guidelines/guideline s.html http://www.iacuc.org http://www4.nas.edu/cls/ilarhome.nsf http://grants.nih.gov/grants/olaw/olaw.htm http ://grants.nih. gov/grants/olaw/references/phspol.htm http://www.aphis.usda.gov/import.html http://www.aphis.usda.gov/ac

T h e B S O is r e s p o n s i b l e for m o n i t o r i n g the facilities a n d practices to assure c o m p l i a n c e a n d for r e p o r t i n g to and s e r v i n g on the IBC. T h e p r i n c i p a l i n v e s t i g a t o r is also r e s p o n s i b l e for assuring c o m p l i a n c e w i t h the g u i d e l i n e s and m u s t r e q u e s t app r o v a l for the r e s e a r c h a n d r e p o r t a n y p r o b l e m s to the I B C .

REFERENCES

Allen, T. (1995). "Animal Welfare Legislation, Regulations, and Guidelines: January 1990-January 1995." Animal Welfare Information Center, National Agriculture Library, Beltsville, Maryland. Andrews, E. J. (1993). Report of the AVMA panel on euthanasia. J. Am. Vet. Med. Assoc. 202, 229-249. Animal Welfare Act of 1966 (Pub. L. 89-544) and subsequent amendments (1966). US. Code, Vol. 7, Secs. 2131-2157 et seq. Barkley, W. E. (1997). "Occupational Health and Safety in the Care and Use of Research Animals." Committee on Occupational Safety and Health in Research Animal Facilities, Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council. National Academy Press, Washington, D.C. Bernstein, I. S. (1998). "The Psychological Well-being of Nonhuman Primates." Committee on Well-being of Nonhuman Primates, Institute of Laboratory Animal Research, Commission on Life Sciences, National Research Council. National Academy Press, Washington, D.C. Clark, J. D. (1996). "Guide for the Care and Use of Laboratory Animals." Committee to Revise the Guide for the Care and Use of Laboratory Animals, Institute of Laboratory Animal Resources, Commission on Life Sciences, National Research Council. DHHS Publ. (NIH) 96-23. National Academy Press, Washington, D.C. Code of Federal Regulations (1999a). Title 9: Animals and Animal Products; Chap. 1: Animal and Plant Health Inspection Service, Department of Agriculture; Subchap. A: Animal Welfare; Parts 1, 2, 3, and 4. Office of the Federal Register, Washington, D.C. Code of Federal Regulations (rev. 1999b). Title 10: Energy; Chap. 1: Nuclear Regulatory Commission; Part 20: Standards for Protection against Radiation. Office of the Federal Register, Washington, D.C. Code of Federal Regulations (rev. 1998a). Title 9: Animals and Animal Products; Chap. 1: Animal and Plant Health Inspection Service, Department of Agriculture; Subchap. C: Interstate Transportation of Animals (including Poultry) and Animal Products. Office of the Federal Register, Washington, D.C.

Code of Federal Regulations (rev. 1998b). Title 9: Animals and Animal Products; Chap. 1: Animal and Plant Health Inspection Service, Department of Agriculture; Subchap. D: Exportation and Importation of Animals (Including Poultry) and Animal Products. Office of the Federal Register, Washington, D.C. Code of Federal Regulations (rev. 1998c). Title 21: Food and Drugs; Chap. 1: Food and Drug Administration, Department of Health and Human Services; Subchap. A: General; Part 58: Good Laboratory Practice for Nonclinical Laboratory Studies. Office of the Federal Register, Washington, D.C. Code of Federal Regulations (rev. 1998d). Title 29: Labor; Chap. 17: Occupational Safety and Health Administration; Part 1910: Occupational Safety and Health Standards. Office of the Federal Register, Washington, D.C. Code of Federal Regulations (rev. 1997a). Title 40: Protection of the Environment; Chap. 1: Environmental Protection Agency; Subchap. E: Pesticide Programs; Part 160: Good Laboratory Practice Standards. Office of the Federal Register, Washington, D.C. Code of Federal Regulations (rev. 1997b). Title 50: Wildlife and Fisheries; Chap. 1: U.S. Fish and Wildlife, Department of the Interior; Subchap. B: Taking, Possession, Transportation, Sale, Purchase, Barter, Exportation, and Importation of Wildlife and Plants. Office of the Federal Register, Washington, D.C. Curtis, S. E. (1994). Farm animal use in biomedical sciencemmelding the guidelines. ILAR News 36, 35-39. Dennis, M. B., Jr., and Van Hoosier, G. L., Jr. (1994). North American legislation and regulation of the use of live animals for scientific research. In "Handbook of Laboratory Animal Science," Vol. 1, "Selection and Handling of Animals in Biomedical Research" (P. Svendsen and J. Hau, eds.), pp. 23-25. CRC Press, Boca Raton, Florida. Guidelines for Research Involving Recombinant DNA Molecules (1994). Federal Register 59, 34496. Hamm, T. E., Jr., Dell, R. B., and Van Sluyters, R. C. (1995). Laboratory animal care policies and regulations: United States. ILAR J. 37, 75-78. Institutional Administrator's Manual for Laboratory Animal Care and Use (1988). Office for Protection from Research Risks, National Institutes of Health, Public Health Service. DHHS Publ. (NIH) 88-2959. U.S. Department of Health and Human Services, Washington, D.C. Institutional Animal Care and Use Committee Guidebook (1992). Office for Protection from Research Risks/Applied Research Ethics National Association, National Institutes of Health, Public Health Service. DHHS Publ. (NIH) 92-3415. U.S. Department of Health and Human Services, Washington, D.C. Interagency Research Animal Committee (1985). "U.S. Government Principles for Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training." Federal Register (May 20, 1985).

2. LAWS, REGULATIONS, AND POLICIES AFFECTING THE USE OF LABORATORY ANIMALS Kreger, M., Jensen, D., and Allen, T., eds. (1996). "Animal Welfare Act: Historical Perspectives and Future Directions." Working for Animals in Research, Drugs, and Surgery (WARDS). McPherson, C. W. (1984). Laws, regulations, and policies affecting the use of laboratory animals. In "Laboratory Animal Medicine" (J. G. Fox, B. J. Cohen, F. M. Loew, eds.), pp. 19-30. Academic Press, New York. Mench, J. A. (1999). "Guide for the Care and Use of Agricultural Animals in Agricultural Research and Teaching." Guide Revision Committee, Federation of Animal Science Societies, Savoy, Illinois. Nomura, T. (1995). Laboratory animal care policies and regulations: Japan. ILAR J. 37, 60-61. Potkay, S., Garnett, N. L., Miller, J. G. et al. (1997). Frequently asked questions about the Public Health Service policy on humane care and use of laboratory animals. Contemp. Top. in Lab. Anim. Sci. 36, 47-50. Public Health Service Policy on Humane Care and Use of Laboratory Animals

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(1986). Office for Protection from Research Risks, National Institutes of Health, Public Health Service. (Reprinted by U.S. Department of Health and Human Services, Washington, D.C., 1996). Reid, C. S. W. (1995). Laboratory animal care policies and regulations: New Zealand. ILAR J. 37, 62-68. Richmond, J. Y., and McKinney, R. W. (1993). "Biosafety in Microbiological and Biomedical Laboratories." Centers for Disease Control and Prevention and National Institutes of Health, U.S. Public Health Service, Department of Health and Human Services. DHHS Publ. (CDC) 93-8395. U.S. Government Printing Office, Washington, D.C. Townsend, P., and Morton, D. B. (1995). Laboratory animal care policies and regulations: United Kingdom. ILAR J. 37, 68-74. Wong, J. (1995). Laboratory animal care policies and regulations: Canada. ILAR J. 37, 57-59.


Chapter 3 Biology and Diseases of Mice Robert O. Jacoby, James G. Fox, and Muriel Davisson

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Origin and History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Genetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Breeding Systems and Nomenclature . . . . . . . . . . . . . . . . . . . . . . . . . D. Housing and Husbandry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Physiology and Anatomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Reproduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Immunology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Infectious Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Metabolic and Nutritional Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Environmental, Behavioral, and Traumatic Disorders . . . . . . . . . . . . D. Congenital, Aging-Related, and Miscellaneous Disorders . . . . . . . . . E. Neoplastic Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I.

A.

INTRODUCTION

Origin and History

The laboratory m o u s e is assigned to the genus Mus, subfamily Murinae, family Muridae, order Rodentia. Anatomical features of the molar teeth and cranial bones help differentiate it from other murids. The house m o u s e of North A m e r i c a and Europe, Mus musculus, is the species c o m m o n l y used for biomedical research. Laboratory strains were usually derived from mice bred by mouse fanciers and their g e n o m e s are a mixture of M. musculus musculus (from eastern Europe) and M. m. domesticus (from western Europe). Since the mid-1980s, strains have LABORATORYANIMALMEDICINE,2ndedition

35 35 36 36 40 41 41 48 51 52 53 53 105 107 108 110 113

been developed from Asian mice (M. m. castaneus from Thailand and M. m. molossinus from Japan) and from M. spretus. The laboratory m o u s e was e m p l o y e d in comparative anatomical studies as early as the seventeenth century, but accelerated interest in biology during the nineteenth century, a renewed interest in M e n d e l i a n genetics, and the research requirement for a small, economical m a m m a l that was easily housed and bred were instrumental in the d e v e l o p m e n t of the "modern" laboratory mouse. These studies have grown exponentially during the current century with the recognition of the power of the m o u s e for gene and comparative mapping and have made the laboratory mouse, in genetic terms, the most thoroughly characterized m a m m a l on earth (Morse, 1979; Silver, 1995; L y o n et al., 1996). The current ability to create highly sophisticated, genetically Copyright2002,ElsevierScience(USA).Allrightsreserved. ISBN0-12-263951-0

36

ROBERT O. JACOBY,JAMES G. FOX, AND MURIEL DAVISSON

engineered mice by inserting transgenes or targeted mutations into endogenous genes has also made the laboratory mouse the most widely and heavily used experimental animal.

B.

Genetics

Genetic mapping in mice began in the early 1900s. The first autosomal genes, albino and pink-eyed dilution, were linked in 1915 (Haldane et al., 1915). Extensive linkage maps and an impressive array of inbred strains are now available to expedite sophisticated genetic research. Mice have 40 chromosomes that are differentiated by the size and patterns of transverse bands. The chromosomes are designated by Arabic numbers in order of decreasing size. During the 1970s, chromosome rearrangements were used to assign known genetic linkage groups-identified by Roman numerals--to specific chromosomes and for determining locus order with respect to the centromere. Genes can now be located physically on chromosomes by fluorescent in situ hybridization (FISH), and the Genome Initiative programs are fostering development of molecular maps of mouse chromosomes. The sequence of the mouse genome is expected to be completed by 2003 (Battey et al., 1999). Inbred mice are valuable for research in virtually all fields of biomedical research, such as immunology, oncology, cardiovascular disease, metabolic disease, microbiology, biochemistry, pharmacology, physiology, anatomy, developmental biology, and radiobiology. Mice carrying spontaneous or induced mutations and strains susceptible to specific diseases provide a wide variety of mouse model systems for basic research, as well as models for biomedical research to understand specific human disorders. For example, several spontaneous mutations in genes affecting pituitary function or producing hormones provide mutant models for human dwarfing conditions. Targeted mutations in the low-density lipoprotein receptor and apolipoprotein genes provide model systems for studying cardiovascular disorders. Mice of the NOD (nonobese diabetic) strain provide a model for human insulin-dependent diabetes. Mice also provide reagents for basic research. For example, inbred histocompatible strains are used extensively as donors of plasma cell tumors to immortalize cell lines (hybridomas) that secrete highly uniform, monospecific immunoglobulins in vitro, theoretically in unlimited quantities. This technology has made a full range of functional mouse antibody molecules available for study. Development of quantitative trait loci (QTL) methodology for mapping genes and the similarity between mouse and human genomes have made the mouse invaluable for identifying genes and underlying complex traits that are inherent to the most common human genetic diseases (Darvasi, 1998; Frankel, 1995). One of the most thoroughly studied genetic systems of the mouse is the histocompatibility complex. Histocompatibility (H) loci control expression of cell surface molecules that modulate major immunological phenomena, such as the recognition

of foreign tissue. For example, the time, onset, and speed of skin graft rejection are controlled by two groups of H loci. The major group is located in the major histocompatibility complex (MHC, H2) on chromosome 17. These genes cause rapid rejection (10-20 days) of grafts that display foreign H2 antigens. Minor H loci groups are scattered throughout the genome and are responsible for delayed graft rejection. Genes associated with the H2 complex also control other immunological functions, such as cell-cell interactions in primary immune responses and the level of response to a given antigen. Immunemediated responses to infectious agents such as viruses and complement activity are influenced directly or indirectly by the H2 complex. The most recent comprehensive review of the H2 complex is by Klein (1986). Because information about this subject is being published so frequently, the reader is advised to consult bibliographic indexes such as MEDLINE for recent updates. Non-MHC or minor histocompatibility systems also are under active study (Roopenian and Simpson, 2000).

C.

Breeding Systems and Nomenclature

1. Breeding Systems

Laboratory mice are identified by strain and by breeding system. A genealogy of most inbred strains is presented in the Mouse Genome Database . Table I summarizes nine breeding systems. Each requires technical skill and a firm understanding of mammalian genetics. Inbred strains were developed first in 1909 by Clarence Cook Little and offer a high degree of genetic uniformity. Mice within an inbred strain, for practical purposes, are genetically identical to other mice of the same strain and sex. They are defined as being produced by brother-sister matings for more than 20 generations. In fact, a strain should not be considered completely inbred until after 40 generations of sibling matings. Inbred strains are valuable because experimental results are reproducible with relatively small sample sizes. They are useful for genetic mapping because they are genetically well characterized, and allelic combinations can be predetermined for linkage crosses. Wild-derived inbred strains of Mus musculus castaneus and M. spretus are used extensively for mapping because of the large number of polymorphic differences from standard inbred laboratory mice. F1 hybrid mice, produced by mating mice of two inbred strains, also are genetically identical to each other and may offer a more robust animal for some studies. For example, inbred strains may differ in behavior and learning abilities, whereas hybrid mice are less likely to have learning deficits or behavioral anomalies. Mutant inbred strains carry spontaneous, targeted, or induced mutations, transgenes, or chromosome aberrations. The genetic backgrounds of such strains are homogeneous like those of regular inbred strains, but some (or all) mice of the strain carry the mutation (or chromosome aber-

37

3. BIOLOGY AND DISEASES OF MICE Table I

Kinds of Mice Used in Research

a

Definition of breeding system Random bred stock: Random mating within a large,

heterogeneous population Inbred strain: Brother- sister mating for more than 20 generations F1 hybrids: Mice from crosses between inbred strains Segregating inbred strain: Brother-sister matings system for more than 20 generations with heterozygosity for the mutations forced by (1) backcrossing, (2) intercrossing, (3)crossing and intercrossing,or (4) backcrossing and intercrossing Coisogenic inbred strains: Occurrence of a mutation within a strain

Congenic inbred strains: (A) Repeatedbackcross

of mutation-bearingmice for 10 or more generations or (B) cross-intercross system for the equivalent of 20 or more cycles with an inbred parent strain Recombinant inbred strains: Brother-sister matings for >20 generations after crossing two inbred strains and their F1 to obtain and F2 Recombinant congenic strains: Same as above except one or more backcrosses of F1 to one parent strain before beginning brother-sister matings Advanced intercross lines: Nonsibling matings from an F2 of a cross between two inbred strains a

Perpetuation of breeding system

Reference

Continue random mating, selectionpairs with random numbers method Continue brother-sister mating

Poiley (1960) Kimura and Crow (1963) Green (1981a)

Cannot be perpetuated Continue brother-sister mating with heterozygosity forced by one of the four methods at left or with homozygosity forced by intercrossinghomozygotes

Green (1981a) Green (1981a)

Perpetuate the mutation by (1) brother-sister mating within strain of origin, (2) backcross or cross-intercross system with strain of origin as parent strain, (3) brother-sister mating with heterozygosity forced by back- or intercrosses, or (4) brother-sister mating between homozygotes Perpetuate the transferred mutation by (2), (3), or (4) above. (1) may be used after 10-12 generations of backcrossing with periodic backcrosses to background strain Continue brother-sister matings

Flaherty (1981) Green (1981a)

Continue brother-sister matings

Flaherty (1981) Green (1981a) Bailey (1971) Demant and Hart (1986)

Continue nonsibling matings

Modified from Green (1981a).

ration). In segregating inbred mutant strains, mutant mice differ from their nonmutant siblings only by the mutation. Therefore, littermates can serve as controls in experiments. In homozygous mutant strains, controls must come from the same or a closely related nonmutant inbred strain. For example, C57BL/6J mice provide controls for the homozygous mutant C 5 7 B L / 6 J - m / m strain. A strain is said to be c o i s o g e n i c if the mutation of interest occurred in that strain. A strain is c o n g e n i c if the mutation or gene of interest was transferred from another strain or stock by repeated backcrossing. Detailed descriptions and diagrams of mating schemes have been provided by Green (198 l a,b), and additional contemporary information on mouse genetics and breeding can be found by consulting Table II. In contrast to inbred mice, r a n d o m bred, or outbred, mice are genetically heterogeneous and are often produced by breeding systems that intentionally minimize inbreeding. Outbred mice may be used when high genetic heterogeneity is desired or for experiments requiring large numbers of mice. However, it is preferable to ensure genetic heterogeneity by intercrossing multiple inbred strains to achieve heterogeneity with known genetic input. Individual random bred mice within a colony may differ

in coat color, histocompatibility loci, enzyme and DNA polymorphisms, and other characteristics. Random breeding requires the statistically random selection of breeders by using a random numbers table or computer program. Random breeding, or outbreeding, can be achieved only in a large colony. A small breeding population or passage through the genetic "bottleneck" of rederivation to improve health status will reduce genetic heterogeneity and lead eventually to some degree of inbreeding. In fact, supposedly "random bred" stocks are often genetically quite homogeneous. In a population of 25 breeding pairs, for example, heterozygosity will decrease at 1% per generation with standard randomization techniques. A random breeding program that is easy to manage is the circular pair mating system, in which each pair is mated only once. Conceptually, cages are visualized in a circle, and each cage contains one breeding pair in the nth generation. Another "circular" set of cages serves as the breeding nucleus for the n + 1 generation. Each mated pair in the nth generation contributes one female and one male to the n + 1 generation. Random breeding is accomplished by assigning the female and male derived from each nth generation cage to different cages in the n + 1 generation.

38

ROBERT O. JACOBY, JAMES G. FOX, AND MURIEL DAVISSON Table II Databases and Websites for Information about Mice Internet resource

Comprehensive database sites and mouse sources Mouse Genome Database (MGD) JAX Mice MRC Mammalian Genetics Unit, Harwell, United Kingdom The Whole Mouse Catalog ORNL Mutant Mouse Database Genetically engineered mouse sites and sources Induced Mutant Resource TBASE European Mouse Mutant Archive (EMMA) BioMedNet Mouse Knockout and Mutation Database Cre Transgenic and Floxed Gene Databases University of California Resource of Gene Trap Insertions Database of Gene Knockouts The Big Blue Web Site The Mouse Brain Library Mouse biology Mouse Tumor Biology Database (MTB) The Mammary Transgene Database Gene Expression Database (GXD) NetVet and the Electronic Zoo The Dysmorphic Human-Mouse Homology Database (DHMHD) The Mouse Atlas and Gene Expression Database Project UCD Medpath Transgenic Mouse Searcher 2.0 Mouse 2-D PAGE Database Body map Human and Mouse Gene Expression DB UNSW Embryology Mouse Development Dynamic [Embryonic] Development Zygote: A Developmental Biology Website Mouse genomics Mouse Nomenclature Guidelines and Locus Symbol Registry Trans-NIH Mouse Initiative Gene Dictionary of the Mouse Genome Genetic and Physical Maps of the Mouse Genome Mouse Backcross Service (U.K. HGMP Resource Centre) The Jackson Laboratory Mapping Panels WashU GSC Mouse EST Project Japanese Animal Genome Database NCBI LocusLink UniGene Mouse Sequences Collection TIGR Mouse Gene Index NIA/NIH Mouse Genomics Home Page WICGR Mouse RH Map Home Page Mammalian Genetics Laboratory, National Institute of Genetics (Japan) Care and use Guidelines for Ethical Conduct in the Care and Use of Animals The Ethics of Using Transgenic Animals Institute for Laboratory Animal Research Laboratory Registration Code Database Research Genetics, Genomic Tools General American Fancy Rat and Mouse Association

Web address

http://www.informatics.jax.org/ http://j axmice.jax.org/index.shtml http://www.mgu.har.mrc.ac.uk/ http://www.rodentia.com/wmc/ http://bio.lsd.ornl.gov/mouse/ http ://lena.j ax. org/resources/documents/imr/ http://tbase.j ax.org / http ://www.emma.rm. cnr.it / http://research.n.com/mkmd http://www.mshri.on.ca/nagy/cre.htm http://socrates.berkeley.edu/--~skarnes/resource.html http ://www.bioscience. org/knockout/knochome.htm http://eden.ceh.uvic.ca/bigblue.htm http://www.nervenet.org/mbl/mbl.html http ://tumor.informatics.j ax. org/cancer links.html http://bcm.tmc.edu/ermb/mtdb/mtdb.html http://www.informatics.jax.org / http://netvet.wustl.edu/vet.htm http://www.hgmp.mrc.ac.uk/dhmhd/dysmorph.html http://genex.hgu.mrc.ac.uk/ http://www-mp.ucdavis.edu/personaltgmouse 1.html http://biosun.biobase.dk/---pdi/jecelis/mouse_data_select.html http://bodymap.ims.u-tokyo.ac.jp/ http://anatomy.med.unsw.edu.au/cbl/embryo/otheremb/mouse.htm http://www.acs.ucalgary.ca/---browder/mice.html http://zygote.swarthmore.edu/info.html http://www.informatics.jax.org/mgihome/nomen/ http://www.nih.gov/science/models/mouse / http://www.nervenet.org/main/dictionary.html http://www-genome.wi.mit.edu/cgi-bin/mouse/index http://www.hgmp.mrc.ac.uk/goneaway/mbx.html http ://www.j ax.org/resources/documents/cmdata / http ://genome.wustl, edu/est/mouse_esthmpg.html http://ws4.niai.affrc.go.jp/ http://www.ncbi.nlm.nih.gov/focuslink/

http://www.ncbi.nlm.nih.gov/.unigene/mm.home.html http://www.tigr.org/tdb/mgi/index.html http://lgsun.grc.nia~nih.gov/ http://www-genome.wi.mit.edu/mouse_rh/index.html http ://www.shigen.nig.ac.jp/mouse/mouse.default.html http://www.apa.org/science/anguide.html http://oslovet.veths.no/transgenics/references.html http://www4.nationalacademies.org/cls/ilarhome.nsf http://www4.nas.edu/cls/afr.nsf/labcodesearch?openform http://www.resgen.com/index.php3 http://www.afrma.org /

3. BIOLOGY AND DISEASES OF MICE

Recombinant inbred (RI) strains are sets of inbred strains developed by single-pair random matings of mice from an F2 generation created by crossing mice of two inbred strains. Lines are propagated by brother-sister matings for more than 20 generations to obtain homozygosity. Recombinant inbred strains may take as long as 7 years to produce. RI strains are valuable for mapping phenotypic or quantitative traits that differ between the progenitor strains. Because each line is inbred, genotyping and phenotyping data are cumulative. RI sets are especially valuable for controlling for environmental variability in a trait, because several genetically identical mice from each line in a set can be typed to score the line for a trait (Bailey, 1971; Taylor, 1996). Recombinant congenic strains are sets of inbred strains derived in a manner similar to that for RI sets, except that one or more backcrosses to one parental strain (designated the background strain) are made after the F1 generation, before inbreeding is begun. The other parental strain is designated as the donor strain. The proportion of background and donor genomes is determined by the number of backcrosses preceding inbreeding (Demant and Hart, 1986). Advanced intercross Lines (AILs) are a third type of RI line. AILs are made by producing an F2 generation between two inbred strains and then, in each subsequent generation, intercrossing mice but avoiding sibling matings. The purpose is to increase the possibility of recombination between tightly linked genes. For further information on this topic, consult Genetic Guidelines, Mouse Genome Database .

2.

Nomenclature

There are currently more than 1000 separate outbred stocks and inbred strains, some with multiple sublines. In addition, there are thousands of mutant strains. Therefore it is critical that strain or stock designations be complete and accurate to avoid semantic and genetic confusion. As an example of subline variation that makes precise nomenclature important, CBA/J carries the gene for retinal degeneration, while the CBA/CaJ subline does not. Specific nomenclatures have been developed for inbred and noninbred strains and stocks. Strains are designated by a series of letters and/or numbers, which frequently provide a shorthand description of the origin and history of the strain (Table III). For example, the inbred strain C57BL/6J originated from female 57 at the Cold Spring Harbor Laboratory (C), was the black (BL) line from this female, and is subline number 6. Sublines of an inbred strain are designated using Laboratory Registration Codes (Lab Codes), unique-2- to 4-letter codes that may be obtained from a central registry maintained at the Institute for Laboratory Animal Research in Washington, D.C. . The J in C57BL/6J means it is the subline maintained at the Jackson

39

Laboratory (J). A new type of strain designation has been created for new inbred strains made by intercrossing mice of two existing inbred strains. This is essential because many engineered mutations are made in 129-derived embryonic stem cells, recovered in C57BL/6-129 chimeras, and then maintained by brother-sister matings after the first generation. For example, an inbred strain derived by sibling matings from a C57BL/6 • 129 chimera is designated B6129. A noninbred stock that meets specific criteria is designated by placing the Lab Code before the stock symbol, separated by a full colon. For example, Hsd:ICR designates an ICR outbred stock maintained by Harlan ("International standardized nomenclature," 1972). Specific designations also distinguish coisogenic, congenic, segregating inbred, and various RI strains. The type of strain or stock often can be recognized from the correct symbol. For example,. BXD-1/Ty is line 1 in a set of RI strains derived from a C57BL/6J (B) female mated to a DBA/2J (D) male and made by Taylor (Ty). Mutant genes are designated by a brief abbreviation for the mutation (e.g., shi for shiverer). When a mutant gene is cloned, the symbol for the parent gene is used and the mutant allele is designated in superscript. For example, Mbp shi is the shiverer mutant allele in the myelin basic protein gene. Nomenclature for genetically engineered mice can be complex and may eventually require simplification. Currently, a transgenic strain is designated by a symbol for the strain followed by a symbol for the transgene. Transgene symbols take the form Tg(YYYYY) #Zzz, where Tg is the transgene, (YYYYY) is a brief description of the inserted DNA (such as a gene symbol), # is the assigned number in the series of events generated using a given construct, and Zzz is the Lab Code. When a transgene causes an insertional mutation in an endogenous gene, the mutant allele of the gene is designated by using the gene symbol and an abbreviation for the transgene as a superscript (e.g., inrg/zz~). A targeted mutation, or knockout, is designated by the mutated gene with the identification of the mutational event as a superscript. For example, C f t r fml Unc is the first mutation in the cystic fibrosis transmembrane regulator gene created at the University of North Carolina. A gene replacement, or knockin, uses similar nomenclature; Myf5 My~ indicates that the Myf5 gene was replaced by the Myod gene. The International Committee on Standardized Genetic Nomenclature for Mice, established in the early 1950s, is responsible for genetic nomenclature rules. The rules are available online at the Mouse Genome Database (MGD) website . They are published periodically in print copy, the most recent being in Davisson (1996). The committee also maintains a list of inbred strains at the MGD site. The reader is refered to Chapters 27 and 28 for further discussion of nomenclature and to Table II for selected databases and websites relevant to mouse genetics and biology that are available as of this writing.

40

ROBERT O. JACOBY, JAMES G. FOX, AND MURIEL DAVISSON Table IIl Examples of Mouse Strain Nomenclature Strain name

Definition

DBA/2J

C3H/HeSn-ash/+ C57BL/6J-TyrC-2J/+ AEJ/GnJ-ae/A w-J AKR.B6-H2b

B6.CBA-D4Mit25-D4Mit80 B6.Cg m Leprdb/+ + B6.Cg-m +/+ Lepr db BXD-1/Ty CcS1 CcSI(N4) B.A-Chr 1 C57BL/6J_mt BALB/c B6; 129-Cfir tmlv"c B6.129-Myf5My~ FVB/N-TgN(MBP) 1Xxx FVB /N_mTglzzz

B6C3F1 B6EiC3-Ts65Dn

Hsd:ICR Pri:B6,D2-G#

0

1.

Inbred strain named for its characteristic coat color genes (using their original gene symbols), dilute (d), brown (b), and nonagouti (a); it is the second of two sublines separated before 20 generations of brother • sister breeding and is the subline maintained at the Jackson Laboratory (J) Coisogenic segregating inbred mutant strain carrying the ashen (ash) mutation, which arose on C3H/HeSn Coisogenic segregating inbred mutant strain carrying the albino 2J mutant allele of the cloned tyrosinase gene (tyr) Inbred strain segregating for two alleles at the agouti gene Congenic inbred strain in which the b haplotype at the H2 complex was transferred from C57BL/6J (B6) to the AKR background Congenic strain in which the chromosomal segment between D4Mit25 and D4Mit80 was transferred from CBA to B6 Congenic inbred strain in which the linked mutant genes misty (m) and diabetes (Lepr db) were transferred from multiple, mixed, or unknown genetic backgrounds to B6 and are carried in coupling, i.e., on the same chromosome Congenic inbred strain in which the m and Lepr db mutations are carried in repulsion Recombinant inbred (RI) strain number 1 in a set of RI strains derived from a C57BL/6J (B) female mated to a DBA/2J (D) male and made by Taylor (Ty) Recombinant congenic (RC) strain number 1 in a set made by crossing the BALB/c (C) and STS (S) strains, backcrossing 1 or 2 times to BALB/c and then inbreeding as with RI strains Recombinant congenic (RC) strain number 1 in a set made by crossing the BALB/c (C) and STS (S) strains, backcrossing N4 times to BALB/c and then inbreeding as with RI strains Chromosome substitution (CSS) or consomic strain in which Chr 1 from A/J has been transferred to the B6 background Conplastic strain with the nuclear genome of C57BL/6J, and the cytoplasmic genome of BALB/c, developed by crossing male C57BL/6J mice with BALB/c females, followed by repeated backcrossing of female offspring to male C57BL/6J First targeted mutation of the cystic fibrosis transmembrane regulator gene created at the University of North Carolina, Unc, and carried on a mixed B6 and 129 background Congenic strain carrying a replacement or "knockin" in which the Myf5 gene was replaced with the Myod gene in 129 ES cells and backcrossed onto the B6 genetic background Transgene in which the human myelin basic protein (MBP) gene is inserted into the genome of the National Institutes of Health (N) subline of the FVB strain originally maintained at the National Institutes of Health Insertional mutation caused by the TglZzz transgene made on the FVB/N genetic background F1 hybrid made by crossing a C57BL/6 female to a C3H male Strain maintained by backcrossing mice with the Ts65Dn chromosome aberration to F1 hybrid mice made by crossing females of the Eicher (Ei) subline of C57BL/6 • C3H; note that these mice are not true F1 hybrids, and the F1 designation is omitted ICR outbred stock maintained at Harlan (Hsd) Advanced intercross line (AIL) created at Princeton (Pri) from the inbred strains C57BL/6 • DBA/2; AlL are made similar to RI strains except mice are intercrossed, avoiding sibling matings, to increase the possibility of tightly linked genes recombining

H o u s i n g and H u s b a n d r y

Housing

H o u s i n g (and h u s b a n d r y ) for m i c e are often g u i d e d by microb i o l o g i c a l r e q u i r e m e n t s . A c o l o n y can be m a i n t a i n e d in a "conv e n t i o n a l " e n v i r o n m e n t or b e h i n d a barrier w h e r e the m i c e are p r o t e c t e d f r o m specific m i c r o o r g a n i s m s . E x a m p l e s of barrier h o u s i n g include positive pressure isolators and m a s s airflow racks that p ro v i d e sterile air t h r o u g h high-efficiency particulate air (HEPA) filters or individually ventilated caging. T h e integrity of the m i c r o e n v i r o n m e n t is m a i n t a i n e d by servicing and

usually m a d e of p o l y c a r b o n a t e , p o l y p r o p y l e n e , or p o l y s t y r e n e plastic (in order of d e c r e a s i n g cost and durability). M i c e are s o m e t i m e s h o u s e d in s u s p e n d e d cages with o p e n - m e s h b o t t o m s that allow e x c r e m e n t to fall t h r o u g h to a col l ect i n g pan. Susp e n d e d caging is rarely used for b r e e d i n g b e c a u s e n e o n a t a l therm o r e g u l a t i o n is difficult to m a i n t a i n w i t h o u t nest in g material. C a g e lids should be stainless steel to facilitate c l e a n i n g and inhibit rust. C a g e s should k e e p animals dry and clean, m a i n t a i n a c o m f o r t a b l e a m b i e n t temperature, allow f r e e d o m of m o v e m e n t

c h a n g i n g cages in specifically d e s i g n e d hoods. M o u s e cages vary in design, size, and c o m p o s i t i o n . T h e

and n o r m a l postural adjustments, avoid u n n e c e s s a r y p h y s ic a l restraints, provi de c o n v e n i e n t access to feed and water, and prevent overcrowdi ng. S o l i d - b o t t o m cages should contain sanitary b e d d i n g , such as

p o p u l a r s h o e b o x cage u s e d for h o u s i n g and b r e e d ing m i c e is

w o o d chips or g r o u n d corncob. Criteria for selecting b e d d i n g


41

Table IV

et al., 1999). However, this type of caging employs passive air exchange and is prone to accumulate CO2 and NH3, thereby requiring frequent sanitation and bedding changes (see Chapter 29). The recent large-scale introduction of individually ventilated cages obviate, to a large extent, the elevated levels of noxious gases. However, purchase of these systems requires substantial expense (see Chapter 21).

Desirable Criteria for Rodent Contact Bedding a Moisture absorbent Dust-free Does not promote microbial growth Nonstaining Atraumatic Ammonia binding Sterilizable Deleterious products not formed as a result of sterilization Easily stored Uniform from batch to batch No microbial or chemical contamination Nonpalatable Nonallergenic Nontoxic Non-enzyme-inducing Nestable Readily available Inexpensive Chemically stable during use Animal behavior is not adversely affected

aModified from Kraft (1980).

Table V Tests of Bedding Qualitya Chemical properties Pesticides and polychlorinated compounds Mycotoxins Nitrosamines Detergent residues Ether-extractable substances Heavy metals Physical properties Particle uniformity Absorbtivity Ammonia evolution Visible trauma and irritant potential Microbiological properties Standard plate count Yeasts and molds Coliforms and Salmonella Pseudomonas

aModified from Kraft (1980). vary with experimental and husbandry needs (Table IV). It is preferable to autoclave bedding prior to use, but if this is not convenient, the bedding should be used only after its origin and microbial content have been evaluated (Table V). Several caging systems with tops containing filters are routinely in use in many academic settings. Their use has been popularized because of evidence that they substantially reduce or prevent airborne transmission of microbial agents (Lipman et al., 1993) and minimize caretaker exposure to allergens (Reeb-Whitaker

2. Husbandry Nutrient requirements for the mouse are influenced by genetic background, disease status, pregnancy, and environment. The best current estimate of nutritional requirements is shown in Table VI. Nutritional requirements for laboratory mice are also published periodically by the National Research Council and have been reviewed by Knapka and coworkers (Knapka, 1983; Knapka et al., 1974). Feed intake and weight gain data are used to estimate the nutritional needs of a particular stock or strain. Mice consume about 3 - 5 gm of feed per day after weaning and maintain this intake throughout life. Outbred mice tend to gain weight faster than inbred mice and are heavier at maturity (Figs. 1 and 2). Diet is often neglected as a variable in animal-related research. Diet can influence responses to drugs, chemicals, or other factors and lead to biased research results. Therefore, diet must provide a balance of essential nutrients, and contaminants must be kept to a minimum (see also Chapter 29). Naturalproduct commercial diets for mice are satisfactory for breeding and maintenance. Fresh produce, grains, fish meal, or other supplements may expose colonies to pathogenic bacteria or harmful chemicals and should be avoided. Mice should have continuous access to potable water even if a high-moisture diet is fed. Water is needed for lubrication of dry food and for hydration. Adult mice drink 6 - 7 ml of water per day. Decreased water intake will decrease food consumption. Water imbalance may occur during disease, because sick mice commonly drink very little water. Therefore, it may be unsuitable to administer medicine orally to affected mice. However, antimicrobials can be administered in the drinking water prophylactically, a measure used commonly to prevent infection in immunodeficient mice.

II.

A.

BIOLOGY

Physiology and Anatomy

Unless otherwise indicated the information in this section is from Cook (1983) and Kaplan et al. (1983). Normative data on the mouse are presented in Table VII, and clinical chemistry reference ranges are summarized in Table VIII.

42

ROBERT O. jACOBY, JAMES G. FOX, AND MURIEL DAVISSON Naturalingredient, openformula diet e

Table VI Nutrient Requirements of Mice a Nutrient

Concentration in diet (%)

Protein (as crude protein) Fat b Fiber Carbohydrate

20-25 5-12 2.5 45-60

A (IU/kg)

Estimated Dietary Amino Acid Requirement

Amino acid

Natural -ingredient, open-formula diet (%)c

Arginine Histidine Tyrosine Isoleucine Leucine Lysine Methionine Phenylalanine Threonine Tryptophan Valine

0.3 0.2 ~ 0.4 0.7 0.4 0.5 0.4 0.2 0.1 0.5

,

Purified diet (%)d

0.12 0.2 0.25 0.15 0.3 0.25 0.22 0.05 0.3

Mineral Calcium (%) Chloride (%) Magnesium (%) Phosphorus (%) Potassium (%) Sodium (%) Sulfur (%) Chromium (mg/kg) Cobalt (mg/kg) Copper (mg/kg) Fluoride (mg/kg) Iodine (mg/kg) Iron (mg/kg) Manganese (mg/kg) Molybdenum (mg/kg) Selenium (mg/kg) Vanadium (mg/kg) Zinc (mg/kg)

1.

1.23 ~ 0.18 0.99 0.85 0.36 ~ ~ 0.7 16.1 ~ 1.9 255.50 104.0 -~ -50.3

Purified dietY 0.52 0.16 0.05 0.4 0.36 0.1 __ 2.0 ~ 6.0 m 0.2 35.0 54.0 ~ 0.1 ~ 30.0

10 0.03 5000 37 3 0.2 2009 4

Purified dietg 0.81 ~ 0.073 0.42 0.89 0.39 m 1.9 ~ 4.5 ~ 36.0 299.0 50.0 ~ ~ -31.0

Chemically defined diet h 0.57 1.03 0.142 0.57 0.40 0.38 0.0023 4.0 0.2 12.9 2.3 3.8 47.6 95.2 1.55 0.076 0.25 38.0

Temperature and Water Regulation

Mice have a relatively large surface area per gram of body weight. This results in dramatic physiologic changes in response to fluctuations in the ambient temperature (TA). The

4000 7 0.01 50 0.05 0.2 1000 2 30

21 8 17

16 6 6 ,

Chemically defined diet h

Purified dietg

1100 1730 22.5 6.0 0.023 0.58 1100 1.71 32 1514 18 10.7 0.2 1 750 2375 0.45 1.43 -248 22.5 35.6

1000

82

,

Mineral and Vitamin Concentrations of Adequate Mouse Diets Naturalingredient, openformula diet e

15,000

B 6 (mg/kg) B12 (mg/kg) D (IU/kg) E (IU/kg) KI equiv. (mg/kg) Biotin (mg/kg) Choline (mg/kg) Folacin (mg/kg) Inositol (mg/kg) Niacin (mg/kg) Calcium pantothenate (mg/kg) Riboflavin (mg/kg) Thiamin (mg/kg)

,

,

Vitamin

Purified dietI

37.5 7.5 22.5

47.5 7.1 4.8

,

a Modified from Knapka (1983). bLinoleic acid: 0.6% is adequate. cJohn and Bell (1976). dTheuer (1971). eKnapka et al. (1974). IAIN (1977). gHurley and Bell (1974). hpleasants et al. (1973).

mouse responds to cold exposure, for example, by nonshivering thermogenesis. A resting mouse acclimated to cold can generate heat equivalent to about triple the basal metabolic rate, a change that is greater than for any other animal. A mouse must generate about 46 kcal/m 2 per 24 hr to maintain body temperature for each I~ drop in TA below the thermoneutral zone.

I CD | 1

4 0

O CFI| A DBA / ZN [] Cs7BI / 6

30

E 20

lO

I

21

I

28

I

35

I

42

I

49

I

56

Age (Days) Fig. 1. Growth comparison: female outbred (CD1 and CF1) and inbred mice. (Courtesy of Charles River Breeding Laboratories.)

43


Table VII Normative Data for the Mouse

40

30 E tO} .--

20

10

A DBA / ZN r-1 Cs~BI / 6 I 21

,

I 28

I 35

I 42

I 49

I 56

Age (Days)

Fig. 2. Growthcomparison: male outbred (CD 1 and CF1) and inbred mice. (Courtesy of Charles River Breeding Laboratories.)

Mice cannot tolerate nocturnal cooling as well as larger animals that have a greater heat sink. Therefore, it is not advisable to conserve energy in animal quarters at night by lowering thermostats. Because of its great ratio of evaporative surface to body mass, the mouse has a greater sensitivity than most mammals to water loss. Its biological half-time for turnover of water (1.1 days) is more rapid than for larger mammals. Water conservation is enhanced by cooling of expired air in the nasal passages and by highly efficient concentration of urine. The conservation of water can preempt thermal stability. If the mouse had to depend on the evaporation of body water to prevent elevations of body temperature, it would go into shock from dehydration. The mouse has no sweat glands, it cannot pant, and its ability to salivate is severely limited. Mice can partially compensate for changes in TA increases from 20 ~ to 35~ It adapts to moderate but persistent increases in environmental temperature by a persistent increase in body temperature, a persistent decrease in metabolic rate, and increased blood flow to the ears to increase heat loss. Its primary means of cooling in the wild is behavioral--retreat into a burrow. In the confinement of a cage, truck, or plane, mice do not survive well in heat and begin to die at an ambient temperature of 37~ or higher. Thus, the mouse is not a true endotherm. In fact, the neonatal mouse is ectothermic and does not have well-developed temperature control before 20 days of age. The thermoneutral zone for mice varies with strain and with conditioning but is about 29.6~176 narrower than that of any other mammal thus far measured. Thermoneutrality should not be equated with comfort or physiological economy. There are repeated studies to show that mice in a TA range of 2 1 ~ 25~ grow faster, have larger litters, and have more viable pups than those maintained in the thermoneutral zone.

Adult weight Male Female Life span Usual Maximum reported Surface area Chromosome number (diploid) Water consumption Food consumption Body temperature Puberty Male Female Breeding season Gestation Litter size Birth weight Eyes open Weaning Heart rate Blood pressure Systolic Diastolic Blood volume Plasma Whole blood Respiration frequency Tidal volume Minute volume Stroke volume Plasma pH CO2 CO2pressure Leukocyte count Total Neutrophils Lymphocytes Monocytes Eosinophils Basophils Platelets Packed cell volume Red bl6od cells Hemoglobin Maximum volume of single bleeding Clotting time PTT Prothrombin time

2.

20-40 gm 18- 35 gm 1-3 years 4 years 0.03-0.06 cm2 40 6.7 ml/8 weeks age 5.0 gm/8 weeks age 98.8~ 99.3~ (37~ 37.2~ 28-49 days 28-49 days None 19-21 days 4-12 pups 1.0-1.5 gm 12-13 days 21 days 310- 840 beats/min 133-160 mm Hg 102-110 mm Hg 3.15 ml/100 gm 5.85 ml/100 gm 163/min 0.18 (0.09-0.38) ml 24 (11-36) ml/min 1.3-2.0 ml/beat 7.2-7.4 21.9 mEq/L 40 _ 5.4 mm Hg 8.4 (5.1-11.6) • 103/~tl 17.9 (6.7-37.2)% 69 (63-75)% 1.2 (0.7-2.6)% 2.1 (0.9-3.8)% 0.5 (0-1.5)% 600 (100-1000) • 103/ktl 44 (42-44)% 8.7-10.5 x 108/mm3 13.4 (12.2-16.2) gm/dl 5 ml/kg 2-10 min 55-110 sec 7-19 sec

Respiratory System

The respiratory tract has three main portions: the anterior respiratory tract consists of nostrils, nasal cavities, and nasopharynx; the intermediate section consists of larynx, trachea, and

44

ROBERT O. JACOBY, JAMES G. FOX, AND MURIEL DAVISSON Table VIII Clinical Chemistry Reference Ranges for Adult Mice a CD-1

Analyte Serum Glucose Urea nitrogen Creatinine Sodium Potassium Chloride Calcium Phosphorus Magnesium Iron Alanine aminotransferase Aspartate aminotransferase Alkaline phosphatase Lactate dehydrogenase Protein, total Albumin Cholesterol Triglycerides Bilirubin

Units

M

F

IU/liter

99 ___86.3

49 ___22.6

41.4 ___ 16.4

29.3 +__7.1

IU/liter IU/liter IU/liter g/liter g/liter mg/dl mg/dl mg/dl

196 +_ 132.6 39 ___25.7

128 + 60.6 51 _+ 27.3

99.5 ___33.4 59 ___ 11.4

73.6 _+ 15.3 118 ___ 15.9

44 _+ 11.0

48 ___8.5

114 + 56.3 91 _+ 58.5 0.4 +__0.2

72 ___20.1 53 ___23.6 0.5 ___0.35

53.9 36.7 94.8 97

63.5 46.4 92 78

Male

Female

Female

1 0 - 40

2 0 - 40 (basal)

1500-2000 (proestrus)

8 0 - 1 2 0 (basal)

250-300 (proestrus/estrus)

Luteinizing hormone

ng/ml

Follicle stimulating hormone

ng/ml

Prolactin Growth hormone Thyroid stimulating hormone Thyroxine Cortico sterone

ng/ml ng/ml

Epinephrine Norepinephrine Progesterone

pg/dl pg/dl ng/ml

Estradiol Testosterone Urine Volume Specific gravity pH Osmolality Creatinine Glucose Protein Albumin

pg/ml ng/ml

ng/ml gg/dl gg/dl

125

10%) Fever (> 103~ Persistent refractory diarrhea Opportunistic infections Noma (cancrum oris) Retroperitoneal fibromatosis Hematologic abnormalities Anemia (PCV < 30%) Neutropenia (< 1700) Lymphopenia (< 1600) Thrombocytopenia (< 50,000) Pancytopenia Bone marrow hyperplasia Characteristic lymph node lesions aFrom Lackner (1988).

intersecting fascicles of spindle-shaped cells that infiltrate along serosal surfaces and encompass normal abdominal structures, usually accompanied by lymphoplasmacytic inflammation (Mansfield and King, 1998). In severely immunosuppressed animals, marked lymphoid depletion with effacement of normal architecture occurs in the spleen, thymus, and lymph nodes. Histiocytes replace depleted plasma cells and lymphocytes in the paracortex, and follicles contain hyalinized arterioles (Osborn et al., 1984). Establishment of SRV/D-free colonies by identification of SRV/D-negative animals is difficult because serologic testing is inadequate. Apparently healthy, seronegative monkeys with persistent infections can only be identified by virus isolation or PCR for viral RNA. A successful protocol combining viral isolation and antibody screening has been described by Lerche et al. (1994). Simian immunodeficiency viruses (SIVs) are lentiviruses closely related to human immunodeficiency viruses HIV-1 and HIV-2. Serologic studies have revealed that seropositive animals are frequently found among wild and captive African monkey populations, while seropositive animals are infrequently found in captive, but not wild Asian monkey (macaque) populations. Simian immunodeficiency virus isolates are identified by subscript to indicate the species of origin, i.e., SIVmac was isolated from a rhesus monkey and SIVcpz from a chimpanzee. Simian immunodeficiency viruses in African species are of relatively low pathogenicity, sexual transmission is most likely (Phillips-Conroy et al., 1994), and transmission from dam to infant has also been proposed (Fultz et al., 1990). In macaque species, SIVs produce devastating AIDs-like disease; horizontal transmission by biting or being bitten, sexual transmission, and dam-to-infant transmission have been demonstrated in natural or experimental infections. Common lesions of uncomplicated

SIV infection in macaques are listed in Table LVI. Opportunistic infections occur with Mycobacterium avium complex, CMV, adenovirus, papovavirus, Pneumocystis carinii, Cryptosporidium spp., Cryptococcus neoformans, toxoplasmosis, and candidiasis. Development of malignant lymphoma among SIVinfected macaque populations has been described (Lowenstine et al., 1992). Unlike SRV infection, seronegative, viral-positive SIV infection in macaques is rare. Serologic testing of macaque colonies and removal of reactors is an effective way to eliminate infection from the population (Lowenstine et al., 1986). Asian monkeys should not have direct contact with African species or their tissues.

d.

Other RNA Viruses

Rabies has been reported in tamarins, squirrel monkeys, macaques, and chimpanzees (Richardson and Humphrey, 1971; Fiennes, 1972). Although current housing practices minimize the possibility of contact with carrier species, possible rabies exposure should be considered when primates are housed outdoors in rabies endemic areas. Reported clinical cases in nonhuman primates are extremely rare. Clinical signs include irritability, self-mutilation, and paralysis of pharyngeal and pelvic

Table LVI Lesions Associated with SIV Infection in Macaques a System

Comment

Lymphoid

Follicular hyperplasia Follicular involution ___expanded paracortical regions Depletion of follicular and paracortical regions Granulomatous (giant cell) lymphadenitis Generalized lymphoproliferative syndrome

Nervous

Nonsuppurative histiocytic meningoencephalitis Multifocal perivascular aggregates of giant cells and histiocytes

Gastrointestinal

Enteropathy; disseminated giant cell disease involving the lamina propria

Cardiopulmonary

Arteriopathy with medial and intimal proliferation of pulmonary arteries Thrombosis, hemorrhage, consolidation, and infarction of lung

Skin

Viral exanthema in experimental infections Perivascular lymphocytic dermatitis

Respiratory

Giant cell interstitial pneumonia

aAdapted from Mansfield and King (1998).

16. NONHUMANPRIMATES muscles. Nonhuman primates may be vaccinated with killed vaccine; however, the efficacy of the vaccine is unknown. Use of attenuated rabies vaccine is contraindicated as vaccine-induced disease is believed to have occurred in New World species (McClure et al., 1972). Montali et al. (1993) reported on the development of a rapidly progressive viral hepatitis in callitrichids in zoological collections due to infection with lymphocytic choriomeningitis virus (LCMV). Infection has been shown to occur following feeding of neonatal mice infected with LCMV and is postulated to occur from consumption of naturally infected wild mice (Montali et al., 1993). Horizontal transmission between callitrichids does not occur; however, vertical transmission has been demonstrated. Clinical signs include anorexia, dyspnea, lethargy and weakness, ataxia or incoordination, and in some instances, seizures (Montali et al., 1993). Affected animals may be jaundiced. Elevated liver enzymes, bilirubin, and alkaline phosphatase levels have been reported. Mortality can be high although serologic evidence of infection has been demonstrated in animals without history of clinical disease (Potkay, 1992). Mortality is related to the amount of infected mouse tissue consumed. Gross lesions include hepatomegaly, splenomegaly, jaundice, subcutaneous and intramuscular hemorrhages, and pleural or pericardial effusions (Montali et al., 1993). Liver lesions include multifocal hepatocellular necrosis with lymphocytic and neutrophilic infiltrates. Acidophilic apoptotic hepatocytes are found within sinusoids and Kupffer's cells (Montali et al., 1993). Necrosis of abdominal lymph nodes, adrenal glands, spleen, and gastrointestinal tract may be seen. Lymphocytic choriomeningitis virus is a zoonotic disease, and veterinarians in contact with infected marmosets developed antibody titers to LCMV (Montali et al., 1995). Naturally occurring cases of hepatitis A virus have been described in chimpanzees, owl monkeys, African green monkeys, and cynomolgus monkeys. Serologic testing indicates that infection occurs in both wild and captive nonhuman primate populations, including many New and Old World species. Transmission is fecal-oral. Infection is usually self-limiting with no clinical disease. Elevated serum alanine aminotransferase and aspartate aminotransferase, 2-10 times above normal levels, with mild elevations of bilirubin are characteristic (Mansfield and King, 1998). Microscopic lesions include focal hepatocellular necrosis with nonsuppurative inflammatory infiltrates in the portal areas. Bile duct hyperplasia and necrosis of bile duct epithelium has been described in chimpanzees. Brack (1987) reported cases of human infection with hepatitis A virus contracted from nonhuman primates, particularly from chimpanzees. Fatal infection with encephalomyocarditis viruses has been reported in owl monkeys, squirrel monkeys, baboons, rhesus macaques, and chimpanzees (Gainer, 1967; Blanchard et al., 1987; Hubbard et al., 1992; Baskin, 1993). Wild mice and rats are the reservoir hosts for this group of viruses, and fecal con-

757

tamination of feed, water, or enclosures has been postulated as the source of infection in nonhuman primates. Death with no premonitory clinical signs is usual in naturally infected monkeys. Pericardial effusion, white-tan mottling of the myocardium, and pulmonary congestion may be observed at necropsy. Nonsuppurative necrotizing myocarditis is the most important microscopic lesion. Placental infection and subsequent abortion can occur (Hubbard et al., 1992). Elimination of feral rodents and cleaning of facilities are essential for prevention and control. Naturally occurring infections with poliovirus have been reported for the great apes and rhesus monkeys. Infection may cause no clinical disease, or infected animals may develop paresis and paraplegia and then die. Lesions are located in the gray matter of the central nervous system and include perivascular inflammatory cell aggregates and meningeal infiltrates with neuronal necrosis and glial nodules. Vaccination of great apes with oral trivalent polio vaccine is recommended. Vaccination has been used effectively in wild chimpanzee populations to prevent disease (Morbeck et al., 1990).

D.

Parasitic Diseases

Nonhuman primates, particularly feral animals, may be infested with a variety of parasites. Many of these infections are incidental or subclinical in nature and will not be presented in detail. Tables LVII-LXIV list parasites that produce clinical disease in nonhuman primates; disease, pathology, diagnosis, and treatment are briefly described. Figures 27-42 illustrate ova, adult parasites, or gross or microscopic lesions.

E.

Nutritional Diseases

Some nutritional diseases of nonhuman primates are summarized in Table LXV (see next page). Most are due to vitamin deficiencies, with one instance of vitamin overfeeding. Simian bone disease, also known as nutritional hyperparathyroidism due to insufficient calcium or improper calcium-phosphorus ratio in the diet, is included although its occurrence is now rare with the use of commercially formulated feeds. Errors can and have occurred during feed manufacture, and a specific batch of a commercial diet may be misformulated (Ratterree et al., 1990; Eisele et al., 1992). Table LXV is not inclusive of nutritional diseases but provides a synopsis of those commonly reported. References in the table footnotes can be used to find more detail on these conditions. In general, young and rapidly growing animals are more likely to develop clinical disease due to nutritional deficiencies. Clinical signs of nutritional disorders may overlap or be sufficiently nonspecific to preclude diagnosis, as in vitamin C and D deficiencies. Radiography to determine specific lesion site and

cr

Table LVII

Enteric Protozoa Parasite

Affected species a

Location

Clinical disease/pathology

Diagnosis

Treatment

Flagellates

Giarclia spp.

Small intestine OWM Apes

Frequently subclinical Diarrhea and vomiting ___steatorrhea

Saline wet mount, fresh feces Fecal concentration for cysts

Metronidazole b,c 3 0 - 5 0 m g / k g BID X 5-10 days Furozolidine ~

1.5 mg/kg X 7 days (marmosets)

Trichomonas spp.

NWM OWM

Intestine Stomach, pelvic cavity (invasive disease in rhesus)

Diarrhea reported for callitrichids

Saline wet mount, fresh feces Rectal swab and culture

Metronidazole a 17.5-25mg/kg BID • 10 days

NWM OWM Apes

Colon, cecum

Varies, more severe in young monkeys and NWM Anorexia, vomiting Severe diarrhea _ hemorrhage Necroulcerative colitis Flask-shaped ulcers Amebic abscesses in liver, lungs, or central nervous system

Saline wet mount Iodine, trichrome, or Giemsa stain Trophozoite 2 0 - 3 0 Ixm diameter Organism in lesioned organs Giemsa, trichrome, PAS stains

Metronidazole c

Amoebas

Entamoeba histolytica

30mg/kg TID X 5 - 1 0 days or in combination with Diiodohydroxyquin c

3 0 - 4 0 mg/kg TID Tetracycline r

2 5 - 5 0 m g / k g 5 - 1 0 days Chloroquin c

5 mg/kg x 14 days Chloramphenicol e

50-100 mg/kg BID Paromomycin f

12.5-15 mg/kg BID x 5-10 days Coccidians

Isospora spp.

Callitrichids

Intestine

Diarrhea

Fecal flotation Saline wet mount

Sulfamethoxine f

50 m.g/kg/day 1, then 25 mg/kg/day

(continues)

Table LVII (Continued) Parasite Cryptosporidium

spp.

Affected species a

Location


Diagnosis

Treatment

Prosimians NWM OWM

Intestine Bile duct, pancreatic duct, conjunctiva, trachea, bronchioles in immunosuppressed animals

Intractable diarrhea Profuse, watery diarrhea Depression, weight loss Hypothermia, anorexia Dehydration Fluid and gas distension of intestine Mesenteric LN enlargement Blunting and fusion of villi Villous atrophy (Fig. 27) Increased mitotic index in crypts Hyperplasia of biliary and pancreatic duct epithelium; periductal fibrosis

Stain fecal smears or concentrates Flotation Direct or indirect FAS Stool antigen detection assay Formalin-fixed feces Histology 4 - 5 p~m oocysts on brush border of enterocytes (Fig. 28)

Supportive care Replace fluid and electrolytes Antidiarrheals Antibiotics

NWM

Cecum, colon

Usually nonpathogenic Can cause severe, ulcerative colitis in apes Anorexia, weight loss, weakness, lethargy Watery diarrhea, tenesmus, rectal prolapse

Large ciliated ovoid organisms 30-150 X 25-120 Ixm

M e t r o n i d a z o l e c,g

Ciliates

Balantidium coli

OWM Apes

aNWM, New World monkeys; OWM, Old World monkeys. bFrom Peisert et al. (1983). CFrom Lehner (1984). dFrom Brady et al. (1988). eFrom Renquist and Whitney (1987). IFrom Wolff (1993). gFrom Swenson et al. (1979).

35-50 mg/kg/day divided doses TID Tetracycline g

40 mg/kg PO divided dose TID Diiodohydroxyquin

8

40 mg/kg PO divided doses TID x 14-21 days

760

BRUCE J. BERNACKY, SUSAN V. GIBSON, MICHALE E. KEELING, AND CHRISTIAN R. ABEE

Fig. 27.

Fig. 28.

Fusion of villi due to cryptosporidial infestation.

Cryptosporidium organisms adherent to enterocytes.

Table LVIII Hemoprotozoa Parasite

Trypanosoma cruzi

Host a NWM OWM Apes

Location


Blood (trypomastigote)

Subclinical Anemia, hepatosplenomegaly Lymphadenitis Generalized edema Anorexia, depression, weight loss Right bundle branch block-ECG

Skeletal and cardiac muscle, reticuloendothelial system (amastigote)

Myocarditis Pseudocysts within myocardial fibers contain 1.5- 4.0 p~m round-to-oval organisms

Treatment

Diagnosis Smear, blood or body fluid, thick and thin

None

Serology EIA or CF Histology

Chloroquine c 2.5-5 mg/kg IM X 4 - 7 days followed by Primaquine 0.75 mg/kg P O x 14 days

Plasmodium brazilianum

NWM

Erythrocytes

Usually persistent low parasitemia Can cause severe, fatal disease Anemia, cyclic pyrexia Hepatosplenomegaly Depression, death

P. knowlesi, cynomolgi, fieldi, fragile, inui

Macaques Leaf monkeys

Erythrocytes

Usually subclinical unless splenectomized or immunosuppressed Anorexia, fever, weakness, splenomegaly

As above

P. pitheci, rodhairi, reichenowi, schwetzi

Great apes

Erythrocytes

Usually subclinical parasitemia Some strains cross-infective with humans

As above

P. hylobati, youngi, eylesi

Gibbons

Erythrocytes

Fever with parasitemia, pathogenic

As above

Hepatocystis spp.

OWM Apes

Blood, liver

Subclinical infection Numerous, random, gray-white foci on surface of liver (merocyst) Eosinophilic granulomatous reaction to ruptured cysts Focal fibrosis when healed

Thick or thin blood smears Typical hepatic lesions

Thick blood smear Serology (FA)

None Vector control

Histology

Babesia pitheci

NWM OWM

Erythrocytes

Severe anemia, death with splenectomy Mild disease in intact monkeys

Blood smear Pyriform 2 - 6 ~tm organisms

None

Entopolypoides macacai

OWM Apes

Erythrocytes

Usually subclinical Fever, anemia, monocytosis Hemolytic anemia and icterus following splenectomy or immunosuppression

Blood smear

None

aNWM, New World monkeys; OWM, Old World monkeys. bEIA, enzyme-linked immunoassay; CF, complement fixation. CFrom Lehner (1984).

762


Table LIX Disseminated Protozoal Infestations Parasite

Toxoplasma gondii

Host a

Location


Prosimians NWM OWM Apes

Lymph nodes, liver, lung, spleen, intestine, brain, heart

Death, no clinical signs Anorexia, listlessness, weakness Depression, somnolence Emesis, diarrhea Oculonasal discharges Dyspnea, tachypnea Neurologic signs Circling, head holding, head hitting, incoordination, paresis, convulsions Hepatosplenomegaly Ulcerative enteritis Lymphadenopathy Pulmonary edema

Encephalitozoon cuniculi

Squirrel monkeys

Brain, kidney, lung, adrenal glands, liver, placenta

None or nonspecific, death Granulomatous inflammation (Fig. 30)

Diagnosis Serology (CF, IFA, HA1)b Impression smears of spleen, lung, lymph nodes

Treatment No treatment reported for nonhuman primates

Pediatric treatment regimen (humans) c Histology, Gram-, PAS- 4 - 8 I~m Sulfadiazine banana-shaped tachyzooites in 100 mg/kg/day; in divided 60 ~m cyst (Fig. 29) doses BID x 1 month Animal inoculation Pyrimetharnine Load with 1 mg/kg/day x 2 - 4 days; then 1 mg/kg/day Folio acid 1 mg/day to prevent bone marrow depression

2.5 X 1.5 lxm oval organisms in 60-120 I~m pseudocysts in tissues (Fig. 31) Gram+, PAS + Serology IFA, ELISA Organisms in urine

None

NWM, New World monkeys; OWM, Old World monkeys. bCF, complement fixation; IFA, indirect fluorescent antibody assay; and HAI, hemagglutination inhibition test. CFrom Lehner (1984).

Table LX Acanthocephalans

Parasite

Host a

Prosthenorchis elegans, P. spirula

NWM

Location Ileum, cecum, colon

Prosimians


Diagnosis

Anorexia, dehydration Abdominal distension Diarrhea Debilitation Weight loss Death

Fecal smears Formalin ether sedimentation (standard flotation not effective) Flexible fiberoptic proctoscopy Palpation of abdominal masses Necropsy (Fig. 32)

Treatment Carbon tetrachloride 0.5 ml/kg PO once

(Efficiency and long-term safety of this treatment is not established)b

aNVC'M, New World monkeys. bFrom Toft and Eberhard (1998).

Table LXI Scabies Parasite

Sarcoptes scablei

Hosta

Location


Diagnosis

Treatment

OWM

Skin

Severe pruritis Anorexia, weight loss, weakness Tremors Alopecia, scaling and thickening of skin Self-mutilation Bacterial dermatitis Death

Deep skin scrapings for parasites and eggs (Fig. 33)

Ivermectin 200mcg/kg; repeat in 3 weeks b

Apes

Old World monkeys. bFrom Toft and Eberhard (1998). a OWM,

16. NONHUMAN PRIMATES

763

Fig. 29. Hepatocellular necrosis due to Toxoplasma gondii infestation. Note toxoplasma tachyzoites within hepatocytes.

Fig. 30. Microgranuloma in the brain of an infant squirrel monkey, Saimiri spp., associated with Encephalitozoon spp.

763

764


Fig. 31. Encephalitozoon spp. pseudocyst, squirrel monkey cerebellum.

Fig. 32.

Thorny-headed worms, Prosthenorchis elegans, attached to the ileal mucosa of a tamarin, Saguinus mystax.

Table

LXII

Nematodes Parasite

Strongyloides cebus

Host a NWM

Location Intestine


Fecal flotation, larvae, or larvated ova Necropsy, histology (Fig. 34, 35, and 36)

Ivermeetin b 200 mcg/kg IM

Diarrhea _ hemorrhage _ mucus Dermatitis, urticaria Vomiting, dehydration Debilitation, emaciation Cough, dyspnea Fatalities in apes, patas Enterocolitisi catarrhal, hemorrhagic, or necrotizing Peritonitis Pulmonary hemorrhage

Fecal flotation, larvae, or larvated ova Necropsy, histology

Ivermectin b

Usually subclinical Death in spider monkey due to overwhelming infestation

Observation of adults at anus Perianal tape test or swab (Fig. 37) Fecal flotation Ellipsoid, asymmetric ova

Thiabendazole e

May be subclinical Anal pruritis and irritation Self-mutilation Aggressiveness Fatalities in chimpanzees Ulcerative colitis, peritonitis, lymphadenitis

Observation of adults at anus Perianal tape test or swab Fecal flotation Ellipsoid, asymmetric ova

Mebendazole d

Weight loss, diarrhea Unthrifty Subserosal nodules 2 - 4 mm in colon and mesentery

Fecal flotation, hookworm like ova Identify larvae following stool culture (Fig. 38)

Thiabendazole a

Usually none, eosinophilia Fatality in a woolly monkey

S. fulleborni, S. stercoralis

Trypanoxyuris sp.

OWM Apes

NWM

Intestine Filariform larvae in lungs and other parenchymous organs

Cecum, colon

Enterobius ve rmicularis, E. anthropopitheci

OWM Apes Chimpanzees

Cecum, colon

Oesophagostomum spp.

OWM Apes

Cecum, colon

Treatment

Diagnosis

Thiabendazole c

50-100mg/kg PO for 1, 2, or 5 days 200 mcg/kg IM, PO Thiabendazole c

5 0 - 1 0 0 mg/kg PO for 1, 2, or 5 days Mebendazole d

22 mg/kg/day PO or SQ x 2 - 3 days Levamisole d 10mg/kg PO or SQ • 2 - 3 days Pyrantel pamoate d

11 mg/kg PO once 5 0 - 1 0 0 mg/kg PO

100mg/kg PO adult ape 10mg/kg PO infant or smaller species P y r a n t e l pamoate d 11 mg/kg PO once

2 5 - 1 0 0 m g / k g PO for 1-2 days Levamisole d 10mg/kg PO or SQ once Mebendazole d

40 mg/kg PO, divided dose TID X 3 - 5 days Repeat all treatments at 10 -14 days

Molineus elegans, M. torulosis

NWM

Pylorus, duodenum Pancreatic ducts

M. elegans usually subclinical Ulcerative enteritis _ hemorrhage with M. torulosis Serosal nodules in duodenum Chronic pancreatitis

Fecal flotation Necropsy

None

(continues)

Table LXH (Continued)

Parasite

Hosta

Location

OWM Apes

Intestine

Trichospirura leptosoma

NWM

Pterygodermatites nycticebi, P. alphi

Prosimians NWM Gibbons

Ascaris lumbricoides

Trichuris spp.

Anatrichosoma cynomolgi


Treatment

Usually subclinical Deaths associated with intestinal blockage due to heavy parasitism and migration to liver, bile ducts

Fecal flotation, roundworm ova (Fig. 39)

Mebendazole f

Pancreatic ducts

Weight loss, wasting disease Acute/chronic pancreatitis Jaundice due to bile duct obstruction

Fecal flotation Thick-shelled, larvated ova

None

Intestine

Watery diarrhea, anorexia, weakness (tamarins) Anemia, leukopenia, hypoproteinemia Pseudomembranous necrotizing enteritis

Fecal flotation, spirurid ova Adults, larvae in feces Necropsy, histology

Ivermectin g

Anorexia, mucoid or watery diarrhea, and occasionally death may occur with heavy infestations

Fecal flotation Bipolar operculated ova (Fig. 40)

Subclinical nasal infestation or mild serous discharge Pruritis of hands and feet Vesicle/pustules in skin Regional lymph node enlargement

Nasal or epidermal swabs or s c r a p i n g s i ova (Fig. 41)

NWM OWM Apes

Cecum, colon

NWM OWM Apes

Nasal mucosa Secondary infestation of hands and feet (creeping eruption)

New World monkeys; OWM, Old World monkeys. bFrom Brack and Rietschel (1986) and Battles et al. (1988). CFrom Bingham and Rabstein (1964), Flynn (1973), Swenson et al. (1979), Lehner (1984), and Abee (1985). dFrom Swenson et al. (1979). eFrom Lehner (1984). fFrom Toft and Eberhard (1998). gFrom Blampied et al. (1983). hFrom Welshman (1985). /From Kumar et al. (1978). JFrom Harwell and Dalgard (1979). a NWM,

Diagnosis

22 mg/kg PO • 3 days Pyrantel p a m o a t e f 11 mg/kg PO once, repeat in 10-14 days

0.5 mcg/kg SQ x 3 days predilute in sterile water for smaller primates (marmosets) Mebendazole g

40mg/kg x 3 days Mebendazole d

40mg/kg PO BID x 5 days Dichlorvos d 10mg/kg PO SID, 1-2 days Levamisole h 7.5 mg/kg SQ x 2 at 2-week interval F l u b e n d a z o l e (5 %)i 27-50mg/kg BID x 5 days (baboons) Fenbendazole /

10-25 mg/kg PO SID x 3-10 days

767


Fig. 33.

Sarcoptes scabiei mite from a rhesus monkey, Macaca mulatta.

Fig. 34.

Larvated Strongyloides egg, fecal flotation.

768


Fig, 35.

Fig. 36.

Strongyloides cebus larva, fecal flotation.

Strongyloides cebus, adult parasite in the small intestine of a squirrel monkey, Saimiri spp.

769

16. N O N H U M A N PRIMATES

Fig. 37.

Pinworm eggs on a perianal tape test from an owl monkey, Aotus spp.

Fig. 38.

Oesophagostomum sp. or nodular worm in intestine of rhesus monkey.

770


Fig. 39.

Fig. 40.

Egg ofAscaris lumbricoides, fecal flotation from a chimpanzee.

Trichuris trichiura egg, fecal flotation from a baboon, Papio spp., iodine stain.

771


Fig. 41.

Fig. 42.

Anatrichosoma cynomolgi egg, fecal flotation.

Cysticercus in the brain of a stump-tailed macaque, Macaca nemestrina.

772

BRUCE J. BERNACKY, SUSAN V. GIBSON, MICHALE E. KEELING, AND CHRISTIAN R. ABEE Table L X I I I

Cestodes Parasite

Location

Host a


Diagnosis

Treatment

All

Small intestine

Abdominal pain, tucked abdomen, crouching Anorexia, vomiting Catarrhal enteritis Abscessed mesenteric lymph node

Fecal flotation Ova Proglottids in feces Necropsy

Niclosamide b 100 mg/kg PO once Bunamidine b 25-100 mg/kg PO once Praziquantel c 0.1 mg/kg IM

Cysticercosis Taenidae

All

Abdominal or thoracic cavities Muscle Subcutaneous tissues Central nervous system (CNS)

Clinical signs dependent on number and location of cysticerci

Necropsy (Fig. 42)

None

Coenurosis Multiceps spp.

Prosimians OWM

Subcutaneous tissue Peritoneal cavity Brain Liver

Dependent on number and location of coenuri Usually subclinical except in CNS

Radiography Palpation of mass in SQ tissues

None

Hydatidosis Echinoccocus granulosus

All

Abdominal or thoracic cavities Liver Lungs Retrobulbar Subcutaneous tissue

Usually subclinical, may mimic neoplasic disease Abdominal distension Exophthalmia Localized subcutaneous mass Anaphylactic shock, death following cyst rupture in lungs

Radiography Ultrasound Serology: intradermal skin test HA d

None

Hymenolepis nana

Larval Diseases

All, all nonhuman primates; OWM, Old World monkeys. bFrom Swenson et al. (1979). cFrom Welshman (1985). dHA, hemagglutination assay. a

Table L X I V

Trematodes Parasite

Host a

Location


Treatment

Diagnosis

Gastrodiscoides hominis

OWM

Cecum, colon

Mucoid diarrhea Mild chronic colitis

Ova in feces

None

Watsonius spp.

OWM

Intestine

Diarrhea, severe enteritis Death

Ova in feces

None

Paragonimus westermanii

OWM

Lung Ectopic sites include brain and liver

Cough, wheezing Blood in sputum Moist rales Progressive weight loss

Ova in feces Necropsy

None

Schistosoma mansoni, S. haematobium, S. matheei

NWM OWM Apes

Mesenteric veins (S. mansoni and S. matheei) Portal veins (S. haematobium)

Usually subclinical Fever, hemorrhagic diarrhea Hematuria Ascites

Ova in feces or urine Necropsy, adults in veins

Praziquantal (56.8 mg/ml) b 0.2 cm3/kg if < 1 kg body weight; 0.1 cm3/kg if > 2 kg body weight

a NWM, New World monkeys; OWM, Old World monkeys. bFrom Toft and Eberhard (1998).

Table L X V Nutritional Diseases Condition

Common name

Species affected

Clinical signs

Hypovitaminosis A

Rhesus monkeys a

Abortion

Hypervitaminosis A

Callitrichids b

Musculoskeletal lameness, paresis cachexia, debilitation, alopecia

Rhesus monkeys c (experimental)

Visual impairment Spastic paralysis of hindlimbs Decreased range of motion in knees Painful, swollen joints Contracted tendons Hand walking Megaloblastic anemia

Hypovitaminosis B 12

May contribute to "cage paralysis"

Chimpanzees Hypovitaminosis C

Scurvy

All primates (young animals most affected)

Squirrel monkeys

Reluctance to move Joint pain and tenderness Lameness, abnormal locomotion Gingival swelling, hyperemia, petechia Bruising Microcytic anemia Acute cephalohematoma

Lesions

Treatment Correct diet

Spinal hyperostosis and spinal ankylosis

Correct diet Irreversible

Subperiosteal hemorrhage Epiphyseal fracture Periodontal bone resorption Long bone fractures Periosteal elevation long bones

25 mg/kg ascorbic acid IM BID X 5 days e

Periosteal elevation skull (Fig. 43)

50mg ascorbic acid IM single dose f or 10mg/kg body weight/day g

250 mg ascorbic acid IM x 2 days and oral 30-100 mg/kg/day d

Hypovitaminosis D 2

Rickets

Old World monkeys

Failure to grow Wrists and knee enlargement Bowing of long bones

Cupping of epiphyses Decreased bone density

Provide D2 in diet Ultraviolet light

Hypovitaminosis D 3

Rickets

New World monkeys (young animals)

Growth retardation Impaired ambulation Fractured long bones Masticatory weakness, difficulty chewing Inanition, death

Metaphyseal cupping and fraying of femur and tibia

1 IU/gm diet D 3 preventive h Provide source of ultraviolet B radiation /

(continues)

Table LXV ( C o n t i n u e d )

Condition

Common name

Folic acid deficiency

Species affected Monkeys

Squirrel monkeys Hypovitaminosis E

Calcium deficiency

Nutritional cardiomyopathy

Baboons l

Vitamin E-responsive anemia

Owl monkeys m (gray-necked)

Contributes to marmoset wasting syndrome

Tamarins n

Simian bone disease; osteomalacia

Prosimians and simians ~

aFrom O'Toole et al. (1974). bFrom Demontoy et al. (1979). CFrom Kark et al. (1974). Agamanolis et al. (1976). aFrom Eisele et al. (1992). eFrom Ratterree et al. (1990). fFrom Kessler (1970). gFrom Lehner et al. (1968). hFrom Lehner et al. (1967). i From Gacad et al. (1992). J From Wixson and Griffith (1986). kFrom Rasmussen et al. (1980). lFrom Liu et al. (1984). mFrom Sehgal et al. (1980) and Weller (1994). "From Baskin et al. (1983). ~ Krook and Barrett (1962) and Snyder et al. (1980).

Clinical signs

Lesions

Megaloblastic anemia j Weight loss, petechia Anorexia, gingivitis, dehydration Diarrhea Alopecia, scaly dermatitis Megaloblastic anemia of pregnancy k Death, no clinical signs Heart failure Dyspnea Weakness, pallor, heart murmur Icterus Hemolytic anemia, hematocrit ~< 15 Weight loss, muscle atrophy Anemia Decreased locomotion Impaired mobility

Treatment

109 lxg folic acid/day preventive White areas of myocardium Acute myocytolysis, fibrosis

Vitamin E

Splenomegaly

Vitamin E and selenium, 02, supportive therapy, transfusion (rarely) Not responsive to treatment but vitamin E was preventive

Myositis, steatitis

Kyphosis Bowing, fractured long bones Thickened jaw Dental displacement

Correct calcium: phosphorus in diet

16. NONHUMANPRIMATES appearance, diet analysis to determine if there is an adequate amount of the nutrient in question, necropsy and histologic diagnoses, and response to treatment can contribute to making a specific diagnosis. Management practices, feeding varied diets of multiple foods, and lack of knowledge of nutritional requirements for a specific animal may interact and result in a nutritional deficiency. (Fig. 43). An example is wasting marmoset syndrome. Affected callitrichids have chronic diarrhea, colitis, and in some cases, hemolytic anemia. Animals experience marked weight loss and muscle atrophy and develop alopecia. Both managerial and dietary factors probably contribute to this syndrome. The practice of feeding groups of marmosets or tamarins on the cage floor or at one feed site has been considered contributory to the development of wasting marmoset syndrome (Tardif and Richter, 1981). The dominant callitrichid may guard the food station and prevent or limit access to other group members. A single commercial diet has not proven to be adequate for callitrichids, so many laboratories feed a varied diet that may or may not be nu-

Fig. 43. Hyperostosisof a squirrel monkeyskull subsequentto cephalohematoma formationfrom vitaminC deficiency.

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tritionally adequate. Primary nutritional vitamin E deficiency and protein deficiency due to chronic diarrhea have been proposed as etiologies for this syndrome (Baskin et al., 1983; Tucker, 1984). Other investigators have suggested that the etiology of this syndrome is inadequate calories per gram of diet, combined with a high daily gross energy requirement for callitrichids (Barnard et al., 1988).

F. 1.

Miscellaneous Disorders

Trauma

Trauma is a common medical problem requiring treatment in pair or group-housed primates. Aggression between animals can cause severe wounding or death. Trauma frequently involves injury to tissues beneath the skin; associated tissue damage may require treatment for shock, sepsis, or multiple organ failure. Severely wounded animals require vigorous treatment, including intravenous fluid and electrolyte therapy. Crush injuries to the skin are commonly associated with bite wounds. The extent of injury can easily be underestimated because there are no visible lacerations and external hemorrhage may be minimal or nonexistent. Laboratory values may be within normal limits. Several days later injured areas have developed ischemic necrosis characterized by dry, blackened, devitalized skin. Underlying tissues may be devitalized and require debridement. Secondary infection is common. In severe cases, damage to underlying tissues can result in myoglobinuria, myoglobin casts in the kidney, and resultant fatal acute renal disease. Accurate early diagnosis of crush injury and aggressive fluid therapy can ameliorate these sequelae. Another cause of traumatic injury in macaques is self-mutilation or self-injurious behavior (SIB). In its mildest form, SIB is expressed as hair pulling; head banging and self-biting are the more severe manifestations of SIB. From 5 to 12% of individually housed rhesus macaques have been reported to engage in SIB (Bayne et al., 1995; Novak et al., 1998). Wounds usually consist of bites or slashes to skin and muscle. Episodes of selfbiting behavior are more frequent than actual wounding. Attempts to decrease the occurrence of SIB through environmental enrichment with puzzle feeders or other manipulanda have been unsuccessful (Novak et al., 1998). Cutting or blunting of the canine teeth decreases wounding but does not stop selfbiting behavior (Bayne et al., 1995). Tail injuries due to tail chewing are a major problem in grouphoused squirrel monkeys. Occasionally 1 or 2 animals within a group will bite the tails of other members. Once injured, infection or irritation of the tail can lead to self-mutilation of the existing wound (Abee, 1985). Blood supply to the tail is poor, and gangrene or infection are frequent sequelae. Tail injuries may be self-inflicted; squirrel monkeys with abscessed teeth or other

BRUCE J. BERNACKY,SUSANV. GIBSON, MICHALE E. KEELING, AND CHRISTIANR. ABEE

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dental problems may gnaw the tail in order to alleviate the primary source of pain (Abee, 1985). Treatment of severely injured or infected tails is partial amputation of the tail to remove the damaged segment.

2.

Hypoglycemia

Squirrel monkeys, marmosets, and owl monkeys may develop hypoglycemia as a primary disorder. Hypoglycemia occurs more commonly in young animals, particularly infants, but can also be seen in older, debilitated animals. Predisposing factors include higher basal metabolic rates, lower percentage of body adipose tissue, limited glycogen reserves, limited gluconeogenic enzymes, and limited ability to utilize ketones or fatty acids (Abee, 1985; Baer, 1994). Separation from the social group, accidental feed deprivation, complications during weaning, prolonged research procedures, fasting, or anorexia due to an underlying disease condition can place monkeys at risk for hypoglycemia. Severity of clinical signs is related to the degree of hypoglycemia. Clinical signs include weakness, lethargy, disorientation, seizures, or unconsciousness (Baer, 1994). Glucose meters developed for human diabetics are useful in diagnosis as they require only one to two drops of blood to make a fast and accurate determination of blood glucose levels. Squirrel monkeys with blood glucose levels less than 40 mg/dl and owl monkeys with glucose levels less than 50 mg/dl are considered hypoglycemic. Conscious hypoglycemic animals can be given oral glucose, sucrose solutions, or fruit juice. Alternatively, warmed 5% dextrose solutions may be administered intravenously at 5 - 8 ml/100 gm body weight (Abee, 1985; Baer, 1994). Use of concentrated dextrose solutions intravenously in New World monkeys is contraindicated (Brady, 2000). Treatment of unconscious squirrel monkeys with 20% dextrose orally by stomach tube at approximately 1 ml/100 gm body weight is effective (Brady, 2000). Hypoglycemia is not likely to be a primary disease condition in larger, Old World monkeys. Treatment with an intravenous bolus of 10 ml of 50% dextrose is recommended for hypoglycemic animals weighing > 5 kg (Rosenberg, 1995). If peripheral circulation and hydration are poor, then 1020% dextrose solution can be administered intravenously. Solutions may be given orally if the monkey has a good swallowing reflex. 3.

Hypothermia and Hyperthermia

Primates housed outdoors are susceptible to hypothermia and hyperthermia associated with extremes in the weather. Particular care should be taken in the introduction of animals to outdoor housing; acclimatization should take place when temperatures are not extreme. Shelter and supplemental heat are required in areas where temperatures drop below freezing. Simi-

larly, shade and shelter are necessary to provide relief during the summer. In areas with more severe summers, water misters may need to be provided to help keep animals cool. Temperature extremes may also occur in indoor housing due to a failure of environmental systems. Hypothermia is frequently encountered in neonatal monkeys that have been separated from or rejected by the dam. These animals are also likely to be hypoglycemic. Moribund infants may appear lifeless when discovered; vigorous toweling, gradual warming, and correction of hypoglycemia, if present, can revitalize them. Treatment for hypothermia is best accomplished by placing the animal in a lukewarm water bath and monitoring body temperature every 4 - 5 min. Alternative methods of warming include use of recirculating water pads, warm-air blankets, warmed fluid enemas, and warmed intravenous fluids, but they do not warm the animal as rapidly. Electric heating pads and heat lamps are contraindicated as extreme temperatures and subsequent burns or overheating of the animal can result. Treatment for hyperthermia is also best accomplished by a cool- to room-temperature water bath with frequent monitoring of body temperature. 4.

Acute Gastric Dilatation

Acute gastric dilatation or bloat in nonhuman primates has occurred following overeating and drinking, following alteration of gastric flora from antimicrobial therapy, or following anesthesia, transportation, or other change in routine (Soave, 1978; Stein et al., 1981). Bloat has been associated with gastric proliferation of Clostridium perfringens (Bennett et al., 1980; Stein et al., 1981), although a definitive causal relationship has not been proven. In one review, Bennett et al. (1980) reported that none of the monkeys involved had had a disruption in schedule nor had they been recently anesthetized or tranquilized. Clostridium perfringens was isolated from gastric contents of 21 of the 24 monkeys and from monkey diet biscuits fed to the animals. Individual monkeys may have a predisposition for developing acute gastric dilatation and experience multiple episodes (Soave, 1978). Early clinical signs include discomfort, as indicated by frequent grimacing and reduction in activity (Soave, 1978). As the disease progresses, monkeys may crouch or lie prone in the cage (Newton et al., 1971; Soave, 1978). Marked abdominal distension, shallow labored respiration, and coma occur terminally. Frequently monkeys are found dead with no clinical signs. Acute gastric dilatation is a medical emergency and must be treated promptly. Soave (1978) reported the following procedures for treatment of bloat in macaques: sedation with ketamine hydrochloride (10-15 mg/kg body weight IM); gastric intubation to relieve intragastric pressure; administration of an agent to control gas formation; oral administration of ampicillin


777

REFERENCES

Fig. 44. Swelling of the sinus due to an abscessed canine tooth in an adult male Bolivian squirrel monkey, Saimiri boliviensis boliviensis. Sinus has been olSened to facilitate drainage following extraction of the tooth.

(30,000 I U / k g ) ; intravenous administration of lactated Ringer's solution ( 2 0 - 3 0 m l / k g ) ; and cortisone (1 m g / k g ) administered IV or I M to counter shock. Gas and fluid should be r e m o v e d slowly to m i n i m i z e vascular collapse following release of gastric pressure.

5.

Dental Abscesses

Severe wear and abscessation of teeth are c o m m o n in adult squirrel m o n k e y s and also occur in other species of nonh u m a n primates. A b s c e s s e s of the u p p e r canine teeth present as swellings b e n e a t h the eye and will rupture if left untreated (Fig. 44). A b s c e s s a t i o n of m o l a r teeth in squirrel m o n k e y s usually extends into the infraorbital region of the eye and can lead to e x o p h t h a l m o s and blindness (Abee, 1985). E n u c l e a t i o n of the eye m a y be required.

ACKNOWLEDGMENTS

We a c k n o w l e d g e the following p e o p l e for their assistance and support during the p r e p a r a t i o n of this d o c u m e n t : Ms. L i n d a Karnstadt, Ms. R e g i n a M c C r e e r y , Ms. L a u r a Zapalac, Ms. Susan L a m b e t h , Dr. David Elmore, Dr. Pat Frost, Dr. G e n e Hubbard, Dr. M e l a n i e Ihrig, Dr. D. R i c k Lee, Dr. M i c h e l l e Leland, Dr. Steve Schapiro, and Dr. Jim Weed.

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Chap ter 17 Biology and Diseases of Amphibians Dorcas P. O'Rourke and Terry Wayne Schultz

I.

II.

III.

Introduction .................................................

793

A.

Taxonomy

793

B.

U s e in R e s e a r c h . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C.

Availability and S o u r c e s

D.

L a b o r a t o r y M a n a g e m e n t and H u s b a n d r y

Biology

..............................................

795

...................................

796

......................

796

....................................................

801

A.

A n a t o m y and Physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

801

B.

Nutrition

...............................................

803

C.

Behavior ................................................

806

D.

Reproduction

807

E.

M a n a g e m e n t and R e p r o d u c t i o n o f X e n o p u s

F.

P h y s i c a l E x a m i n a t i o n and T e c h n i q u e s

............................................ ....................

808

........................

813

Diseases ....................................................

814

A.

Infectious Diseases

814

B.

Metabolic/Nutritional Diseases ..............................

822

C.

Traumatic Disorders

823

D.

Toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

823

E.

Neoplasms

823

....................................... ......................................

..............................................

References ..................................................

I.

INTRODUCTION

Amphibians are unique among vertebrate species in that they represent the transition between ancestral aquatic life-forms and more recently evolved terrestrial existence. The word amphibian is derived from the Greek "amphibios," which means "double life." This "double life" accurately describes the aquatic larval stage and postmetamorphic terrestrial lifestyle of many amphibians. This chapter presents an overview of amphibian biology and husbandry, followed by a specific section


823

on Xenopus management, and concludes with a discussion of amphibian diseases.

A.

Taxonomy

Class Amphibia is represented by approximately 4300 species contained in 3 orders: Gymnophiona, Caudata, and Anura (Table I). Caecilians comprise the order Gymnophiona. Caecilians are legless, burrowing amphibians that inhabit wet, tropical areas of Asia, Africa, and the Americas. Most are less than


794

DORCAS P. O'ROURKE AND TERRY WAYNE SCHULTZ Table I

Scientific and CommonNames of Selected Amphibian Species Common names Hellbender Mudpuppy Axolotl Tiger salamander Red-backed salamander Red-spotted newt Fire-bellied toad African clawedfrog Bullfrog Leopard frog Green tree frog Green poison dart frog Giant toad

Scientific names Cryptobranchus alleganiensis Necturus maculosus Ambystoma mexicanum A. tigrinum Plethodon cinereus Notophthalmus viridescens Bombina orientalis Xenopus laevis Rana catesbeiana R. pipiens Hyla cinerea Dendrobates auratus Bufo marinus

50 cm in length and resemble earthworms, with blunt heads, degenerate eyes, and annular grooves along the body (Zug, 1993). Little is known about caecilian biology, and caecilians are rarely used in a research setting. Salamanders are in the order Caudata. There are approximately 400 species in Caudata, divided into three groups: sirens (eel-like amphibians), primitive salamanders (hellbenders and other related species), and advanced salamanders (mudpuppies, amphiumas, axolotls, newts, and many terrestrial species) (Zug, 1993). Sirens have external gills, no hindlimbs, and reduced forelimbs. They are totally aquatic and inhabit sluggish waterways of southern North America. There are 2 genera in the family Sirenidae, Siren and Pseudobranchus. Siren (true sirens) contains 2 species, and Pseudobranchus (dwarf sirens) is represented by a single species (Zug, 1993). There are 2 families of primitive salamanders. Cryptobranchidae includes hellbenders (Cryptobranchus sp.) of the United States and the giant salamanders (Andrias sp.) of Asia. Cryptobranchids are neotenic (condition where salamanders retain larval characteristics while becoming fully functional, reproducing adults). They are aquatic and have gill slits but no external gills. Respiration is almost exclusively cutaneous, and the skin lies in extensive, fleshy folds on the sides of the body. The head and body are flattened. Andrias can reach a length of 1.5 meters (5 feet); it is the largest salamander in the world. Hynobiidae, an exclusively Asian family comprising over 32 species, is the second family of primitive salamanders. Most members of this group are smaller than the cryptobranchids, have stout bodies, and undergo complete metamorphosis (Conant and Collins, 1991; Zug, 1993). Six families make up the advanced salamanders. Amphiumidae contains 3 species of Amphiuma, which superficially resemble sirens. Amphiuma, however, lack external gills and can reach an adult length of over 1 meter. Proteidae contains 2 genera, Proteus and Necturus. Proteus is a cave-dwelling salaman-

der found in Europe. Necturus maculosus, the mudpuppy, has a broad, flat head and well-developed external gills. Mudpuppies are aquatic and are found in east and central North America. There are 2 genera and over 34 species represented in Ambystomatidae. Several species demonstrate neoteny, including Ambystoma mexicanum (the axolotl) and A. tigrinum (the tiger salamander) (Fig. 1). Ambystomatids are predominantly terrestrial, with strong limbs and functional lungs. They are robust animals, and adults of many species can exceed 14 cm in length. Dicamptodontidae comprises 2 genera, Dicamptodon and Rhyacotriton. These salamanders resemble the ambystomatids but are found in moist forests of the Pacific coast. More than 300 species in North and South America and Italy make up the family Plethodontidae. Plethodontids occur in a wide variety of sizes and shapes; however, all are lungless, are quadrupedal, and possess a nasolabial groove. Included in this family are the genera Plethodon, Desmognathus, Eurycea, Gyrinophilus, Pseudotriton, Aneides, and Batrachoseps. Members of the family Salamandridae share some characteristics with Plethodontidae; however, salamandrids possess lungs and have numerous poison glands in their skin. Additionally, they may be brightly colored, an advertisement of their toxicity. Salamandra, Taricha, and Notophthalmus are representative genera of this family. Notophthalmus (newt) has an aquatic larval stage, terrestrial juvenile period (during which the animals are termed "efts"), and aquatic adult stage (Conant and Collins, 1991; Zug, 1993). There are over 3800 frog species in the order Anura, ranging from the Arctic Circle to extreme points in the southern hemisphere (Conant and Collins, 1991; Zug, 1993). All frogs share a common body plan designed for jumping, which allows movement an average of 2 to 10 times the body length. Classification of frog species remains controversial; however, frogs can be di-

Fig. 1. Ambystomatigrinum, the tiger salamander,is a native North American species used in research.

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17. BIOLOGY AND DISEASES OF AMPHIBIANS

vided basically into primitive frogs (3 families), transitional frogs (2 families), and two groups of advanced frogs (2 families in one and 16 in the other). In the primitive frogs, family Discoglossidae contains species used in research, including the fire-bellied toad, Bombina orientalis, and the midwife toad, Alytes obstetricans. The spadefoot toad, Scaphiopus, is in the family Pelobatidae, a member of the transitional frog group. The first group of advanced frogs contains the family Pipidae, in which the genera Pipa and Xenopus are found. Xenopus, although highly specialized, is the most primitive of the pipids, first occurring in the Cretaceous period. The second group of advanced frogs is represented by 16 families (Zug, 1993). Of these, Bufonidae, Dendrobatidae, Hylidae, Leptodactylidae, and Ranidae have species that can be encountered in a research environment. Bufonidae contains the true toads, including the genus Bufo. Toads have warty, thick skin with well-developed parotoid glands (a raised cluster of granular glands located on the head behind the eyes), and males have a Bidder's organ (ovarian tissue located on the cranial pole of the testis). Poison dart frogs (Dendrobates and Phyllobates) are members of the family Dendrobatidae. They are small, active frogs with bright color patterns, which alert would-be predators to the presence of highly toxic skin secretions (a characteristic termed aposomatism). The family Hylidae contains over 250 species of the genus Hyla. Hyla are tree frogs, recognizable by their slender bodies, their long limbs, and the expanded tips of their digits (Fig. 2). Ceratophrys, the horned frogs of South America, are large animals with fleshy protuberances over the eyes and phenomenally wide mouths. These members of the family Leptodactylidae are

Fig. 2. The barking tree frog, Hyla gratiosa, has expanded digit tips characteristic of the genus.

voracious terrestrial predators (Zug, 1993). Ranidae ("true frogs") includes members of the genus Rana. Species used in research are R. catesbeiana (bullfrog), R. grylio (pig frog), R. clamitans (bronze frog), and R. pipiens (northern leopard frog). Ranids are medium to large frogs with smooth skin. In some species (bullfrog, pig frog, and bronze frog), the tympanum of the male is larger than its eye, while the tympanum of the female is the same diameter as its eye (Conant and Collins, 1991).

B.

Use in Research

Amphibians have been used in research for many years. Xenopus laevis, the African clawed frog, is probably the most widely used and easily recognized amphibian research subject. Xenopus was originally used in pregnancy assays, when it was discovered that injection of a pregnant woman's urine into the dorsal lymph sac of a female Xenopus caused the frog to begin laying eggs. This method of pregnancy detection was soon replaced; however, the clawed frog remained popular with developmental biologists because of its ability to reproduce yearround when injected with commercially available hormones. Xenopus has also been used in cell and developmental biology research (Gurdon, 1996). Once normal clawed-frog development was documented (Nieuwkoop and Faber, 1994), Xenopus became the subject of developmental toxicology investigations and standardized as the FETAX (frog embryo teratogenesis assay: Xenopus) system (Dumont et al., 1983; Dawson and Bantie, 1987; Burkhart et. al., 1998). Currently, the most common use of Xenopus involves cell and molecular biology research. Oocytes of clawed frogs, when injected with foreign DNA or messenger RNA, will allow expression of this genetic material. Thus, Xenopus use has become an integral part of molecular biology laboratories (Sive et al., 2000), and clawed frogs are present in virtually all animal facilities supporting this type of research. Other amphibian species are used in research and teaching. Mudpuppies (Necturus) have traditionally been the subjects of comparative anatomy laboratories. Axolotls (Ambystoma mexicanum) are used in developmental research. Limb regeneration in axolotls and newts has been heavily investigated (Brockes, 1994). Frogs (usually bullfrogs and leopard frogs) have been extensively utilized in teaching physiology and in conducting physiology research (Karnes et al., 1992; Williams, 1997). Because of their ability to regurgitate easily, these species have also been used to study the effects of antiemetics (Kawai et al., 1994; Tai et al., 1995). A frog model has been developed and is being used to test antinociceptive (pain-relieving) effects of analgesics (Stevens, 1992). The pharmacologic and chemical properties of compounds secreted by amphibian skin are widely studied and characterized (Shen, 1995; Daly, 1995). Proteins isolated from frog eggs and embryos demonstrate anticancer activity (Rybak et al., 1996). Frogs have also been used aboard the

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space station M i r to explore the effects of microgravity posture, behavior, and motion sickness (Suzuki et al., 1993; IzumiKurotani et al., 1997). In addition to being used as research animal models, amphibians are studied extensively in the laboratory and the field. These studies focus on the ecology, behavior, and conservation of amphibians themselves, and are critically important as biologists strive to understand and correct the worldwide decline of amphibian species. It is essential that the laboratory animal veterinarian be familiar with the biology, husbandry, behavior, and medicine of amphibians in order to provide necessary support for these ongoing research projects.

C.

Availability and Sources

It is widely recognized that many amphibian populations throughout the world are drastically declining in numbers. Therefore, when choosing an amphibian as a model for teaching or research, special consideration should be given to acquiring animals that have been raised in captivity. Commercial vendors such as NASCO, Xenopus Express, Carolina Biological Supply, the University of Indiana Axolotl Colony, and Xenopus I sell colony-reared amphibians. A list of vendors can be accessed through the Institute of Laboratory Animal Resources (ILAR)

website. Using purpose-bred animals will protect wild populations and provide the researcher with a healthier, less stressed, and better-characterized animal model.

D.

Laboratory Management and Husbandry

Amphibians occupy a variety of niches in the wild, and laboratory housing requirements are equally varied. This section will describe general principles of amphibian husbandry, with emphasis on frogs and salamanders used commonly in research. Sources containing-in-depth information on biology and husbandry of a particular species should always be consulted prior to attempting to house that species.

1. PrimaryEnclosures Glass aquaria work very well as primary enclosures for amphibians, especially aquatic species. Plastic shoe boxes and sweater boxes also provide appropriate housing and have the advantage of being stackable. Small terrestrial salamander species such as P l e t h o d o n have been successfully maintained in medium and large plastic petri dishes (Jaeger, 1992). Larger aquatic frogs and salamanders are frequently housed in stainless steel, fiberglass, and Plexiglas tanks (Fig. 3). All cages should be constructed of impermeable, easily sanitized material, and

Fig. 3. EnclosedPlexiglashousing with a slanted floorworkswellfor Rana pipiens and R. catesbeiana.

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17. BIOLOGYAND DISEASES OF AMPHIBIANS should ideally be able to withstand multiple cage washings. Cages should be of adequate height to accommodate behavioral needs of climbing and jumping species, such as tree frogs and bullfrogs. Fitted, nonabrasive lids are required for most terrestrial and many aquatic species to prevent escape. Several commercial vendors, such as Pharmacal Research Laboratories, Inc., Marine Biotech, and Aquatic Habitat, Inc., offer customdesigned Xenopus housing with either flow-through or recirculating systems. Likewise, commercially available research-fish housing units can be adapted for Xenopus or other amphibian species. Amphibians require a moist habitat and can rapidly desiccate if left in a dry environment. Moistened sphagnum moss is a good substrate for many species. Some authors suggest using a layer of heat-treated soil covered with leaves or sphagnum moss. Leaves can be frozen for several days to eliminate arthropod parasites (Wright, 1996). Soil pH is important. In one study, the red-backed salamander (Plethodon cinereus) preferred the most basic pH range offered (pH 6-6.5). Juveniles avoided soils with a pH of less than 3.7. Very acidic pH ranges (pH 2.5-3) were acutely lethal, and a range of pH 3 - 4 caused death within 8 months (Wyman and Hawksley-Lescault, 1987). Jaeger (1992) recommends three layers of moistened filter paper for petri dish primary enclosures, and soft, moistened paper towels for larger containers (Fig. 4). Echternacht (personal communciation) uses precut, fitted pieces of foam rubber or sponge on the tank bottom, soaked with dechlorinated water (water level should be even with the top of the sponge) (Fig. 5). Because many terrestrial salamander and frog species are secretive, pieces of bark, polyvinyl chloride (PVC) pipe, or other types of hiding places should be provided. Many amphibians do not drink; water is absorbed through the skin (Pough, 1991). Water should be pro-

vided to terrestrial species in shallow dishes, through the moistened substrate, or by misting (Pough, 1991; Wright, 1996). Aquatic amphibians can be housed with or without substrate (depending on species). Aquatic species should also be given hiding places. Polyvinyl chloride pipe works well as a retreat. Semiaquatic frog and salamander species should be provided with a sloping floor or other means of facilitating emergence from the water (Culley, 1992; Wright, 1996). Bullfrogs and leopard frogs do very well in this type of environment. 2.

Water Quality

Fresh, dechlorinated water is preferred for amphibians. Although some species may tolerate low levels of chlorine, many are quite sensitive and will die from exposure to chlorinated water. Allowing open containers of water to age for 2 4 - 4 8 hr, aerating the water, adding sodium thiosulfate, and passing tap water through activated carbon filters are four methods of dechlorination (Fig. 6). Chloramines may be used in place of chlorine in some municipal water systems. Chloramines can be more toxic than chlorine and are best removed with an unused, activated charcoal filter or sodium thiosulfate. When sodium thiosulfate is added to chloramines, ammonia will be released. Zeolites can be used to remove excess ammonia (Gratzek, 1992). An analysis of local water quality should always be obtained prior to establishing an amphibian housing facility. Aquarium or swimming pool test kits will also provide approximate chlorine levels. Copper is toxic to amphibians, and care should be taken to avoid use of pipes made with this metal. Maintaining correct pH of water in the tank or cage is very important. If the preferred pH of a given species is not known, Wright (1996) recommends starting with a pH of 6.8-7.1 (neutral to slightly acidic), then adjusting to a more basic pH if the

Fig. 4. Smallspecies of salamanderscan be housed on moist filter paper in large petri dishes.

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Fig. 5. A saturatedpiece of foam serves as a substrate and keeps humidityelevated in amphibian cages.

animal appears irritated or is anorectic. Verhoeff-de Fremery et al. (1987) prefer housing amphibians at a pH of 7.5 - 8.5, and Horne and Dunson (1994) demonstrated that chronic exposure to low pH affected whole body water and sodium in a terrestrial salamander. Other parameters that can affect amphibian health are dissolved oxygen and ammonia. Test kits are commercially available to monitor water quality. Amphibians can be kept in static, recirculating, or flow-

through systems (Fleming, 1990; Sibold et al., 1993; Bartholomew et al., 1993; Stewart, 1994). Static systems work well for both small and large groups of animals, and many tanks can be plumbed to facilitate draining and refilling. A major disadvantage of this type of system is the need for frequent cleaning. Recirculating systems use filters to remove debris and nitrogenous waste from the water. Although less frequent cleaning is necessary with recirculating systems, filters can easily become overtaxed by high population densities and species that generate large amounts of waste. Flow-through systems run a constant stream of water into and out of the tank. Fresh water is always available with this method; however, a mechanism for dechlorination should be built in the line to assure removal of chlorine.

3.

Fig. 6. Waterfor aquatic amphibians can be dechlorinatedby allowingit to age in containers.

Temperature

Many amphibians spend their existence beneath leaf litter of forest floors or submerged in cool ponds and fast-moving streams. Consequently, most have preferred thermal zones lower than those of reptiles. Wright (1996) has found that tropical species can be maintained at 21~176 (700-85 ~F), while amphibians from temperate regions do well at 18~176 (65 ~ 72~ Pough (1989) recommends lower ranges: 20~176 (680-77 ~F) for tropical species, and 15~ ~C (590-68 ~F) for temperate species. Animals from temperate zones may require seasonal decreases of 5 ~ 1 7 6 (10~176 (Wright, 1996). Jaeger (1992) warns that temperatures in excess of 20 ~C (68 ~F) will prevent salamanders of the genus P l e t h o d o n from assimilating food rapidly enough to meet the needs of their increased metabolic rate. Mattison (1998) provides recommended tem-

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Table II Recommended Temperatures for Selected Amphibian Species a Species

Pipa pipa Bombina orientalis Rana catesbeiana R. pipiens Dendrobatids Bufo marinus Hyla versicolor Ceratophrys Ambystoma mexicanum A. tigrinum Salamandra salamandra a

Temperature (~ 28 25 15-25 15-25 25-30 23-30 21-27 23-30 10-25 15-25 20 maximum

From Mattison (1998).

peratures for several species (Table II). Other references contain specific information about preferred temperature ranges of less common amphibian species; these should always be consulted to ensure providing the most correct zone for a given species. 4.

Lighting

Most amphibians live in cool, dark environments in the wild; therefore, direct exposure to bright light should in general be avoided. For animal-room lighting, full-spectrum bulbs are suggested, especially if the particular needs of the species are not known (Wright, 1996). Shelter should be provided, so the animal may retreat from light if desired. Light cycles of 12 hr light12 hr dark are satisfactory in most cases. However, if breeding or mimicking the natural habitat of the animal is desired, light cycles will need to be manipulated accordingly. 5.

Airflow

Amphibians require moist habitats, and relative humidities of about 80% work well for most species (Pough, 1991; Wright, 1996). Normal animal-room airflows tend to cause evaporation and dry out wet environments. To prevent this desiccation of habitat and animals, room airflows should be reduced. Alternatively, amphibians can be housed in primary enclosures, which have minimal openings. These will retain moisture; however, care should be taken to ensure that temperatures inside the primary enclosures do not rise to unacceptable levels. 6.

Secondary Enclosures

Conventional animal rooms can be successfully adapted to house amphibians. Walls, floors, and ceilings should be impervious and easy to sanitize. Electrical outlets should have ground-fault interrupters, especially in rooms housing aquatic

species. Individual light timers and thermostats are recommended for each room, since species have different requirements. In cases where very cool and moist conditions are needed and cannot be achieved with normal room manipulations, environmental chambers can be utilized (Jaeger, 1992). 7.

Sanitation

Routine sanitation of amphibian primary enclosures requires special consideration. Many terrestrial species, particularly salamanders, are territorial and mark their environments with pheromones. Excessive cleaning will disrupt normal behavior and can be stressful to the animal (Jaeger, 1992). However, allowing excess buildup of excreta will result in accumulation of toxic metabolites as well as overgrowth of bacteria and fungi. Animals then are placed in a more compromised and stressful environment and can easily succumb to disease. Singly housed, terrestrial animals with ample floor space can be changed every 2 weeks (Jaeger, 1992) and spot-cleaned as necessary between. Group-housed animals require more frequent cleaning. Housing for aquatic species may require cleaning on a daily, weekly, or less frequent basis, depending on stocking density, frequency of feeding, and type of system (filtration, flow-through, etc.). In general, tanks should be cleaned at appropriate intervals to prevent water from fouling. Cleaning solutions should be carefully chosen, and extreme care must be taken to thoroughly rinse away chemical residues. Amphibians are exquisitely sensitive to many compounds, and their permeable skin makes them particularly susceptible to toxins. Phenolics are highly toxic and should not be used around amphibians. 8.

Handling

Amphibians should be handled carefully, and care must be taken to avoid disrupting their protective mucous layer or causing excess secretion of toxins. Gloves should be worn; they must be free of powder and moistened with dechlorinated water. Abrasive paper towels should not be used. Nets must be made of soft fine mesh, and should be an appropriate size to comfortably hold the animal. Aquatic species can be transferred or held in glass jars to prevent removal from water and to protect the sensitive gills. Small terrestrial amphibians can be manually restrained with one hand. Large salamanders should be firmly but gently grasped behind the head and around the pectoral girdle with one hand, and around the pelvic girdle with the other hand (Verhoeff-de Fremery et al., 1987; Crawshaw, 1993; Wright, 1996). Some species will release their tail as a predatoravoidance mechanism; therefore, the tail must not be used for restraint. Frogs, like salamanders, can be held around the pectoral girdle; however, the strong hindlegs must also be restrained to prevent kicking out and slipping through the handler's grasp (Fig. 7). Most amphibians can bite, and some can

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DORCAS P. O'ROURKE AND TERRY WAYNESCHULTZ to the animal using stainless steel or nonabsorbable suture. The suture must pass through a muscle mass in order to permanently anchor the beads; sewing to the skin only can result in sloughing within a few weeks. Passive integrated transponder (PIT) tags are being used with increasing frequency in the field and in the lab (Donnelly et al., 1994; Wright, 1996). Transponders can be placed intracoelomically using a large-diameter needle. Sterile microchip transponders can also be implanted in the dorsal lymph sac (Hoogstraten-Miller and Dunham, 1997). Although this method is more expensive than others, it allows unique identification of animals of virtually any size and body conformation. 10.

Fig. 7. Frogscan be held by supporting the pectoral girdle and restraining the powerful hindlimbs. inflict painful wounds. Additionally, many species secrete skin toxins, and in some species such as Bufo marinus, toxin can be ejected when the parotoid gland is pressed. Therefore, eye protection is recommended when handling these animals (Wright, 1996).

Quarantine

All newly arrived amphibians, especially those that are wildcaught, should undergo a quarantine period. The animals must be housed separately from existing colonies, and their room should be serviced last. Implements should not be shared between quarantine and other rooms. Animals intended for longterm use should be screened and treated, if necessary, for parasites. Diseases should be diagnosed and treated accordingly. The length of quarantine may vary, depending on intended use of the amphibians. Animals for acute studies may be quarantined for a shorter period, but long-term studies dictate a 2- to 4-week minimum quarantine period. Longer periods may be necessary for wild-caught amphibians, due to their questionable health status and unknown exposures.

9. Identification

Each primary enclosure housing amphibians should be labeled with appropriate information. Individual animals can be identified in a variety of ways. In animals with varying color patterns, these patterns can be drawn or photographed and used as unique identifiers (Donnelly et al., 1994). Toe clipping has historically been used in both field and laboratory situations (Donnelly et al., 1994; Wright, 1996). Many amphibians regenerate digits, so this method may be ineffective for long-term studies. Anesthesia (local or general) should be used for toe clipping, and instruments should be sterilized before use. Direct pressure will provide adequate hemostasis in small animals; larger species may require sutures or hemostatic clips. Careful attention must be paid to the animals postamputation for an extended period to ensure that inflammation, infection, and necrosis do not occur (Golay and Durrer, 1994; Wright, 1996). Tattooing and freeze or chemical branding have been used, but are nonpermanent. Skin transplantation (surgically transplanting various-shaped grafts of pale abdominal skin onto the darker dorsal skin) is labor-intensive and can result in graft rejection (Hoogstraten-Miller and Dunham, 1997). Glass or plastic beads have been used to identify individual frogs and salamanders (Verhoeff-de Fremery et al., 1987; Hoogstraten-Miller and Dunham, 1997). These beads are sewn

11.

Zoonoses/Allergies

Amphibians can potentially harbor zoonotic diseases. The most familiar of the amphibian zoonoses is atypical mycobacteriosis, caused by Mycobacterium fortuitum, M. marinum, or M. xenopi. Individuals handling aquatic amphibians that are shedding this disease can develop cutaneous lesions on the fingers and hands. In rare instances, the disease can spread to involve lymph nodes. Immunocompromised individuals are more at risk and can develop severe systemic disease. The best precaution against atypical mycobacteriosis is to wear gloves, especially if there are preexisting cuts or abrasions on the hands. Salmonellosis has been reported in amphibians, but with much less frequency than in reptiles (Woodward et al., 1997). Appropriate precautions include wearing gloves, washing hands thoroughly after handling amphibians or their surroundings, and screening animals periodically for Salmonella. Chlamydia psittaci has been isolated from clinically ill Xenopus laevis (Newcomer et al., 1982; Wilcke et al., 1983; Howerth, 1984). More recently, Chlamydia pneumoniae was identified in a giant barred frog (Berger et al., 1999). Although no reports have been identified that document amphibian-tohuman transmission, both agents should be recognized as potential human pathogens and appropriate precautions taken.


There have been occasional reports of allergies to frog skin and secretions (Armentia and Vega, 1997; Holtz et al., 1993). Individuals experiencing respiratory or cutaneous signs when working with frogs should seek medical advice concerning allergies.

II.

A.

BIOLOGY

Anatomy and Physiology

1. Integumentary System

The skin of most amphibians is smooth, moist, and glandular. Two primary types of skin glands are present in amphibians: mucous glands and granular glands. Mucous glands secrete a slimy protective layer, which prevents mechanical damage to the skin, facilitates retention of body fluids, and provides a barrier against pathogens. Granular glands synthesize and secrete a variety of compounds that protect against predators, as well as chemicals that have antibacterial and antifungal properties. Granular glands are usually found on the head and shoulders but can be scattered over the body (Zug, 1993; Clarke, 1997). The parotoid gland of toads (Bufo), located on the head behind the eyes, is a raised cluster of granular glands. Fire salamanders (Salamandra salamandra) also have parotoid glands. Defensive compounds found in parotoid and other granular glands can have neurotoxic, cardiotoxic, myotoxic, hallucinogenic, hypotensive, and vasoconstrictive activity (Clarke, 1997). Highly toxic alkaloids found in poison dart frogs (Dendrobatidae), Bufo marinus, and other species of frogs and salamanders can cause vomiting, respiratory paralysis, and death in some cases. In addition to antipredator activity, granular glands of some species secrete peptides, which exhibit antimicrobial activity. The magainins, peptides secreted by Xenopus laevis, inhibit growth of gram-positive and gram-negative bacteria, several fungi, and some protozoal species. Bombesin, caerulein, and bradykinin are among the peptides found in other amphibian species, including fire-bellied toads, midwife toads, and tree frogs. Skin secretions of Bufo have also demonstrated antibacterial and antifungal properties (Clarke, 1997). Other chemical compounds secreted by granular glands of various species include pheromones used in courtship and mating; dermorphin, a potent opioid that may function as an endogenous analgesic; and bioadhesives, which allow temporary entrapment of predators (Clarke, 1997), or permit certain species of small male frogs to adhere to females during breeding (Zug, 1993). Amphibians shed their skin in cycles, which may range from days to weeks. The skin commonly splits middorsally, and the

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animal uses its limbs to climb out of its skin. Shed skins are commonly eaten (Zug, 1993). Some amphibian species have a specialized area of permeable skin in the abdominal region, which is pressed against wet substrates and used to absorb water from the environment for rehydration (Jorgensen, 1997). 2.

Musculoskeletal System

The amphibian skeleton has undergone several modifications. The salamander skeleton is largely cartilaginous. Ribs are absent or greatly reduced in most frogs. Anuran adaptations for jumping include fusion of postsacral vertebrae into an elongate bone, the urostyle, which articulates with the sacral vertebra and the ilium; and fusion of the tibia and fibula into a single, strong bone, the tibiofibula. Many salamander species have tail autotomy, and are capable of regenerating a new tail if the original is lost. Moreover, certain species of newts also have the ability to regenerate limbs, jaws, and ocular tissues (Brockes, 1997). 3.

Respiratory System

Larval amphibians breathe primarily through gills. Adults can retain and use gills, lose gills and develop lungs, breathe with both gills and lungs, or have neither (Fig. 8). Adult plethodontids (lungless salamanders) lack both lungs and gills, and rely on cutaneous respiration. Skin, in fact, is the primary respiratory surface in most amphibians and must be kept moist. In species that use lungs for respiration, air is forced in and out of the lungs by movement of the buccopharyngeal floor (Zug, 1993). Lungs lack alveoli and are very fragile and easily ruptured (Wright, 1996). In many frog species, the trachea is short, and bifurcation occurs close to the glottis; this anatomic feature must be taken into account when performing endotracheal intubation. 4.

Cardiovascular System

Larval amphibians, like fish, have a two-chambered heart. Most adult amphibians have a three-chambered heart, consisting of paired atria and a single ventricle (plethodontid salamanders lack an atrial division, since they lack lungs). Hellbenders, mudpuppies, and sirens have a partial interventricular septum (Burggren and Warburton, 1994). Patterns of blood flow and mixing of oxygenated and deoxygenated blood vary among species, depending on degree of pulmonary respiration, physiological state, and anatomic structures (Zug, 1993). Hepatic portal veins drain blood from the rear half of the amphibian's body; this may impact the pharmacokinetics of drugs with hepatic excretion (Wright, 1996). Plasma osmolarity of amphibians is

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Fig. 8. Axolotlsare large, aquatic, neotenous salamandersthat respire through feathery external gills.

200 mOsm/kg (Walker and Whitaker, 2000). This difference from mammalian osmolarity should be considered when preparing media for in vitro work with amphibian tissues, as well as when administering replacement fluids to dehydrated amphibians. Amphibian Ringer's solution contains 6.6 gm NaC1, 0.15 gm CaC12, and 0.2 gm NaHCO3 per liter of water (Walker and Whitaker, 2000).

7. Excretory System

Salamanders and frogs have a mesonephric kidney and lack the ability to concentrate urine in excess of plasma levels. Aquatic amphibians excrete ammonia, and terrestrial amphibians excrete urea. Most amphibians have a bladder, which functions in water conservation. Many frogs, when frightened, will release urine to deter predators (Wright, 1996).

5. Lymphatic System

The lymphatic system of amphibians drains directly into the venous system. At venous junctions, lymph hearts contract and force lymph into the veins. Large sinuses, collection sites for lymph, are found throughout the amphibian's body. In frogs, a pair of these sinuses lies subcutaneously over the sacral area, lateral to midline. Substances injected into these dorsal lymph sacs will be transported to the venous circulation (Fig. 9). 6.

Gastrointestinal System

Adult amphibians are carnivorous and therefore have a relatively short gastrointestinal tract. The tongue is well developed in all species except pipids and is important for prehending food items. Xenopus and other pipids direct food items into the mouth with their front legs. Melanin is commonly found in the amphibian liver and other abdominal organs, and pronounced pigmentation is not unusual. Vomiting in amphibians is a common defensive mechanism, and it is not unusual for some frog species to evert part of the stomach during regurgitation (Bisazza et al., 1998).

8. Nervous System/Special Senses

Cerebral cortical structure in amphibians is dissimilar to that of higher vertebrates, and the function of the various areas is still controversial (Nieuwenhuys, 1994; Bruce and Neary, 1995). Amphibians have 10 cranial nerves. The hypoglossal nerve (cranial nerve XII), is formed by branches of the first 2 spinal nerves (Anderson and Nishikawa, 1997). A lateral line system (similar to that of fish) is well developed in larval amphibians and is retained by adults of many aquatic species (Fig. 10). The lateral line system is recognizable as a linear arrangement of neuromasts on the head and along the body of the animal. Neuromasts detect changes in water pressure and currents, and function in locating prey (Zug, 1993). Amphibians can detect higher-frequency sound transmitted through the air to the tympanic membrane, but low-frequency vibration is transmitted through the forelimbs and the cranium to the ear. The amphibian eye has two types of rods, red and green, which are responsible for color sensitivity (King et al., 1993). Cones detect only presence of light (Zug, 1993). A vomeronasal (Jacobson's) organ is responsible for odor detection.

803


summarizes hematologic values for several amphibian species, and Table IV lists serum chemistry values for Rana catesbeiana. In a recent study involving fall-collected bullfrog samples, Cathers et al. (1997) found significant differences between males and females for plasma proteins, sodium, and calcium. No differences were found in the remainder of the complete blood count (CBC) or serum chemistry values. Pfeiffer et al. (1990) investigated hematologic changes in Japanese newts following tail amputation. They observed a decrease in hematocrit during the first 10 days postamputation; hematocrit was restored by day 30. A transient lymphocytosis was also noted in the first few days following amputation. Basophil percentages were consistently high (49-64%) throughout this study, in contrast to that of Jerrett and Mays (1973), who found no basophils in two populations of hellbenders. Incomplete understanding of the function of the various cell types further complicates interpretation of amphibian hematologic and serum chemistry values. Amphibian species have nucleated red blood cells and thrombocytes. Amphibian lymphocytes, monocytes, and thrombocytes function in a fashion similar to that of their higher vertebrate counterparts. Neutrophils appear to respond to infection in a manner similar to that of mammalian neutrophils and reptilian heterophils. Eosinophil and basophil function is largely unknown, and interpretation of elevated percentages of these cell types cannot be extrapolated from mammalian literature (Campbell, 1991).

B. 1.

Fig. 9. The paired dorsal lymph sacs (arrows) of Xenopus and other frog species are a preferred site for injection. 9.

Normal Values

Longevity data are available for many amphibian species (Bowler, 1977; Kara, 1994; Smirina, 1994). Amphibians from northern climates tend to have longer life spans than those from southern latitudes, and larger aquatic salamanders live longer than their smaller, terrestrial counterparts. Xenopus laevis have been documented to live 15 years; Bombina, 11-13 years; bullfrogs, 16 years; and newts, 9 years (Kara, 1994; Smirina, 1994). Cryptobranchus can exceed 25 years; Desmognathus, 10 years; and Bufo americanus, 5 years (Zug, 1993). Age can be most accurately determined in amphibians by counting the layers in bone (Smirina, 1994). Hematologic and serum chemistry values for amphibians can be affected by a number of variables, including season, sex, environmental factors, and method of sample processing. Table III

Nutrition

Adult

Adult amphibians are carnivorous, and many are opportunistic feeders. In the wild, salamanders feed on a variety of vertebrate and invertebrate species. Ambystoma tigrinum has been documented to ingest worms, insects, snails, young field mice, and lizards. Plethodon cinereus eats ants, spiders, flies, beetles, and other small invertebrates. Notophthalmus viridescens feeds on aquatic insects and mollusks. Adult amphibians will frequently cannibalize larvae of their own and other species. Large salamanders such as Cryptobranchus and Amphiuma eat crawfish, fish, frogs, and mammals. Necturus feeds on both small and large prey items (Petranka, 1998). Frogs also feed on a variety of invertebrates, and larger species such as Rana catesbeiana have been noted to eat salamanders, snakes, turtles, and small birds and mammals. Bufo marinus has been reported to eat dog food (Bartlett and Bartlett, 1996). Most species will adapt to dietary modifications required for housing in a laboratory animal facility. Jaeger (1992) successfully kept several salamander species using a diet of Drosophila for smaller individuals, and crickets and earthworms for larger animals. Newts will eat chopped earthworms, fly larvae, and

804

DORCAS P. O'ROURKE AND TERRY WAYNE SCHULTZ

Fig. 10. Manyaquatic amphibians,includingXenopuslaevis, havea lateral line system(arrow). Tubifex worms. Many terrestrial and semiaquatic frog species orient visually to prey and require moving food. Rana, Bufo, Bombina, and most other frog species will take crickets, earthworms, and waxworms. Dendrobates requires pinhead crickets (newly hatched). Mudpuppies and axolotls have been maintained on diets of beef muscle and organ meat; however, vitamins and minerals should be supplemented if these are fed (Verhoeff-de Fremery et al., 1987). Raw meat and organs should not be fed to young, growing amphibians, or calcium deficiency will result. Salmonella contamination is also a concern when feeding raw meat and organs. Axolotls will readily eat earthworms and adapt to commercially prepared diets. Most whole vertebrate prey items, if properly nourished, will constitute a balanced diet for amphibians. Bones provide calcium, phosphorus, and magnesium; liver and kidneys provide vitamins; pancreas provides zinc; and thyroids provide iodine. In contrast, the chitinous exoskeleton of many invertebrates is, for the most part, indigestible and contains little to no calcium or other nutrients (Donoghue, 1996). Because insects lack a calcium-rich skeletal structure, a vitamin-mineral mix should be dusted on the prey before feeding (Bartlett and Bartlett, 1996). Alternatively, insects can be fed a diet that is vitaminmineral rich ("gut loaded") immediately before being fed to amphibians. To avoid feeding prey items of poor nutritional quality, crickets, mealworms and similar species can be raised in-house on nutritious diets. Crickets can be kept in a large, deep container on a substrate of sawdust or vermiculite. A shallow dish with

moistened cotton balls or vermiculite serves as both a water source and a place to lay eggs. Crumpled newspaper, egg crates, or paper-towel tubes should be placed about the cage for hiding places. Crickets can be fed laying mash or crushed dog food (Fig. 11). Vegetables such as broccoli, carrots, and alfalfa sprouts can be added, and food can be sprinkled with calcium. Mealworms can be raised in a ventilated container with a fitted lid. Laying mash or chick starter and bran can be used for substrate, and should be covered with a paper or cloth towel. Vegetables and fruit can be added to provide moisture. Food should be replenished periodically (Bartlett and Bartlett, 1996; Mattison, 1998). Adult amphibians should be fed anywhere from daily to twice weekly, depending on species, age of animal, and ambient temperature. 2.

Larvae

Larval salamanders are carnivorous and eat a variety of prey items. Ambystoma tigrinum larvae in the wild consume aquatic mollusks, nematodes, insects, and eggs and larvae of their own and other amphibian species. Notophthalmus viridescens larvae eat small invertebrates, including copepods, snails, and water mites. Hatchling Cryptobranchus have large yolk sacs and apparently rely on yolk for nutrition for the first few months of life (Petranka, 1998). Tadpoles of several frog species tend to be more herbivorous initially after hatching, then convert to an omnivorous and later, a carnivorous diet.

Table Ill

CBC Values for Various Amphibian Species

Rana catesbeiana

Fall a Parameter Red Blood Cells (105/txl) White Blood Cells (103/1~1) Hemoglobin (g/dl) Packed Cell Volume (%) Plasma protein (g/dl) Segs (%) Bands (%) Lymphs (%) Monos (%) Eosinophils ( % ) Basophils (%) Differential/ WBC count method

Spring b

M

F

q

2.4-11.8

3.2-6.0

3.2-6.0

14-27

14-27

2.5-5.2

4.1-4.8

2-52 0-2 36-90 0-3 0 - 18 0-8 Natt & Herrick (direct)

2-52 0-2 36-90 0-3

9.5 40.4

Winter M

Spring F

4.57

4.06

5.98

5.71

10.08

9.65

41.5

31.7

Summer

Fall

Spring

M

F

M

F

M

F

3.17

2.64

6.04

4.61

6.57

5.91

6.4

7.6

12.72

12.30

47.4

44.4

16.1

17.9

6.59

4.80

29.0

24.1

11.6

10.2 8.44

11.04

44.9

33.7

Cryptobranchus alleganiensise

22.8

0.28-1.5 0.04 -0.3

6.2 22.4

m

6.63-12.16 40

30-55

28

2.7-44.8

m m

m

m

m m

m

m h

0 - 18

0-8 Natt & Herrick (direct)

From Cathers et al. (1997). ~ Carmena-Suero et al. (1980). CFrom Harris (1972). aFrom Pfeiffer et al. (1990). eFrom Jerrett and Mays (1973). a

M,F

m

2.4-11.8

Hyla Cynops septentrionalisb pyrrhogaster d

Rana pipiens c

m

m

m

m

m

m

m m

3 6 4 57 Wright/ Leishman (indirect)

46.4-83.7 3.0-19.7 1.0-9.7 0 Neutral red/ Formalin (indirect)

806

DORCAS P. O'ROURKE AND TERRY WAYNE SCHULTZ Table IV

Serum Chemistry Values for Rana catesbeianaa Parameter Sodium (mEq/liter) Potassium (mEq/liter) Chloride (mEq/liter) Total CO2 (mEq/liter) Albumin (gm/dl) Calcium (mg/dl) Creatinine (mg/dl) Aspartate Amino Transferase (U/l) Lactate Dehydrogenase (U/l) Phosphorus (mg/dl) Magnesium (mg/dl) Uric acid (mg/dl) Urea (mg/dl) Anion gap

Male

Female

100 -114 2.0-3.2 65 -86 15-32 1.0-2.1 6.5-8.5 0.7-3.0

107-115 2.0-3.2 65 - 86 15-32 1.0-2.1 8.2-9.6 0.7-3.0

22-91 10 -68 2.5-5.2 1.4-2.57 0-0.1 3 -6 1.3-24.2

22-91 10 - 68 2.5-5.2 1.4-2.57 0-0.1 3- 6 1.3-24.2

Instrument: Monarch Plus, Model 2000. From Cathers et al. (1997). aFall season.

In the laboratory animal facility, tadpoles of Rana pipiens, R. catesbeiana, Bombina, and other species will eat boiled romaine or other leafy dark green lettuce, ground rabbit chow mixed with gelatin, and in some cases, flaked fish food (Nace et al., 1974; Mattison, 1998). Spinach can cause oxalate toxicity and should be avoided. A standard diet has been developed

Fig. 1I.

for bullfrog tadpoles (Culley, 1992). Flores-Nava and GascaLeyva (1997) demonstrated that a Purina shrimp f e e d - g e l a t i n mixture provided best growth rates in bullfrog tadpoles when fed on a vertical platform. Dendrobates larvae are carnivorous and can be fed brine shrimp, flaked fish food, and egg yolk. Most small salamander larvae require young Daphnia, brine shrimp nauplii, and other very small prey items. As larvae grow, larger prey can be fed. Axolotl larvae will eat sectioned earthworms, Daphnia, and Tubifex (Nace et al., 1974; Mattison, 1998). Larval amphibians are voracious feeders and should be fed more frequently than adults. Some species require several small feedings throughout the day; others can be fed once or twice daily. Uneaten food must always be removed to prevent fouling of water.

C.

Behavior

Many flogs and salamanders, particularly terrestrial species, are territorial and should not be housed together. Both males and females of Plethodon cinereus are territorial and will vigorously defend their habitats. Fecal pellets and granular gland secretions are routinely used to mark home ranges. If another animal is encountered, agonistic posturing such as raising the tail and trunk can result. Aggression can escalate to biting. Plethodon cinereus bites the nasolabial groove of its competitor, thereby decreasing that animal's ability to locate food, and

Cricketscan be fed chicken laying mash or dog food to increase their nutritive value.

807

17. BIOLOGY AND DISEASES OF AMPHIBIANS ultimately affecting its survivability (Jaeger, 1981; Petranka, 1998).

D.

Reproduction

1. ReproductiveAnatomy and Physiology Sexual dimorphism exists in many amphibian species; this can be particularly evident during the breeding season. In general, female amphibians are larger than males. Male bullfrogs, pig frogs, and bronze frogs have a tympanum that is larger than the eye; the female counterpart is the same diameter as the eye. Differences in coloration between males and females exist in some frog and salamander species (e.g., Bufo and Triturus). The vocal sacs of male frogs become larger and more pigmented in breeding season. Hyla and related species often have yellowish vocal sacs, and the sacs of Bufo tend to be blackish. Cloacal glands in male salamanders become swollen, resulting in enlarged cloacal lips. Male plethodontid salamanders develop enlarged hedonic glands on the chin; secretions of this gland are rubbed on the female during courtship. Most male frogs develop keratin pads on their thumbs to assist in gripping females during amplexus (a characteristic prolonged breeding embrace). Enlarged teeth can be found in certain frog and salamander males in breeding readiness. In some plethodontid species, these teeth are used to abrade the skin of females and allow introduction of chin gland secretions into her bloodstream (Conant and Collins, 1991; Zug, 1993; Petranka, 1998).

Courtship and reproduction in amphibians range from simple to very elaborate. Internal fertilization occurs in many salamander species. Salamanders may engage in ritual behavioral displays such as the "hula" of Notophthalmus viridescens. In this dance, the male undulates his tail and body while swimming in front of a potential mate. If the female shows interest, the male deposits a spermatophore, and the female picks the packet up with her cloaca. Plethodon cinereus has a more complex courtship. Males use pheromone trails to locate receptive females. The male approaches the female, arches and undulates his tail, then rubs his hedonic gland secretions over her body (using his enlarged teeth to abrade her skin and introduce secretions into her system). The male next aligns himself along the female's body, keeping his tail arched and curled. The female places her chin on his dorsal surface above the vent, and the couple performs a "tail-straddle walk." Finally, the male deposits a spermatophore, the couple moves forward, and the female picks the packet up. The pair separates, the female deposits the fertilized eggs in clusters suspended by a pedicle, and she coils around the eggs until hatching (Petranka, 1998). Frog courtship is no less colorful than that of salamanders. In general, frogs have external fertilization. Males attract mates by vocalizing. When a receptive female is located, the male grasps her with his forelimbs in amplexus. The eggs are expelled, and the male releases sperm to fertilize them. Various adaptations of this basic plan include the courtship of the Surinam toad (Pipa pipa), a relative of Xenopus (Fig. 12). During amplexus, the pair swims in an upside-down circle, with the female releasing eggs

Fig. 12. The female Surinamtoad, Pipapipa, incubates her eggs in the skin of her back. (Photo courtesyof S. Echternacht.)

808


at the top of the circle. The male releases sperm, and the fertilized eggs drop and land on the back of the female, who is now at the bottom of the circle. The female's skin grows over the eggs, and the eggs and larvae are carried in this fashion until metamorphosis, when tiny froglets emerge. The midwife toad, Alytes obstetricans, exhibits another breeding strategy. After amplexus and fertilization, the male toad wraps the strands of eggs around his legs. He carries the eggs with him in this fashion, visiting ponds periodically to keep them moist until hatching (Mattison, 1998). The most unusual reported reproductive behaviors include the Darwin's frog and the gastric brooder, Rheobatrachus. The male Darwin's frog takes his newly hatched tadpoles into his mouth, where they migrate to the vocal sacs. The tadpoles remain in the vocal sacs until metamorphosis, then emerge from their father's mouth as froglets. The Rheobatrachus female swallows her fertilized eggs. The eggs and tadpoles are brooded in the female's stomach, where they feed on yolk, undergo metamorphosis, and emerge as froglets. During this brooding period, the female ceases all gastric activity (due to the release of prostaglandin E). Normal gastric activity resumes after the froglets have emerged (Duellman, 1992). 2.

Husbandry

Many amphibian species have been successfully bred in the laboratory. Arginine vasotocin regulates reproductive behavior in amphibians (Boyd and Moore, 1992). In some species, temperature plays a more important role than photoperiod in certain aspects of reproduction (Paniagua et al., 1990). When breeding axolotls, both males and females are kept separately at 22~ (72~ for a week, then placed together in 12~ (54~ water in a shaded container and left undisturbed. Breeding and egg deposition will occur within 2 days. A substrate should be provided for egg attachment. After oviposition, eggs should be removed and incubated in separate containers (Verhoeff-de Fremery et al., 1987; Mattison, 1998). Specific detailed information is also available for laboratory breeding of other amphibian species, including induction of ovulation and artificial insemination (Nace et al., 1974). 3.

Larval Amphibians and Metamorphosis

With few exceptions, larval amphibians are aquatic. Their skin is thin, fragile, and well vascularized to assist in respiration. Gills (internal or external) are present. All larvae lack eyelids. The skeleton is primarily to completely cartilaginous. Dorsal and ventral fins are present on the tail, and both the tail and body are heavily muscled for swimming. Lateral line systems are well developed in all amphibian larvae (Duellman and Trueb, 1986; Zug, 1993). Salamander larvae closely resemble adult animals, having four limbs and other common features. Premetamorphic tad-

poles (frog larvae), in contrast, appear very different from adult frogs. Most tadpoles have a fleshy oral disc surrounding their mouths. This disc can be located dorsally, ventrally, or anteriorly, depending on method of feeding. Teeth are not present; tadpoles have horny beaks and denticles that rasp algae and cut food into smaller pieces. Gills are initially external but are soon covered, along with the forelimbs, by an operculum. Hindlimbs appear late in the larval period (Zug, 1993). Larvae should be housed in well-aerated aquaria in appropriate stocking densities. Care should be taken to separate large from small larvae, especially in cannibalistic species. Pfennig and Collins (1993) discovered that cannibalism develops more slowly if sibling larvae are housed together exclusively. Metamorphosis requires the presence of thyroid hormone and iodine. Species such as the spadefoot toad, which lives in ponds prone to drying, can accelerate metamorphosis by environmentally induced release of corticotropin-releasing hormone (Denver, 1997). As larval development progresses (prometamorphosis) in tadpoles, external gills are resorbed and limbs develop. Immediately before emergence (metamorphic climax), the tail is resorbed, forelimbs break through the operculum, and the hindlimbs become functional (Duellman and Trueb, 1986; Zug, 1993). Water levels should be decreased as larvae undergo metamorphic climax and prepare to emerge. In many cases, a ramp or other object should be placed on the water to facilitate emergence to a terrestrial existence. Newly transformed amphibians can drown if this assistance is not provided. Metamorphosis is a time of immune stress in amphibians, and larvae that undergo metamorphosis at less-than-optimal size become immune-compromised (Rollins-Smith, 1998). Every effort should be made to prevent undue stressors and exposure to infectious agents during the metamorphic period.

E.

M a n a g e m e n t and Reproduction of Xenopus

As previously noted, the African clawed frog, Xenopus laevis, is used extensively in developmental, cellular, and molecular biology research. Current use of Xenopus has expanded into other areas as a result of several factors. First, Xenopus is easier to maintain than terrestrial amphibians. The species is hardy and thrives in captivity. The life cycle is relatively short, making it useful for studies involving multiple generations. The embryos are large and have a high yield of genetic material. Embryo development is well characterized and responses to environmental toxins make it ideal for teratogenesis studies. Finally, oocytes will express foreign nucleic acids when injected, making Xenopus invaluable in expression cloning research. These characteristics have resulted in Xenopus becoming a study animal in many facilities (Gurdon, 1996). Xenopus tropicalis, the only diploid Xenopus species, has re-

809


cently gained popularity in genetic and molecular research. Xenopus tropicalis is much smaller than X. laevis, has half the haploid genome size per nucleus, and has 20 chromosome pairs (X. laevis has 36 chromosome pairs). The generation time of X. tropicalis is 4 to 6 months. Females lay up to 3000 oocytes per ovulation, and embryos are less variable in development (Amaya et al., 1998). 1.

Natural History

The African clawed frog belongs to the family Pipidae. There are over 17 species of Xenopus and 6 subspecies of X. laevis, which occur throughout Africa. The subspecies are morphologically distinct, with X. laevis laevis being notably larger than the others. Color patterns and digit length also distinguish subspecific differences (Kobel et al., 1996). The clawed frog ranges from central to South Africa in a wide variety of habitats, including rivers, lakes, swamps, ditches, and wells. It appears to prefer still, cloudy water, although populations are found in clear streams. In lakes devoid of fish, Xenopus has evolved to occupy the fish niche. This species occurs at altitudes up to 3000 meters (Tinsley et al., 1996). Xenopus tolerates a fairly wide water-temperature range. Adults become stressed at prolonged temperatures less than 14 ~C (57 ~F) and greater than 26 ~C (81 oF). At these extremes, a decrease in oocyte quality is observed (Wu and Gerhart, 1991). Individuals have been known to survive in ice-covered ponds and desert ponds. When temperatures become too hot (30 ~C, 86 ~F), X. laevis will excavate pits in the cool mud on the bottom of ponds. In drought conditions, it will estivate (Tinsley et al., 1996). The preferred temperature range for this species is approximately 20~176 (68~176 (Etheridge and Richter, 1978). In the wild, Xenopus will breed in both acidic and alkaline water (however, tadpole survival rates decrease in water with a pH of 5). Xenopus laevis will also tolerate elevated salinity (40% seawater) for a short time (Tinsley et al., 1996). 2.


Xenopus laevis laevis has a yellowish to darker, spotted to marbled dorsal coloring. The frog's ventral surface is solid yellowish white to spotted. This subspecies has a fifth toe that is much longer than the tibia. Female X. laevis laevis are larger than males and average 110 mm in length (Kobel et al., 1996). Females have large cloacal papillae, and males develop dark inner surfaces on their forearms (nuptial pads). The skin secretions of Xenopus include thyrotropin releasing hormone, caerulein, and xenopsin. Antimicrobial compounds (magainins) are also found in skin secretions (Kreil, 1996). Xenopus lacks a tongue; when feeding, it shreds prey with its hind claws and uses its front feet to shove food into its mouth (Tinsley et al., 1996). The eyes of the clawed frog are located

more dorsally on the head, are lidless, have a convex cornea, and are adapted for vision in air rather than water. Xenopus floats at the water's surface, and vision is directed upward; therefore, objects passing above will elicit a hiding response from the frog. There are two separate olfactory cavitiesmone for detecting scent in water, and one for airborne odors. The lateral line system is located dorsally and ventrally, and is retained in adult animals. The vocal apparatus of the clawed frog is designed for underwater sound production (Deuchar, 1975). The lungs, heart, and liver of Xenopus are large, and the urinary bladder is spherical. Xenopus must come to the water's surface and gulp air, because cutaneous respiration is not as well developed as in other species. Even tadpoles develop and utilize lungs as well as gills for breathing (Deuchar, 1975). During times of drought, Xenopus adapts physiologically by producing urea rather than ammonia. When ample water is available, the frog reverts to production of the more toxic ammonia, which is rapidly dissipated in the water (Tinsley et al., 1996). Life span of X. laevis in the wild is reported to be greater than 10 years. In captivity, the clawed frog can live 15 years or more (Deuchar, 1975; Tinsley et al., 1996). 3.

Housing and Husbandry

When acquiring Xenopus for the laboratory, it is important to verify the species of the animals, as different species may require different temperature and housing conditions. X. laevis are hardy frogs and can be kept successfully in a variety of housing situations. Tanks can be constructed of fiberglass, glass, plastic, or stainless steel (Fig. 13). Unless the sides of the tank are tall, lids should be provided. Screen, metal grills, and perforated plastic lids are commonly used. Xenopus will jump out of tanks if water levels are low (such as during cleaning) and when they are startled. Stackable plastic containers with lids that allow adequate air exchange are useful if space is limited in the facility. In the wild, X. laevis is commonly found in murky water. This dark water provides a visual barrier to predators (Tinsley et al., 1996). In the laboratory animal facility, clawed frogs prefer opaque containers to clear containers; one study demonstrated that darker tank colors actually enhanced frog growth (Hilken et al., 1995). Additionally, partially covered tanks and/or retreats such as PVC pipe, stainless steel rabbit feeders, styrofoam, sponge, round ceramic tiles, terra-cotta pots, and sandstone can provide adequate hiding places and decrease stress (Kaplan, 1993; Major and Wassersug, 1998). Both static and flow-through systems are used. To prevent fouling of water, static systems should be changed after animals have fed. Inexpensive modified, nonrecirculating systems have been described (Dawson et al., 1992; Rogers et al., 1997). Recirculating systems may incorporate charcoal or sand filters to clean and reuse water. Flow-through systems frequently contain a standpipe to drain accumulated water and feces, and a hose or

810


Fig. 13. Xenopuslaevis can be group-housedin stainless steel or fiberglasstanks.

other constant-drip water source. Both flow-through and recirculating systems are now commercially available through several companies. Regardless of the system, water should be dechlorinated before itis added to the tank. Water depth can vary from 5 to 20 cm. Although water that is too shallow will increase stress and escape response, no difference in growth was documented between water that was 5, 10, and 20 cm deep (Hilken et al., 1995). Population density within the Xenopus tank is a critical factor in growth and productivity. Suggested stocking densities of adult breeding frogs range from 1 frog per 3 liters to 4 frogs per 5-10 liters (McBride, 1978; Dawson et al., 1992; Hilken et al., 1995; Major and Wassersug, 1998). Increase in stocking densities will cause decrease in growth. Water temperature should be kept 20~176 (68~176 (Etheridge and Richter, 1978; Dawson et al., 1992; Hilken et al., 1994, 1995; Major and Wassersug, 1998). Ranges of 18 ~ 24~ (64~176 are considered acceptable for adequate growth. Some facilities keep Xenopus on a natural light cycle; however, most facilities use a constant 12 hr light-12 hr dark cycle, especially if breeding the frogs year-round (Major and Wassersug, 1998). Housing Xenopus directly under very bright light should be avoided (Hilken et al., 1994). A recent survey of Xenopus husbandry practices concluded that the most commonly used procedures include housing frogs in opaque plastic containers with static water systems, with approximately 1 frog per 4 liters water. Water temperature is maintained at 19~176 and light cycle is 12 hr light-12 hr dark.

Frogs are fed commercial dry chow 3 times a week, and tanks are changed after feeding. Cover is provided in about half of the facilities surveyed; however, many of the labs, which do not use cover, house the animals in opaque tanks (Major and Wassersug, 1998). Adult Xenopus should always be handled with soft nets or gloved hands to prevent skin abrasions and disruptions of the protective mucous layer. Xenopus are most commonly identified by sewing beads on a thread to the skin of the frog (Fig. 14). It has been found necessary to anchor the suture in a muscle mass of the forearm or hindleg to prevent sloughing of suture when skin is shed. Some laboratories have grafted pale skin from the ventral surface to the back of the frog. Individual animals are given unique graft patterns (Verhoeff-de Fremery and Griffin, 1987). This method is time-consuming and has more potential complications than the bead-and-thread method. Sterile microchip transponders can be implanted into the dorsal lymph sac (Hoogstraten-Miller and Dunham, 1997). 4.

Diet

In the wild, Xenopus eat a wide variety of prey items, including aquatic invertebrates, small crustaceans, and insects. Amphibians (including Xenopus tadpoles), small birds, and fish have also been documented as occasional prey items. Clawed frogs use olfaction rather then vision as the primary means of locating food; therefore, they will also scavenge carcasses (Tinsley et al., 1996).

811


5.

Fig. 14. Xenopus can be individually identified by sewing colored beads to the skin (arrow).

Laboratory-housed Xenopus have been maintained on a variety of diets. Historically, clawed frogs have been fed ground beef liver or heart. Adults can apparently be maintained for several years on these diets; however, tadpoles will develop calcium-phosphorus imbalance if fed only organ meat or muscle (calcium levels are very low and phosphorus levels high in these tissues). This type of food is best used as a supplement for adults fed a more balanced diet. Ground meat or liver can be presented to the frogs on an applicator stick or with a spoon to decrease fouling of the water (Sackin and Sackin, 1991; Dawson et al., 1992). Tanks should also be cleaned after animals have fed, because organ meat and muscle tend to plug drains (Dawson et al., 1992). Several animal facilities have converted from a raw meat diet to commercially prepared diets. Among those in use are salmon chow (Soft-Moist Salmon Diet, Rangen, Inc.), trout chow (Purina), Frog Brittle (Nasco), and Sinking Frog Food (Xenopus Express). Trout chow can be purchased in both floating and sinking forms, comes in various sizes, and does not break down in water. About 5-10 pellets per adult frog are sufficient for a single feeding. Clawed frogs may require a period of several days to 2 weeks to adapt to a new diet (Wu and Gerhart, 1991). Most facilities feed Xenopus from 2 to 5 times a week. Additional foods for clawed frogs include earthworms, mealworms, chick embryos, Tubifex worms, goldfish, and crickets. Feeding chitin-containing animals (crickets, mealworms) excessively can result in intestinal obstruction. Tanks should be cleaned after Xenopus are fed; however, care should be taken not to disturb the frogs for at least an hour, or they may regurgitate their food (Etheridge and Richter, 1978; McBride, 1978; Dawson et al., 1992).

Reproduction

Under ideal conditions, X. laevis will undergo metamorphosis at 2 months and will reach sexual maturity within 8 months of hatching. Cooler temperatures will slow development. In the wild, the breeding cycle corresponds to the onset of the rainy season and subsequent prey abundance (Tinsley et al., 1996). Females reach optimum egg production at 2 - 3 years and continue producing for several years. In captivity, breeding can be accomplished year-round using HCG injections. Females should be bred a maximum of 1 time per month (the ideal induced breeding interval is once every 1 to 4 months). To induce ovulation and breeding, HCG is injected into the dorsal lymph sac of both males and females. Two injections are given to each, spaced 1-5 hr apart. Males receive 400 units of HCG at each injection. Females receive 150250 units on the first injection, followed by 650-850 units on the subsequent injection. Injected frogs are placed into FETAX solution (Table V) and allowed to breed (Dawson and Bantle, 1987). In the laboratory, frogs should be bred in containers with false bottoms, to allow passage of the eggs and prevent ingestion by the parents. Each egg has an individual jelly capsule; therefore, eggs are less likely to clump as they do in other frog species. Larvae hatch 3 days after spawning. 6.

Embryos

Embryos can be collected by gently flushing them from the bottom of the breeding chamber, using a plastic meat baster. They should be placed in a separate container and covered with FETAX solution. Handling large numbers of embryos is easier if their jelly coat is first removed. This can be accomplished by gently swirling them in a 250 ml flask containing 100 ml of 2% (2 g/100 ml) cysteine solution at pH 8.1 (Dawson et al., 1992). A number of factors, including the thickness of the jelly coat and the number of embryos, affects the time it takes to dejelly the embryos (Dawson et al., 1992). Normally, 1-3 min are

Table V FETAX Solution a

Component

Concentration (mg/liter)

NaC1 NaHCO3 KC1 CaC12 CaSO4. H20 MgSO4

625 96 30 15 60 75

aUse deionized, distilled water. From Dumont et al. (1983).

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sufficient, but the process should not go on longer than necessary. After swirling, the cysteine solution is decanted and the embryos rinsed 5 to 8 times with FETAX solution. Dejellied embryos should be placed into a container (e.g., glass petri dish) with a large surface area and covered with FETAX solution. The L-cysteine (the free-base form [Sigma Chemical, St. Louis, Missouri]) should be stored in a refrigerator and cysteine solution prepared fresh just prior to use. Best results are obtained using cysteine powder that is less than 6 months old. 7.

Tadpole Biology

Xenopus tadpoles have functional lungs as well as gills, and will periodically surface to breathe. They grow best when provided access to air, and deprivation of surface air results in increased buccopharyngeal respiration and consequent decreased feeding (Feder et al., 1984). Tadpoles orient themselves parallel to one another and hover in a characteristic head-down fashion, using their undulating tails to direct food particles to their mouths (Fig. 15). They are very efficient filter feeders and eat materials suspended in the water. From hatching through day 4, tadpoles absorb yolk sac; they begin feeding on day 5. In the wild, plankton makes up the bulk of the diet of the Xenopus tadpole. In captivity, strained baby-food green vegetables or algae powder have been used (Dawson et al., 1992; Hilken et al., 1995). Nettle powder, baker's yeast, and bonemeal in a 7:2:1 ratio have also been used successfully (Wu and Gerhart, 1991). A pelleted tadpole diet can be fed to larger larvae. Tadpoles are generally fed daily, and water should be changed after they feed. Xenopus tadpoles can be stocked at a density of 1-10 per liter, with more frequent water changes recommended for higher stocking densities (Wu and Gerhart, 1991). Jars are preferred to

nets when handling or transferring tadpoles, to avoid abrading the skin. When tadpoles are undergoing metamorphosis, they should be placed in shallow water to prevent drowning. 8.

Oocyte Harvest

For nonsurgical oocyte harvest, female frogs can be induced with HCG injections as described previously, then allowed to lay their eggs naturally. Eggs can also be carefully manually expressed from HCG-primed females. Tricaine methane sulfonate (MS222) can be used to anesthetize Xenopus for surgical oocyte harvest. The female frog is immersed in a buffered solution of MS222; a surgical plane of anesthesia is usually reached within 10-15 min. The animal can then be removed and anesthesia maintained if necessary by dripping the solution on the skin. Oocyte harvests can normally be completed in 30 min or less and should not require supplemental anesthesia. Recommended concentrations of MS222 are 0.5-2.0 gm per liter (Crawshaw, 1993), buffered with sodium bicarbonate at 0.42-1.05 gm per liter (Crawshaw, 1992; Schaeffer, 1997). It has been found that 1 gm per liter MS222 buffered with 0.7 gm per liter sodium bicarbonate provides adequate anesthesia for oocyte harvest. Caution should be exercised when handling crystalline MS222, to prevent inhalation and eye and skin exposure. Once the procedure is concluded, the frog can be recovered from anesthesia by rinsing in clean, dechlorinated water and placing into a recovery tank with a very shallow water level. In order to reduce stress, the water used for both induction and recovery can be taken from the Xenopus tank. Frogs should be closely observed during induction and recovery, to ensure that drowning does not occur.

Fig. 15. Xenopustransform from filter-feedinglarvae (A) to carnivorousfroglets (B) during metamorphosis.

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Fig. 16. Surgical harvest of Xenopus oocytes must be done aseptically.

Aseptic technique must be used whenever harvesting oocytes surgically (Fig. 16). A mask and sterile gloves should be used; gloves are changed between frogs. One procedure involves autoclaving instruments prior to use. They can be sterilized in a glass-bead sterilizer between animals to eliminate the possibility of inadvertently introducing toxic cold sterilants into the surgical site or onto the patient's permeable skin. Although Xenopus skin contains antimicrobial agents, performing a single skin prep with dilute povidone iodine or chlorhexidine solution is recommended. Soaps and scrubs must not be used, for they will destroy the protective mucous layer and be systemically absorbed. The animal is draped with a sterile drape, and an incision is made paramedian in the lower abdominal quadrant on either side (sides can be alternated to allow maximum healing time). Forceps are used to grasp the ovary and exteriorize the oocyte masses. The desired number of oocytes are excised, and the remainder carefully replaced in the coelomic cavity. Muscle and skin layers are closed separately with 4-0 nylon in a simple interrupted pattern. Absorbable suture can be used in the muscle layer. Analgesics can be given, but care should be exercised to ensure that the frog's swimming and other motor functions are not impaired to the point of risking drowning. Analgesics that have proven efficacy in leopard frogs and would likely work in Xenopus include morphine (10 mg/kg intracoelomic q12 hr), flunixin meglumine (25 mg/kg intracoelomic q4 hr), butorphanol (25 mg/kg intracoelomic q12 hr), and xylazine (10 mg/kg intracoelomic q12-24 hr). Flunixin causes an increase in activity, while butorphanol and xylazine have a calming effect (Terril-Robb et al., 1996). The frog is then rinsed with clean dechlorinated water and placed into the shallow recovery container. Response to stimuli should return within 15-30 min, and ability to swim and function fully, within a few hours. Once re-

covered, the frog can be returned to its home tank. Frogs are rested for at least a month between surgeries. Sutures should be removed in about 6 weeks if they have not sloughed out with shed skin. No published information was found on complications associated with multiple oocyte harvests in frogs. The Institutional Animal Care and Use Committee at the University of Tennessee currently allows a maximum of six survival surgeries per frog. Frogs have been necropsied after four surgical oocyte harvests, and only occasional minor adhesions have been identified. More controlled studies are warranted to clarify this issue and define specific end points.

F.

Physical Examination and Techniques

1. Physical Examination Physical examination of an amphibian should begin with observing the animal in its primary enclosure. Attitude, posture, equilibrium (especially if in water), locomotion, body color, respiration, and behavior should be noted. The enclosure should be checked for appropriate temperature, humidity, and cleanliness. Presence and nature of feces and vomitus should be recorded. Once this initial assessment has been made, the animal can be removed from the cage for closer examination. Restraint should be done as described previously (Section I,D,8). Care should always be taken to support the animal's entire body, and to avoid disrupting the protective mucous layer. When handling larger or more-aggressive amphibians, it is advisable to have one person restrain while the second performs the physical exam. The surface of the skin should be thoroughly examined for ulcerations, abrasions, redness, or other lesions. Heart rate can be

814


determined while examining the ventral body surface. Abdominal palpation can be attempted; however, many amphibians will inflate the abdomen as a defense mechanism, making palpation difficult. The corneas should be clear, and a blink reflex should be present (except for aquatic species such as Xenopus, which lack eyelids). Nares should be free of exudate. The mouth can be gently opened with a thin, flat plastic speculum, and the oral cavity examined for lesions, excessive salivation, or exudate. Care should be taken to avoid breaking the thin delicate maxillary bones. Withdrawal and righting reflexes can be tested (Raphael, 1993; Wright, 1996). The amphibian should be weighed and its body condition assessed before returning it to its cage. Body condition can be determined by observing the prominence of the skeletal system (particularly the pelvic bones in frogs), and by palpating muscles and abdominal contents (Crawshaw, 1993). 2.

Blood Collection

Blood samples should be collected in lithium heparinized syringes. The midventral abdominal vein can be used to collect blood in both frogs and salamanders. A small-gauge needle appropriate for the amphibian's size (usually a 26- to 27-gauge) is inserted at a point midway between the sternum and pelvis in a cranial direction, and the sample is collected. The ventral caudal vein in salamanders can be used as a phlebotomy site. Frogs have a prominent lingual venous plexus situated beneath the tongue. The tongue can be gently drawn forward with a cottontipped applicator, the plexus punctured with a needle, and the blood sample collected in a heparinized capillary tube (Wright, 1996). Cardiocentesis can be used in both frogs and salamanders. Because of the hazards associated with this technique, animals should be anesthetized. The amphibian must be in dorsal recumbency, and the heartbeat identified. A 25- to 27-gauge needle is inserted beneath the xiphoid at an angle of approximately 10~ ~ to the ventral body wall, and the sample withdrawn from the ventricle (Raphael, 1993; Wright, 1996). If a blood sample cannot be obtained in frogs, lymph should be aspirated from the dorsal lymph sac. Systemic infections can be identified by performing culture and sensitivity on lymph collected in this manner (Raphael, 1993). 3.

also been used in amphibians (Crawshaw, 1993; Raphael, 1993; Wright, 1996). 4.

Injections/Gavaging

Common routes of injection include subcutaneous, intramuscular, intracoelomic, and dorsal lymph sac. Subcutaneous injections can be given in the skin overlying the shoulder or pelvis. The muscles of the forelimbs in frogs and the epaxial muscles in salamanders are locations for intramuscular injections (Wright, 1996). Intracoelomic injections should be given off midline in the lower abdomen, and dorsal lymph sac injections are given subcutaneously in the caudodorsal part of the frog's body (over the pelvic area). Gavaging can be accomplished using standard rodent stainless steel gavage tubes, or with IV Teflon catheters in small species (Wright, 1996). 5.

Euthanasia

Amphibians are most commonly euthanatized by immersion in an overdose of MS222 or other immersion anesthetic. Concentrations exceeding 3gm/liter constitute overdoses for most amphibian species. As done for anesthesia, the acidity of the MS222 should be buffered to prevent irritation. Inhalant anesthetics may be used for euthanasia. Isoflurane, halothane, or methoxyflurane can be delivered into an induction chamber for air-breathing species; they can also be bubbled into aquatic systems. Euthanasia solutions such as sodium pentobarbital can be injected via the coelom or the dorsal lymph sacs. Amphibians are extremely resistant to hypoxia; therefore, certain physical methods that may be permissible in mammals are contraindicated. Decapitation alone is not considered sufficient; animals must be anesthetized prior to decapitation or double-pithed afterward (Andrews et al., 1993; Beaver et al., 2000). Cooper et al. (1989) recommend complete destruction of the brain by concussion if physical means are necessary. Because of the tolerance of many species to cold temperatures, hypothermia is not appropriate as either an anesthetic or euthanasia agent (Schaeffer, 1997; Cooper et al., 1989; Andrews et al., 1993; Beaver et al., 2000). Quick-freezing of anesthetized amphibians in liquid nitrogen may be acceptable (Cooper et al., 1989; Andrews et al., 1993; Beaver et al., 2000).

Other Diagnostic Tests

Fecal examination is performed as for other species. Specimens collected for culture and sensitivity should be incubated at both standard and room temperatures. Impression smears, skin scrapings, and abdominocentesis are performed as for other species. Biopsies can be taken under MS222 anesthesia, and the skin closed with nonabsorbable suture or tissue glue. Radiology is very useful in identifying foreign bodies, impactions, and pneumonia, and for assessing skeletal abnormalities. Dental film works well for small amphibians. Fluoroscopy, endoscopy, and transillumination (using an intense, cool light source) have

lIl.

A. 1.

DISEASES

Infectious Diseases

Bacterial

a.

Redleg

i. Etiology. The organism most commonly isolated from amphibians with redleg is Aeromonas hydrophila, a gram-

815


negative bacterial rod. Aeromonas is frequently found in the aquatic environment and can be a normal inhabitant of the intestinal tract of healthy frogs (Hubbard, 1981; Hird et al., 1981). Stress and subsequent immunosuppression predispose amphibians to colonization by Aeromonas. The organism is transmitted to susceptible animals through the tank water. Aeromonas has been responsible for mass mortality in both wild and captive populations (Hubbard, 1981; Nyman, 1986; Rafidah et al., 1990). ii. Epizootiology and transmission.

iii. Pathogenesis. Waterborne Aeromonas will colonize the skin and visceral organs of frogs and salamanders. Course of the disease can be either acute or chronic (Rafidah et al., 1990). In acute disease, septicemia frequently results (hence the name "redleg").

Signs of acute infection include petechiation and ulceration of the skin, particularly evident on the legs and abdomen (Fig. 17). Lethargy, anorexia, and ascites are also seen. Ocular and periocular inflammation is often noted with this disease. Chronically infected animals exhibit ascites and neurologic signs (Hubbard, 1981; Nyman, 1986; Rafidah et al., 1990; Crawshaw, 1993; Williams and Whitaker, 1994; Wright, 1996). iv. Clinical signs.

vii. Treatment. Appropriate treatment is based on culture and sensitivity. Tetracycline (50 mg/kg PO BID) can be effective against Aeromonas. Valuable animals should be concomitantly treated with aminoglycosides (5 mg/kg IM q48 hr). Chloramphenicol at 50 mg/kg IM, IP q24 hr has also been effective against Aeromonas (Raphael, 1993; Crawshaw, 1993; Wright, 1996). Stoskopf et al. (1987) found that gentamicin at 2.5 mg/kg IM q72 hr provided therapeutic blood levels in Necturus housed at 3 ~C. Another study demonstrated that Rana pipiens required 3 mg/kg IM SID to achieve therapeutic levels when housed at 22~ (Teare et al., 1991). Riviere et al. (1979) showed that immersion in a solution of gentamicin sulfate (1 mg/ml) would provide therapeutic levels in R. pipiens; however, Teare et al. ( 1991) demonstrated that this concentration resuited in increasing serum levels and death after 120 hr. viii. Control. Affected animals should be isolated and husbandry practices reviewed to ensure that appropriate water quality, temperature, stocking density, and food are provided. The environment should be thoroughly cleaned and disinfected.

v. Necropsy findings. Hepatic necrosis, splenic congestion, and other lesions consistent with septic thrombi are commonly seen (Wright, 1996).

ix. Prevention. Amphibians should be housed in clean, dechlorinated water with proper stocking density and temperature. They should be provided nutritious food on a feeding schedule appropriate for the species. Newly arrived animals must be quarantined separately and thoroughly examined before introduction into the existing colony. Animals should be colony-reared rather than wild-caught if at all possible.

vi. Differential diagnoses. Organisms that cause similar lesions include Flavobacterium, Pseudomonas, and Proteus (Crawshaw, 1993; Wright, 1996).

x. Research complications. Redleg can severely decimate research populations of adult and larval amphibians. Data can be affected by using chronically infected animals.

Fig. 17. Bacterialsepticemiacausesthe subcutaneoushemorrhageseen in redleg(arrow).

816

b.


Pseudomonas

i. Etiology. Pseudomonas, a gram-negative rod, is commonly found in the aquatic and terrestrial environment. ii. Epizootiology and transmission. The organism is a waterborne opportunist, which typically causes secondary infections in immunosuppressed animals. iii. Pathogenesis. In Necturus, the organism colonizes the gills (Anver and Pond, 1984). Pseudomonas has also been implicated in ulcerative disease in axolotls (Crawshaw, 1993). iv. Clinical signs. In axolotls and other amphibian species, Pseudomonas is associated with skin sloughing and ulceration. Necturus become septicemic and die (Raphael, 1993; Anver and Pond, 1984). v. Necropsy findings. Lesions in the mudpuppy include necrotic gray foci on the gills and cutaneous hyperemia (Anver and Pond, 1984). vi. Differential diagnoses. Other bacterial agents that may cause ulceration, skin sloughing, and hyperemia include Aeromonas and Proteus. vii. Treatment. Gentamicin and chloramphenicol are the antibiotics of choice in treating Pseudomonas. Doses are the same as for Aeromonas. viii. Control. Control is the same as for Aeromonas and other waterborne opportunistic bacteria. ix. Prevention.

Prevention is the same as for Aeromonas.

x. Research complications. As with Aeromonas, Pseudomonas can cause significant morbidity and mortality in research amphibians, thus preventing accurate data collection.

c.

Mycobacteriosis

i. Etiology. The mycobacteria most frequently responsible for disease in amphibians are Mycobacterium xenopi, M. fortuitum, and M. marinum. More recently, M. chelonae has been reported as a pathogen in a colony ofXenopus laevis (Green et al., 2000). These acid-fast organisms are common saprophytes of soil and water (Crawshaw, 1993; Wright, 1996). ii. Epizootiology and transmission. Transmission of mycobacteriosis is most likely through traumatized skin of an immunocompromised animal. The disease does not appear to be highly infectious from animal to animal (Raphael, 1993).

iii. Pathogenesis. The organisms colonize amphibian skin and form nodules; they can also spread to the viscera and form granulomas in organs (Crawshaw, 1993). A recent study with Rana pipiens demonstrated chronic, nonlethal granulomatous disease in immunocompetent frogs; steroid-treated frogs developed an acute, lethal disease (Ramakrishnan et al., 1997). iv. Clinical signs. Typically, amphibians suffering from mycobacteriosis will demonstrate wasting in spite of a good appetite. Pneumonia may develop. Nodules or ulcers may be seen on the skin surface. Extensive skin lesions can interfere with cutaneous respiration. As the disease progresses, animals become more debilitated and eventually die (Crawshaw, 1993; Raphael, 1993). v. Necropsy findings. Gross lesions include yellowish white dermal and visceral granulomas and ulcerations. Visceral granulomas are most commonly found in the liver. Acid-fast organisms frequently are present in the granulomas (Anver and Pond, 1984). vi. Differential diagnoses. Fungal infections can cause cutaneous ulcers and granulomas. Lesions can be cultured and examined histologically with acid-fast stains to distinguish between mycobacterial and mycotic infections. Mycobacterium fortuitum will grow at both room and human body temperature when cultured. vii. Treatment. Valuable animals can be treated with amikacin (5 mg/kg SC, IM, IP q 2 4 - 4 8 hr) or enrofloxacin (5 mg/kg SC, IM q24 hr) (Raphael, 1993). Because of the zoonotic potential, most affected animals are culled. viii. Control. Disease can be controlled by isolating and treating or culling affected animals, and by cleaning and disinfecting the environment. ix. Prevention. Good husbandry (clean environment, appropriate food and temperature, lack of abrasive objects in tank, and low stocking density) will help prevent mycobacteriosis. x. Research complications. Debilitated animals are inappropriate as research subjects. Additionally, this disease is zoonotic, particularly to immunocompromised humans. d.

Flavobacterium

i. Etiology. Flavobacterium is a gram-negative, aerobic rod that is found in both soil and water. The organism is pigmented yellowish orange. Flavobacterium meningosepticum and F. indologenes have caused disease in leopard frogs, and F. meningosepticum and F. oderans disease outbreaks have been identified in Xenopus laevis colonies (Green et al., 1999).

817


ii. Epizootiology and transmission. The organism is typically present in water and can enter through a wound or abrasion. Disease can occur in animals that are not stressed (Olson et al., 1992). iii. Pathogenesis. Flavobacterium indologenes can spread to multiple organs, ultimately causing septicemia. Flavobacterium meningosepticum affects the nervous system (Olson et al., 1992; Taylor et al., 1993). iv. Clinical signs. Signs of F. indologenes infection include weight loss, edema, ascites, petechia, dyspnea, uveitis, corneal edema, and incoordination. Flavobacterium meningosepticum causes head tilt, circling, ataxia, anorexia, and ocular opacity (Olson et al., 1992; Taylor et al., 1993). Flavobacterium meningosepticum can also cause ascites, petechial hemorrhage, lethargy, and other signs of septicemia (Green et al., 1999). v. Necropsyfindings. Histologic changes associated with F. indologenes are vascular congestion and hemorrhage, panophthalmitis with conjunctival and corneal edema, cardiac and skeletal myositis, and hepatocellular degeneration and necrosis (Olson et al., 1992). Flavobacterium meningosepticum causes panophthalmitis, meningitis, and otitis (Taylor et al., 1993); macrophage and neutrophilic infiltration of liver, spleen, and kidney has also been described (Green et al., 1999). vi. Differential diagnoses. Aeromonas will cause septicemia; however, panophthalmitis and corneal edema are not consistently seen with Aeromonas. Culture and sensitivity will distinguish between the two organisms. vii. Treatment. Flavobacterium can be treated with trimethoprim sulfa (3 mg/kg SC, IM, PO q24 hr) (Raphael, 1993). viii. Control. Sick animals should be isolated and the environment cleaned and disinfected, as for other bacterial diseases. ix. Prevention. The same husbandry practices used to prevent Aeromonas should help prevent outbreaks of Flavobacterium. x. Research complications. This organism has the same epizootic potential as Aeromonas and can seriously affect research colonies. e.

Salmonella

i. Etiology. The genus Salmonella includes a single species (S. choleraesuis) that comprises 2300 serovars and is subclassified into 7 subgroups based on DNA similarity and host range (Miller and Pegues, 2000). Salmonella can cause disease in many vertebrates, including amphibians.

ii. Epizootiology and transmission. The organism is most commonly shed by the fecal-oral route. An aquatic environment facilitates transmission. iii. Pathogenesis. The organism colonizes the intestinal tract; it can also spread via the blood. iv. Clinical signs. Affected amphibians exhibit anemia, lethargy, anorexia, and diarrhea (Raphael, 1993; Crawshaw, 1993). v. Necropsy findings. Gross and histopathologic lesions are consistent with enteritis and septicemia. vi. Differential diagnoses. Other bacteria can cause septicemia. Blood or lymph cultures will identify the causative agent. Cloacal and fecal cultures will help determine the cause of diarrhea (Raphael, 1993). vii. Treatment. Appropriate antibiotics should be selected based on culture and sensitivity results. viii. Control. Isolation and disinfection of the environment should be done as for other bacterial agents. Certain Salmonella species are zoonotic; care should be taken when handling infected animals. ix. Prevention. Amphibians should be obtained from reliable, colony-bred sources; quarantined; and maintained in appropriate conditions. x. Research complications. Anemia and diarrhea may affect results in physiologic and other types of studies. 2.

Viral/Chlamydial

a.

Luckg Tumor Herpesvirus

i. Etiology. The Luck6 tumor herpesvirus (LTHV) has icosahedral morphology and is 95-110 nm. It occurs spontaneously in the northern leopard frog, Rana pipiens. ii. Ep&ootiology and transmission. The virus replicates during cool (hibernation) winter temperatures and is shed during spawning. When warmer temperatures of summer occur, viral replication ceases and tumor growth begins. If summer flogs are cooled down again, the inactive tumors will begin to demonstrate herpesvirus replication (Mizell, 1985; Williams et al., 1996). iii. Pathogenesis. The virus causes renal adenocarcinomas in R. pipiens. Tumor growth is rapid during the warm months of summer but stops during winter virus production. With warmer temperatures, tumor growth resumes, and most frogs die after spawning (Wright, 1996).

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iv. Clinical signs. Affected frogs may not show signs until disease is well advanced. Emaciation, lethargy, ascites, and death are most commonly seen. v. Necropsy findings. At necropsy, one or more whitish tumors can be found on either or both kidneys. Tumors can be quite large and metastasize to other organs. Histologically, the tumor is a papillary adenocarcinoma. Ascitic fluid may contain neoplastic cells. In winter flogs, eosinophilic intranuclear inclusions may be seen in renal cells (Anver and Pond, 1984; Wright, 1996). vi. Differential diagnoses. Lucke tumor herpesvirus can be distinguished from other tumors by history and necropsy. vii. Treatment.

There is no treatment for this disease.

viii. Control. Affected frogs should be culled and not allowed to reproduce.

v. Necropsy findings. Frog necropsies demonstrated petechial and ecchymotic hemorrhage of the skeletal muscle and viscera, dermal ulceration and necrosis, digit necrosis, and erythema of the skin (Cunningham et al., 1996a). Microscopic examination of salamander tissues showed hypertrophy of epidermal, gill, and liver cells, with evidence of viral infection (Jancovich et al., 1997). vi. Differential diagnoses. Lesions appear consistent with those of Aeromonas. This bacterium is present in most described cases of iridovirus infection; however, Jancovich et al. (1997) were unable to reproduce disease using bacteria alone, and Cunningham et al. (1996a) postulate that the iridovirus causes the primary lesions, with secondary invasion by the opportunistic Aeromonas. vii. Treatment. Successful treatment of iridovirus infections in amphibians has not been documented. Secondary Aeromonas infections may be treated as described previously.

ix. Prevention. Purchase of laboratory-reared, disease-free R. pipiens will prevent this disease.

viii. Control. Affected animals should be isolated if possible and given supportive care. Care should be taken not to mechanically transmit virus through handling.

x. Research complications. Asymptomatic animals with early-phase tumors can yield poor research data, particularly if renal physiology studies are being conducted.

ix. Prevention. In laboratory populations, good quarantine and husbandry practices should help in prevention of outbreaks.

b.

Iridovirus

i. Etiology. The organism is an enveloped, icosahedral virus ranging from 140 to 180 nm. Iridovirus or iridovirus-like particles have been implicated in disease outbreaks involving R. temporaria, R. esculenta, and Ambystoma tigrinum stebbinsi (Fijan et al., 1991; Drury et al., 1995; Cunningham et al., 1996a; Jancovich et al., 1997). ii. Epizootiology and transmission. This virus has been associated with epizootics in wild populations of frogs and salamanders. Transmission appears to be horizontal and likely waterborne. Previously described tadpole edema virus and viral hemorrhagic septicemia of frogs demonstrate lesions that closely resemble those of currently described iridoviruses. iii. Pathogenesis. Initial lesions are found on the skin, with progression to viscera. iv. Clinical signs. Affected wild flogs were found emaciated, with varying degrees of cutaneous erythema and ulcerations (Cunningham et al., 1996a). Tiger salamanders initially developed small white polyps, which spread to cover most of the epidermis, then progressed to epidermal hemorrhaging, excess mucus production, sloughed skin, lethargy, and anorexia (Jancovich et al., 1997).

x. Research complications. Epizootics can decimate populations of amphibians and seriously impair accurate data collection. c.

Frog Erythrocytic Virus

i. Etiology. Frog erythrocytic virus is a large (450 nm), enveloped icosahedral virus with double-stranded DNA (GruiaGray et al., 1989). ii. Epizootiology and transmission. The virus is found in the cytoplasm of erythrocytes of wild populations of Rana. iii. Pathogenesis. The organism appears to be transmitted mechanically by mosquitoes and midges. The virus infects the red blood cells, with juvenile frogs being most frequently affected. Adults apparently acquire immunity with repeated exposure to the virus. iv. Clinical signs. anemia.

Affected frogs may be weak due to

v. Necropsy findings. Structural changes in erythrocytes due to presence of virus particles can result in anemia. vi. Differential diagnoses. This virus can be distinguished from other organisms producing similar changes by observing

819


trapezoidal inclusions in the cytoplasm of red blood cells, and by observing structural changes in the cells themselves (GruiaGray and Desser, 1992). vii. Treatment.

No treatment is described.

viii. Control. Exclusion of midges and mosquitoes from frog enclosures should control spread of disease. ix. Prevention. Acquisition of colony-reared frogs and prevention of vector contact should effectively stop disease entry. x. Research complications. Anemic frogs will be less hardy and more prone to developing secondary infections, thereby complicating research projects.

d.

Chlamydia

i. Etiology. The agent most commonly involved is Chlamydia psittaci. Chlamydia is the sole genus of the order Chlamydiales (Howerth, 1984). Chlamydia psittaci is best described as the causative agent of psittacosis in birds, and is a zoonotic disease. ii. Epizootiology and transmission. Chlamydia psittaci is most commonly transmitted through the fecal-oral route. Experimental inoculation into the dorsal lymph sacs of Xenopus laevis has caused disease. iii. Pathogenesis. The organism colonizes the lung, liver, spleen, kidney, and heart of Xenopus and causes a pyogranulomatous inflammatory response. iv. Clinical signs. Signs include lethargy, bloating, disequilibrium, and erythema and patchy depigmentation of skin. v. Necropsy findings. Gross lesions consist of hepatosplenomegaly, cutaneous petechiation and ulceration, coelomic effusion, and subcutaneous edema. Dense, basophilic intracytoplasmic inclusions can be found in liver and spleen cells. Interstitial pneumonia, glomerulonephritis, and endocarditis have also been described (Newcomer et al., 1982; Howerth, 1984). vi. Differential diagnoses. Aeromonas and iridovirus infections can produce cutaneous lesions similar to those of Chlamydia psittaci. Diagnosis can be made by observing typical chlamydial inclusions in liver and spleen cells, and by absence of bacteria in lesions (Crawshaw, 1993; Wright, 1996). vii. Treatment. Oxytetracycline (50 mg/kg PO q12-24 hr) or doxycycline may be effective in treating chlamydiosis in amphibians (Raphael, 1993; Wright, 1996).

viii. Control. Affected amphibians should be isolated, and valuable animals can be treated. Chlamydia psittaci is zoonotic; therefore, appropriate precautions should be taken. ix. Prevention. Amphibians should be purchased from reliable, disease-free sources. Appropriate husbandry should be provided, and sick animals should receive a thorough diagnostic workup. x. Research complications. This disease can cause significant animal loss, as well as interfere with physiologic and reproductive studies. Diseased animals pose a potential threat to researchers and animal-care staff. 3.

Parasitic

Amphibians normally host a variety of parasites, without exhibiting signs of disease (Poynton and Whitaker, 1994; Tinsley, 1995). Determination of a pathogenic state is made by identifying parasite burden, concomitant stressors, and the inherent pathogenicity of the parasite in question. Many species of parasites infest amphibians; the following are common examples from each of the major parasite groups. a.

Protozoal

i. Etiology. Many protozoans infest amphibians. Significant species include Entamoeba ranarum (ameba); Trichodina (ciliate); Oodinium and Trypanosoma (flagellates); and Plistophora (microsporidian). ii. Ep&ootiology and transmission. Fecal-oral transmission is common in enteric protozoal infestations. Species of pathogenic protozoa can be transmitted through water and from aquatic vegetation or feeder fish to amphibians. iii. Pathogenesis. Entamoeba cysts are swallowed and directly colonize the colon. Trophozoites can spread to the kidney and liver. Oodinium and Trichodina are external parasites that affect the skin and gills of aquatic amphibians. Ingestion of infected fly larvae is the likely source of Plistophora myotropica in toads. Trypanosoma infects the blood of amphibians and has an indirect life cycle. iv. Clinical signs. Signs of amebiasis include dehydration, anorexia, and emaciation. Feces are loose and bloody; vomiting may also occur. Ascites may be noted with hepatic and renal involvement. Oodinium causes the skin and gills to become grayish in color. Debilitation occurs in chronic cases. Reddened gills and skin cloudiness and ulceration can be observed with Trichodina and other ciliates. Animals affected by trypanosomiasis may be asymptomatic or may die acutely. Plistophora causes anorexia, muscle wasting, and death.

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v. Necropsy findings. Entamoeba causes lesions of the colonic mucosa, suppurative nephritis, and hepatic abscesses. Cysts may be found in the liver and kidney. Splenomegaly is seen in amphibians that die acutely from trypanosomiasis. Necropsy findings associated with Plistophora include muscle atrophy and pale streaks in myofibers. vi. Differential diagnoses. Enteric protozoa can be identified by fecal examination or colonic wash. Skin scrapings and gill biopsies will demonstrate external protozoa. Plistophora sporocysts can be seen in histologic sections of degenerated myofibers. Trypanosoma and other hemoparasites can be identified on Wright-Giemsa-stained blood smears. vii. Treatment. Amebiasis and other enteric protozoal infections can be treated with metronidazole (100-150 mg/kg PO q14 days or 50 mg/kg PO SID for 3 - 5 days). Aquatic species may be treated with 50 mg/liter bath for 24 hr. Enteric ciliated protozoa may be treated with a combination of tetracycline (50 mg/kg PO BID) and paramomycin (50-75 mg/kg PO SID). Trypanosomiasis may respond to a quinine sulfate bath (30 mg/liter for 1 hr). Oodinium, Trichodina, and other external protozoa can be treated with salt baths (10-25 mg/liter SID for 5 - 3 0 min) or acriflavin baths (constant 0.025% bath for 5 days). Copper sulfate has also been used, but this compound can be toxic in some amphibian species (Crawshaw, 1993; Raphael, 1993; Wright, 1996; Whitaker, 1999; Wright, 1999a). viii. Control. Affected animals should be separated from community groups; they should be handled last, and equipment should not be shared. Tanks should be cleaned and sanitized, and water should be changed more frequently. Vectors should be excluded from animal facilities. ix. Prevention. Incoming animals should be quarantined and evaluated for presence of disease and/or pathogenic organisms. Food items and aquarium plants should be treated before introduction (short salt bath followed by thorough rinsing and 1-2 hr acriflavin bath). Whenever possible, purchase colonyreared animals and food items from reliable sources. x. Research complications. Subclinical infections of hemoparasites can confound hematologic and physiologic data. Overt protozoal disease can decrease research populations and render data questionable. b.

Nematodes

i. Etiology. The three most commonly described nematodes of amphibians are Pseudocapillaroides xenopi, Rhabdias, and Foleyella. Pseudocapillaroides is a major parasite of Xenopus laevis; Rhabdias affects both frogs and salamanders; and Foleyella has been described in frogs (Crawshaw, 1993).

ii. Epizootiology and transmission. Pseudocapillaroides is contracted when the eggs are ingested along with sloughed skin from a host frog. The life cycle is direct, with the nematode living in the epidermis of Xenopus and shedding its eggs directly into the aquatic environment. Rhabdias larvae penetrate the host frog's skin and migrate to the lungs. Eggs are coughed up and swallowed; thus, eggs and larvae are found in the gastrointestinal tract. Larval Foleyella are found in the blood; adults live in the body cavity and lymph spaces. iii. Pathogenesis. Pseudocapillaroides xenopi burrows into the epidermis, causing desquamation, debilitation, and secondary infection. Larval nematodes can be found in the kidney. Rhabdias causes damage to pulmonary tissue, and Foleyella can cause debilitation. iv. Clinical signs. Xenopus affected with Pseudocapillaroides have a rough, thickened, pitted appearance to the skin on their dorsal surface, and large patches slough (Fig. 18). Burrows and parasites can sometimes be seen in the epidermis. Debilitation and invasion by opportunistic bacteria and fungi can follow. Pneumonia may be observed in frogs with heavy Rhabdias infection. Foleyella may be asymptomatic or cause weakness and general malaise (Stephens et al., 1987; Crawshaw, 1993; Wright, 1996). v. Necropsy findings. Pseudocapillaroides xenopi lesions are usually confined to the skin and consist of hyperkeratosis, vacuolation, and a mixed inflammatory cell infiltrate. Severe cases involve epithelial erosion and ulceration. Nematodes are present in the lesions. In severe cases, visceral evidence of septicemia may be present (Ruble et al., 1995). Larval nematodes are sometimes found in Bowman's spaces and wrapped around glomerular tufts in the kidneys (Brayton, 1992). vi. Differential diagnoses. Pseudocapillaroides can be diagnosed by wet-mount preparations of desquamated skin or skin scraping. Bipolar eggs and adults will be detectable with these methods. Rhabdias larvated eggs and larvae can be found by fecal examination. Eggs can also be found in tracheal washes. Foleyella can be demonstrated in fresh blood smears or in Wright-Giemsa-stained samples. vii. Treatment. Pseudocapillaroides was initially treated with thiabendazole, but efficacy problems coupled with reports of adverse reactions have resulted in reduction of its use (Ruble et al., 1995; Iglauer et al., 1997). Ivermectin (0.2 mg/kg into the dorsal lymph sac or IM; repeat in 14 days) has proven effective (Dawson et al., 1992; Wright, 1999a). Levamisole has also been suggested as a treatment for Pseudocapillaroides (Cunningham et al., 1996b; Iglauer et al., 1997). Iglauer et al., (1997) recommends levamisole (12 mg/liter water, with each frog having access to 4.17-6.25 liters of treated water for a minimum of

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Fig. 18. Pseudocapillaroidesxenopi causes epidermalthickening and sloughing.The Xenopus pictured here had concomitantbacterial septicemia. 4 days; treatment repeated in 10-14 days). Wright (1999a) recommends levamisole (8-10 mg/kg IM or intracoelomically q14-21 days or 100-300 mg/liter bath for 24 hr q7-14 days); however, he warns of toxicity problems (flaccid paralysis) at more prolonged exposures. Rhabdias and other nematodes can be treated with ivermectin (2.0 mg/kg topically q14 days, or 0.2-0.4 mg/kg IM or PO q14 days) (Letcher and Glade, 1992; Crawshaw, 1993; Wright, 1999a). Fenbendazole can also be used to treat nematodes (100 mg/kg PO q10-21 days or 50 mg/kg PO SID for 3 - 5 days; repeat in 14-21 days (Wright, 1999a).

viii. Control. Isolation and treatment of affected animals, sanitation of environment, and elimination of vectors will help control nematodes in amphibians. ix. Prevention. zoal diseases.

Prevention can be carried out as for proto-

x. Research complications. Nematode-infested animals can be unthrifty to clinically ill, and therefore poor research subjects. c.

Trematodes and Cestodes

i. Etiology. Amphibians can serve as hosts to both trematodes and cestodes. Polystoma and Gyrodactylus are common trematodes of amphibians, and Nematotaenia is a frequently encountered cestode. Recently, cercariae of the trematode Ribeiroia have been associated with limb abnormalities in Pacific tree frogs (Johnson et aL, 1999).

ii. Epizootiology and transmission. Polystoma is found in the bladder of frogs, and Gyrodactylus is found on the skin and gills of aquatic species of amphibians. Nematotaenia is found in the gastrointestinal tract of amphibians. iii. Pathogenesis. Trematode and cestode infestations may be subclinical. High numbers of cestodes may cause mechanical obstruction of the gastrointestinal tract or wasting and debilitation of the amphibian. iv. Clinical signs and necropsy findings. Polystoma is typically asymptomatic. Gyrodactylus can cause debilitation, dyspnea, anemia, and ulceration of the skin. Nematotaenia can cause unthriftiness and gastrointestinal obstruction. v. Differential diagnoses. Polystoma can be detected by urinalysis. Gyrodactylus requires skin scraping and gill biopsy, and Nematotaenia can be detected by fecal examination (Crawshaw, 1993; Whitaker, 1999; Wright, 1999a). vi. Treatment. Praziquantel (8-24 mg/kg PO, SC, or intracoelomically q14-21 days or 10 mg/liter bath for up to 3 hr; repeat in 14-21 days) has been used to treat trematodes and cestodes in amphibians (Wright, 1996; Wright, 1999a). vii. Control and prevention. Trematodes and cestodes can be controlled and prevented in the same manner as other parasites. viii. Research complications. Debilitated animals make inappropriate research subjects, and subclinical infestations may confound data.

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d.


Other

i. Etiology. Acanthocephalans, copepods, leeches, trombiculid mites, and toad flies are examples of other types of parasites that may infest amphibians. ii. Epizootiology and transmission. Acanthocephalans have an indirect life cycle and require an arthropod host. Copepods are seen in aquatic amphibians, and leeches may be found on wild-caught animals. Trombiculid mites are found in soil and leaf litter, and parasitize terrestrial species. Toad flies infest terrestrial anurans. iii. Pathogenesis. Acanthocephalans inhabit the gastrointestinal tract; the other parasites are external. Toad flies lay eggs in the nasal cavity of frogs; larvae eat the nasal passages and the frog's face until the frog dies. iv. Clinical signs and necropsy findings. Acanthocephalan infections may be subclinical; however, weight loss and enteritis can be seen. If the intestinal wall is perforated, peritonitis will result. Leeches and copepods are visible externally. Trombiculid mites can cause erythematous vesicles on the skin of affected amphibians. Toad fly larvae can be seen in the nasal passages of affected frogs. v. Differential diagnoses. Acanthocephalans are detected by fecal examination. Copepods can be detected on skin scrapings. Leeches, mites, and flies are readily visible. vi. Treatment. Salt baths (10-25 gm salt/liter for 15-30 min) can be used to remove copepods and facilitate removal of leeches. Topical ivermectin may be effective in treating trombiculid mites. Treatment for toad flies and acanthocephalans is generally unrewarding (Crawshaw, 1993; Raphael, 1993; Wright, 1996; Whitaker, 1999). vii. Control and prevention. Excluding vectors and intermediate hosts is effective in controlling toad flies and acanthocephalans. Avoiding wild-caught animals will reduce problems with leeches and copepods, and heat-treating or freezing leaf litter, soil, and other cage accouterments will eliminate trombiculid mites. viii. Research complications. As in all parasitic infestations, compromised research animals are poor subjects and yield questionable data. 4.

Fungal

i. Etiology. Most fungi that affect amphibians are soil and water saprophytes; infection commonly occurs secondary to stress or disease. The fungal infections most frequently iden-

tiffed in amphibians are saprolegniasis, chromomycosis, and phycomycosis.

ii. Epizootiology and transmission. Saprolegniasis is caused by several fungi, including Saprolegnia. Various pigmented fungi cause chromomycosis, and Basidiobolus is the agent most commonly isolated from cases of phycomycosis. iii. Pathogenesis and clinical signs. Saprolegnia colonizes preexisting skin lesions in aquatic amphibians. A cottony mat of fungal hyphae cover the lesion. Paler tufts are indicative of acute infections, while darker mats indicate chronicity (Crawshaw, 1993; Wright, 1996). Lesions of chromomycosis are usually raised dark nodules; however, they may be ulcerated (Ackermann and Miller, 1992; Wright, 1996). Debilitation and weight loss may also be observed. Phycomycosis produces lesions similar to those of chromomycosis (Wright, 1996). iv. Necropsy findings. Lesions tend to remain cutaneous in saprolegniasis, while visceral granulomas can be seen in chromomycosis. v. Differential diagnoses. mounts and fungal cultures.

Diagnosis can be made by wet

vi. Treatment. Saprolegniasis can be treated with saltwater baths (10-25 gm/liter for 5 - 3 0 min SID) or benzalkonium chloride (2 mg/liter bath for 10-60 min) (Wright, 1999a). Groff et al. (1991) successfully treated Basidiobolus ranarum with benzalkonium chloride (2 mg/liter bath for 30 min every other day for 3 treatments; repeat in 8 days). Several treatments for chromomycosis have been tried; results are unrewarding (Ackermann and Miller, 1992; Wright, 1996). vii. Control and prevention. Fungal infections can be minimized by keeping animals healthy and unstressed in a clean environment. viii. Research complications. Saprophytic fungi can colonize surgical wounds and other skin lesions, compromising the health of the research animals.

B.

Metabolic/Nutritional Diseases

Amphibians are susceptible to several metabolic and nutritional diseases, including metabolic bone disease, lipid keratopathy, spindly leg, gas bubble disease, and dehydration. Metabolic bone disease (MBD) is seen in both adult and larval amphibians that have been fed diets deficient in calcium or with an improper calcium-phosphorus ratio. Tadpoles require significant amounts of calcium in their diet. Animals deficient in calcium will mobilize the mineral from bones in order to keep

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serum calcium levels normal. Signs of MBD in tadpoles include folding fractures, scoliosis, and "rubber jaw" (inordinate mandibular flexibility due to calcium loss). Adult frogs exhibit abdominal bloating and tetany following exertional movement. Diagnosis of MBD is based on clinical signs and radiographs (mammography film works well when radiographing small amphibians). Radiographic changes include decreased bone density and pathologic fractures. Treatment of MBD consists of appropriate calcium supplementation (daily 5% calcium gluconate baths, injectable vitamin D/calcium, oral supplementation with tropical fish food slurry), and change to an appropriate diet. The disease can be prevented by feeding larval and adult amphibians appropriate diets (Crawshaw, 1993; Wright, 1996). Lipid keratopathy is seen in female frogs. Affected animals have corneal thickening and opacity, with vascularization, superficial pigmentation, and cholesterol clefts. In some cases, xanthomatosis is associated with corneal changes. Possible etiologies include lipid and cholesterol mobilization associated with egg production, and high levels of dietary fat (from feeding with milk-fed newborn mice) (Williams and Whitaker, 1994). Spindly leg is seen in young frogs, particularly poison dart frogs. Limbs develop abnormally, do not emerge properly at metamorphosis, are thin and poorly muscled, and have angular deformities. Etiology is unknown; theories include genetics, temperature, water quality, oversupplementation of vitamins, and malnutrition. The condition is untreatable, and euthanasia is recommended (Crawshaw, 1993; Wright, 1996). Gas bubble disease is produced by air supersaturation of water. Large amounts of gas accumulate in the vascular system, causing obstruction of blood flow and capillary hemorrhage. Air bubbles are evident in webbing of feet and skin, and permit entry of bacteria, resulting in septicemia. The disease can be prevented by ensuring that water is not supersaturated with air (Colt et al., 1984; Crawshaw, 1993; Raphael, 1993; Wright, 1996). Amphibians require moist environments and are predisposed to dehydration. Signs of dehydration include dull, dark skin, sunken eyes, lethargy, and dry, sticky mucus. Mild dehydration can be treated by immersion in clean, dechlorinated water. Animals in shock can be given dexamethasone (1-2 mg/kg intracoelomically) and hypotonic fluids. Two parts saline to 1 part 5% dextrose is given intracoelomically at 2 - 5 % of the animal's body weight. Subsequent fluid solutions should be 9 parts saline to 1 part sterile water. Antibiotic baths are recommended if epidermis is damaged (Wright, 1999b).

C.

Traumatic Disorders

Traumatic lesions in amphibians are primarily bite wounds caused by cagemates, and abrasions from rough surfaces and

cage tops. Appropriate wound closure (if warranted) and antibiotic therapy are indicated (Wright, 1994). Aggressive animals should be separated, and cages should be free of abrasive surfaces.

D.

Toxins

Amphibians are exquisitely sensitive to a number of toxins, including chlorine and chloramine, nitrite, ammonia, iodine, heavy metals (copper, lead, zinc), PVC adhesives, and pesticides (Whitaker, 1993; Stansley and Roscoe, 1996). Signs associated with toxicities include excess mucus production, irritability, dyspnea, convulsions, paralysis, petechiation, and regurgitation (Wright, 1996). Animals displaying acute signs of toxicity should be removed immediately from their environment and placed in a clean, toxin-free enclosure.

E.

Neoplasms

With the exception of Lucke's renal adenocarcinoma, spontaneous neoplasms are relatively sporadic in amphibians. Ovarian and hepatic tumors are fairly common. Spontaneous tumors are reported more frequently in frogs, and frogs appear more sensitive than salamanders to carcinogen-induced tumors (Anver, 1992). Examples of spontaneous amphibian tumors include skin adenomas, papillomas, fibromas, pulmonary carcinomas, and testicular tumors (Balls and Clothier, 1974).

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Taylor, E R., Simmonds, R. C., and Loeffler, D. G. (1993). Isolation of Flavobacterium meningosepticum in a colony of leopard frogs (Rana pipiens). Lab. Anim. Sci. 43, 105. Teare, J. A., Wallace, R. S., and Bush, M. B. (1991). Pharmacology of gentamicin in the leopard frog (Rana pipiens). Proc. Am. Assoc. Zoo Vet. 128129. Terril-Robb, L. A., Suckow, M. A., and Grigdesby, C. F. (1996). Evaluation of the analgesic effects of butorphanol tartrate, xylazine hydrochloride, and flunixin meglumine in leopard frogs (Rana pipiens). Contemp. Top. Lab. Anim. Sci. 35, 54-56. Tinsley, R. C. (1995). Parasitic disease in amphibians: Control by the regulation of worm burdens. Parasitology 111, S153-S178. Tinsley, R. C., Loumont, C., and Kobel, H. R. (1996). Geographical distribution and ecology. In "The Biology of Xenopus" (R. C. Tinsley and H. R. Kobel, eds.), pp. 35-59. Clarendon Press, Oxford. Verhoeff-de Fremery, R., and Griffin, J. (1987). Anurans (frogs and toads). In "The UFAW Handbook on the Care and Management of Laboratory Animals" (T. B. Poole, ed.), 6th ed., pp. 773-783. Churchill Livingstone, New York. Verhoeff-de Fremery, R., Griffin, J., and Macgregor, H. C. (1987). Urodeles (newts and salamanders). In "The UFAW Handbook on the Care and Management of Laboratory Animals" (T. B. Poole, ed.), 6th ed., pp. 759-772. Churchill Livingstone, New York. Walker, I. D. E, and Whitaker, B. R. (2000). Amphibian therapeutics. In "The Veterinary Clinics of North America: Exotic Animal Practice" (S. P. A. Fronefield, ed.), Vol. 3, pp. 239-255. Saunders, Philadelphia. Whitaker, B. R. (1993). The use of polyvinyl chloride glues and their potential toxicity to amphibians. Proc. Am. Assoc. Zoo Vet. 16-18. Whitaker, B. R. (1999). Parasitic problems of amphibians. Proc. North Am. Vet. Conf., 801-803. Wilcke, B. W., Jr., Newcomer, C. E., Anver, M. R., Simmons, J. L., and Nace, G. W. (1983). Isolation of Chlamydia psittaci from naturally infected African clawed frogs (Xenopus laevis). Infect. Immun. 41, 789-794. Williams, J. H. (1997). Contractile apparatus and sarcoplasmic reticulum function: Effects of fatigue, recovery, and elevated calcium. J. Appl. Physiol. 83, 444-450. Williams, D. L., and Whitaker, B. R. (1994). The amphibian eye--a clinical review. J. Zoo Wild. Med. 25, 18-28. Williams, J. W. III, Tweedell, K. S., Sterling, D., Marshall, N., Christ, C. G., Carlson, D. L., and McKinnell, R. G. (1996). Oncogenic herpesvirus DNA absence in kidney cell lines established from the northern leopard frog Rana pipiens. Dis. Aquat. Org. 27, 1-4. Woodward, D. L., Khakhria, R., and Johnson, W. M. (1997). Human salmonellosis associated with exotic pets. J. Clin. Microbiol. 35, 2786-2790. Wright, K. (1994). Amputation of the tail of a two-toed amphiuma, Amphiuma means. Bull Assoc. Reptil. Amphib. Vet. 4, 5. Wright, K. (1999a). Common bacterial and fungal diseases of captive amphibians. Proc. North Am. Vet. Conf., 810-813. Wright, K. (1999b). Fluid therapy for amphibians. Proc. North Am. Vet. Conf., 814-816. Wright, K. M. (1996). Amphibian husbandry and medicine. In "Reptile Medicine and Surgery" (D. R. Mader, ed.), pp. 436-459. Saunders, Philadelphia. Wu, M., and Gerhart, J. (1991). Raising Xenopus in the laboratory. In "Methods in Cell Biology" (B. K. Kay and H. B. Peng, eds.), Vol. 36, pp. 3-18. Academic Press, San Diego. Wyman, R. L., and Hawksley-Lescault, D. S. (1987). Soil acidity affects distribution, behavior, and physiology of the salamander Plethodon cinereus. Ecology 68, 1819-1827. Zug, G. R. (1993). "Herpetology: An Introductory Biology of Amphibians and Reptiles." Academic Press, San Diego.

Chapter 18 Biology and Diseases of Reptiles Dorcas P. O'Rourke and Juergen Schumacher

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Taxonomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Use in Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Availabilityand Sources . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Laboratory Management and Husbandry . . . . . . . . . . . . . . . . . . . . . . II. Biology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Anatomy and Physiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Nutrition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Reproduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Behavior . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Physical Examination and Diagnostic Techniques . . . . . . . . . . . . . . . III. Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Infectious Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Metabolic/Nutritional Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Traumatic Disorders . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Toxins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Neoplastic Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

I.

INTRODUCTION

Reptiles are the first class of vertebrates to evolve an amniotic, shelled egg; therefore, they no longer require an aquatic e n v i r o n m e n t for reproduction. Furthermore, in contrast to superficial external appearance, reptiles are more closely related to endothermic birds than to ectothermic amphibians.

A.

Taxonomy

M e m b e r s of class Reptilia are derived from two lineages, Anapsida and Diapsida. Turtles are anapsids, unmistakable due LABORATORY ANIMAL MEDICINE, 2nd edition

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to the presence of a b o n y shell covering the b o d y Diapsids include the saurians (crocodilians and, according to m a n y taxonomists, birds) and the lepidosaurians (tuataras, lizards, and snakes) (Zug, 1993). Chelonians (turtles) are represented by over 240 species occurring worldwide. They are divided into two broad taxonomic groups based on m e t h o d of head retraction. Pleurodira or sideneck turtles withdraw their head and neck and fold it onto the shoulder. T w o families of pleurodires are found in freshwater in the Southern Hemisphere: Pelomedusidae from South America, Africa, Madagascar, and the Seychelles Islands; and the more advanced Chelidae from South America, Australia, and New Guinea. The most familiar of the chelids is Chelus fimbriatus, Copyright 2002, Elsevier Science (USA). All rights reserved. ISBN 0-12-263951-0

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DORCAS P. O'ROURKE AND JUERGEN SCHUMACHER

the matamata. This unusual turtle with fleshy head protuberances and pronounced keels on its shell inhabits the freshwater streams of South America (Ernst and Barbour, 1989; Zug, 1993). Cryptodira contains several families, all of which withdraw the neck into the shell in a vertical, S-shaped fashion. Cheloniidae and Dermochelyidae are the sea turtles; representatives of this group can weigh more than a ton. A second group of cryptodires includes the softshell turtles (Trionychidae) and the mud and musk turtles (Kinosternidae). Softshell turtles have a leatherlike shell that lacks typical horny scales. Mud and musk turtles are smaller, have a domed shell, and exude an unpleasant odor when disturbed. A third group of cryptodires, Chelydridae, is represented by the snapping turtle (Chelydra) and alligator snapping turtle (Macroclemys). Snapping turtles have large heads, long tails, and powerful jaws (Fig. 1). The final group of cryptodires contains the "pond" turtles (Emydidae) and the tortoises (Testudinidae). Representatives of this group include the North American sliders (Trachemys), painted turtles (Chrysemys), box turtles (Terrapene), and the European Hermann's tortoise (Testudo hermanni) (Ernst and Barbour, 1989; Zug, 1993). Crocodilians are medium to large, quadrupedal reptiles adapted to an aquatic habitat. The order Crocodylia comprises three families: Alligatoridae, Crocodylidae, and Gavialidae. Two species of alligators and 6 species of caimans make up Alligatoridae. Caiman crocodilus (common caiman) and Alligator mississippiensis (American alligator) have been most commonly used in research. The narrow-snouted gharial is the sole representative of Gavialidae. Crocodylidae is the largest family, containing 8 genera and 22 species that occur throughout the world in tropical regions (Ross and Magnusson, 1989; Zug, 1993). The remainder of reptiles are classified as lepidosaurs. Included in this group are the orders Sphenodontida and Squa-

mata. The tuatara, a unique, lizard-like reptile, is the sole representative of Sphenodontida; the only 2 species of tuatara are both found in New Zealand. On the other hand, squamates (lizards and snakes) are the largest group of reptiles, with over 5000 species occurring worldwide. Lizard families composing Squamata include Gekkonidae (geckos), Iguanidae (iguanas, anoles), Chamaeleonidae (chameleons), Helodermatidae (Gila monsters), Varanidae (monitors), Scincidae (skinks), and Teiidae (tegus) (Zug, 1993). Snake families commonly seen in research settings are Boidae (boa constrictors, anacondas), Pythonidae (reticulated pythons, Burmese pythons, ball pythons), Elapidae (coral snakes, sea snakes, cobras), Viperidae (rattlesnakes, copperheads, puff adders, Gaboon vipers), and Colubridae (king snakes, rat snakes, garter snakes, water snakes). Boas and pythons are large, primitive snakes; some species can exceed lengths of 20 feet. Vipers are a group of venomous snakes with large erectile fangs. Elapids have smaller, fixed fangs and venom that is primarily neurotoxic. Colubridae is the largest family, with 290 genera and approximately 1700 species. Most colubrids are nonvenomous; however, some venomous species, including the rearfanged brown tree snake (Boiga irregularis) are members of this family (Zug, 1993; Greene, 1997). A list of common and scientific names is presented in Table I.

B.

Use in R e s e a r c h

For many years, herpetologists have studied reptiles in the field and maintained captive populations in the research laboratory. Most of these investigations have focused on the natural history, behavior, and reproduction of animals in their native habitats. Specific questions that cannot be answered in a field

Fig. 1. The snappingturtle has powerfuljaws and can inflict a seriousbite.

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18. BIOLOGY AND DISEASES OF REPTILES

Table I Commonand Scientific Namesof Selected Reptile Species Common names

Scientific names

Green iguana Anole Broad-head skink Leopard gecko Red-eared slider Painted turtle Box turtle Hermann's tortoise Gopher tortoise Garter snake Banded water snake Rat snake Corn snake King snake Boa constrictor Ball python American alligator Common caiman

Iguana iguana Anolis carolinensis Eumeces laticeps Eublepharis macularius Trachemys scripta elegans Chrysemys picta Terrapene carolina Testudo hermanni Gopherus polyphemus Thamnophis sirtalis Nerodia fasciata Elaphe obsoleta Elaphe guttata Lampropeltis getula Boa constrictor Python regius Alligator mississippiensis Caiman crocodilus

situation are addressed by bringing specimens into the laboratory for more intensive study. Thus, a substantial amount of research involving reptiles is dedicated to understanding and conserving the species themselves. A number of reptiles have been used as animal models and for teaching purposes. Red-eared sliders (Trachemys scripta elegans) and painted turtles (Chrysemys picta) are commonly used to teach physiology and anatomy (Fig. 2). Turtles have also been used to investigate effects of microgravity on orientation (Mori, 1995). Lizards have been the subjects of numerous investigations into stress and behavior, and snakes are commonly

used for chemoreception and behavior studies (Greenberg et al., 1989). Snake venoms have been intensively studied and their various components identified and used in production of antivenoms, as therapeutic agents, and for development of models of disease, such as myoglobinuria (Ponraj and Gopalakrishnakone, 1996). Neuroanatomy and neurophysiology research has frequently used crocodilians as animal models. Use of alligators has resulted in development of artificial blood and perfection of transmyocardial perfusion techniques (Dyer, 1995; Kohmoto et al., 1997). Alligator mississippiensis has also been the subject of environmental studies, ranging from examination of effects of mutagens (Winston et al., 1991) to assessment of endocrine disruptors on reproduction (Crain and Guillette, 1998) (Fig. 3). C.

Availability and Sources

Acquisition of reptiles for research and teaching has traditionally involved capturing animals from the wild. With chronic overharvesting of species such as the red-eared slider (Trachemys scripta elegans), many wild populations are suffering severe declines. When choosing a reptile species for research or teaching, reputable breeders should be given preference whenever possible. Animals purchased from these individuals are generally healthier and better adapted to captivity. A list of vendors is available through the Institute for Laboratory Animal Research website; reptile hobbyist magazines also contain names of suppliers and products. Many reptiles are captured in the wild by investigators and brought back to the laboratory for study. Alternatively, a species may be manipulated (sometimes rather extensively) in its natural habitat. Federal, state, and local permits may be required for collection and field studies, depending on location and the species involved. Investigators should be aware of and abide by all regulations governing the reptile they are studying (Greene, 1995). D.

Laboratory Management and Husbandry

Reptiles represent a diverse group of animals with speciesspecific husbandry requirements. Several excellent texts (referred to throughout this chapter) are available that address anatomy, physiology, behavior, reproduction, and captive maintenance of various species. These texts should be consulted prior to acquiring a given species of reptile in the laboratory animal facility. 1. Primary Enclosures

Fig. 2. The painted turtle, Chrysemys picta, is frequently used in research.

Reptiles can be maintained in a variety of primary enclosures. Glass aquaria are the most commonly used type of housing. Aquaria are readily available, come in a variety of sizes, and are easily sanitized. The two major drawbacks to glass aquaria are

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Fig. 3.

Alligatorsare used for transmyocardialperfusion and environmentaltoxicologystudies.

that they are breakable and bulky. A workable alternative to glass aquaria is plastic shoe boxes and sweater boxes. Flexible plastic brands such as Rubbermaid withstand repeated cage washing and do not warp if lids are replaced immediately after removal from the cage washer. These cages are unbreakable, and with perforations for ventilation made in the sides, are stackable and occupy less space than aquaria. Several types of reptile housing are commercially available. Many of the hard

Fig. 4.

plastic cages do not withstand cage-washer temperatures. Fiberglass and Plexiglas housing units are also available commercially. These will withstand repeated cage washing, but the triangular-shaped fiberglass cages with sliding Plexiglas front entry take up more space than aquaria (Fig. 4). Additionally, approaching many reptiles directly from the front evokes more defensive responses than reaching in quietly from above. Stainless steel cages can be adapted for larger species of reptiles, and

Snakescan be maintainedin shoe-boxcages and fiberglasshousingunits.

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turtles and crocodilians can be housed in galvanized stainless steel or fiberglass tanks. With few exceptions, reptiles are escape artists and must be housed in cages with secure lids. Lids can be made of screen or a solid impervious material that is partially screened or otherwise ventilated. Lids should fit snugly, and for many species, should be secondarily secured with latches or weights. Many substrates can be used for reptiles. Newspaper or brown paper work very well and are fairly absorbent. Hardwood shavings and paper bedding are very absorbent, easy to spot-clean, and most important, allow the animals to burrow. Indooroutdoor carpet can be precut to fit cages, is easy to remove and sanitize, and works especially well for species such as garter snakes, which are very active and generate relatively large amounts of waste. Additionally, carpet offers traction for normal locomotion (Rossi, 1992). Hardwood chips are sometimes used but may inadvertently be ingested during feeding. Use of corncob bedding is strongly discouraged by many authors, because of accidental ingestion and development of gastrointestinal impaction. Corncob is also hygroscopic and will desiccate young animals. Likewise, kitty litter should never be used as a substrate (Page and Mautino, 1990; Anderson, 1991; Rossi, 1992; Boyer, 1991). Occasionally, a fastidious species may require a specific substrate such as sand, soil, sphagnum moss, or cypress mulch (Rossi, 1992) (Fig. 5). Sphagnum and cypress will inhibit growth of pathogens; sand and soil should be heattreated or washed prior to use. Most aquatic species do not require a substrate in the aquarium or tank; in fact, substrates can make cleaning more difficult (Boyer and Boyer, 1992). Terrestrial reptiles must be provided with a water bowl. Species that lap drops of water from leaves in the wild should be misted in addition to offered water in a container (Rossi, 1992).

Water bowls should be shallow enough to allow easy access (especially for terrestrial turtles), heavy enough to prevent tipping, and wide enough to accommodate all occupants of the cage simultaneously (Fig. 6). Reptiles spend time soaking, particularly prior to shedding, and should have access to fresh water at all times. In order to decrease stress and allow normal behavioral activity, reptile cages should contain certain accessories. Reptiles must have a hide box in which to retreat and feel secure (Page and Mautino, 1990; Anderson, 1991; Boyer, 1991; Rossi, 1992). Sanitizable, plastic hide boxes are available commercially in a variety of sizes. Polyvinyl chloride (PVC) pipe cut lengthwise in half works well, especially for larger species. Ceramic pots, heat-treated pieces of bark, and cardboard containers also make excellent refuges. Items that cannot be sanitized should be discarded when soiled (Fig. 7). Arboreal (treedwelling) reptiles should be provided with branches or dowels on which to climb. Species that bask should be provided with basking platforms. This is particularly important for many aquatic reptiles. A haul-out area or platform, easily accessible from the water and large enough to comfortably hold all tank occupants, must be provided to aquatic species in order to allow normal drying and behavioral thermoregulation (Boyer and Boyer, 1992). 2.

Water Quality

Aquatic reptiles are more tolerant than amphibians of chlorinated water; therefore, dechlorination of aquarium water is not necessary. In fact, chlorine may help retard growth of pathogens in the aquatic environment. When aquatic turtles are being housed, water should be at least as deep as the width of the

Fig. 5. Speciesof crawfish snake, Regina, require speciallyprepared tannin water and a cypress mulch basking spot.

832


shell; this will allow an overturned animal to right itself and not drown (Boyer and Boyer, 1992). Turtles and crocodilians generate large amounts of waste; therefore, static systems with frequent complete water changes or flow-through systems are preferred over recirculating systems. If recirculating systems are used, water quality should be monitored on a routine basis. 3.

Temperature

Reptiles tend to prefer warmer temperatures than do amphibians. Warm temperatures are necessary for normal physiologic processes, such as digestion, growth, reproduction, and immune function (Kanui et al., 1991; Dickinson and Fa, 1997). Each reptile species has a preferred optimal temperature range at which it should be maintained (Mattison, 1998). Examples of these ranges are provided in Table II. A thermal gradient should be provided within the cage. Low-wattage incandescent bulbs focused over a basking area can provide a site for behavioral thermoregulation (Fig. 8). Heat pads or strips placed under part of the cage will result in a temperature gradient. Aquarium heaters will increase water temperature for aquatic species, and a haul-out basking site will serve as the warm spot. "Hot rocks" (electrically heated basking surfaces that directly contact the animal) should be avoided because thermal burns have been reported with use of these devices (Boyer, 1991; Barten, 1996a). Reptiles should never be allowed to come into direct contact with any heat source; life-threatening thermal burns can result. 4.

Fig. 6. Waterbowls should be large enough for reptiles to soak in.

Lighting

Many reptile species need exposure to ultraviolet light in the appropriate UVB spectrum (290-320 nm) in order to endogenously manufacture vitamin D 3 (Gehrmann, 1996). Several species of lizards and turtles will develop metabolic bone disease if

Table II

Preferred Temperatureand Humidity for Selected Reptilian Speciesa Species Iguana iguana Anolis carolinensis Eublepharis macularius Boa constrictor Elaphe sp. Lampropeltis sp. Thamnophis sirtalis Testudo sp. Terrapene carolina Trachemys scripta Fig. 7. All reptiles should be provided with a hide box or other retreat,

a

From Divers (1996).

Temperature (~ 29-35 23-29 25-30 28-30 25-30 25-30 21-28 20-28 24 - 30 20-24

Humidity(%) 60-85 70-80 20-30 50-80 30-70 30-70 50-80 30-50 50 - 80 60- 90

18.

BIOLOGY AND DISEASES OF REPTILES

Fig. 8.

833

Baskinglights shouldbe providedfor manyreptile species.

deprived of ultraviolet light (Harcourt-Brown, 1996), particularly if an inappropriate diet (low in calcium or improper calcium-phosphorus ratio) is being fed. The ultraviolet light source should be placed 18-24 inches above the reptile and should not pass through plastic or glass (this will absorb the UV radiation) (Fig. 9). Examples of UV lights include Vitalites (Duro-Test), Chroma-50 (General Electric), and Colortone 50 (Philips). These may also be used in combination with a

Fig. 9.

black light (Boyer, 1991; Boyer and Boyer, 1992; Boyer, 1992); however, prolonged exposure to black light can result in ocular problems (Anderson, 1991; Fletcher, 1994; Divers, 1996). Artificial ultraviolet light sources should be replaced approximately every 6 months (Divers, 1996). Some species do not appear to require UV light if a proper diet is fed (Henkel and Schmidt, 1995). The biology, natural history, and nutritional requirements of a given species should be carefully

Manyreptiles require ultravioletlights to manufactureendogenousvitaminD 3.


834

researched when deciding on whether or not to supplement with UV light. Most reptiles do well with a 12 hr light-12 hr dark cycle. Breeding animals should have their light cycles adjusted accordingly.

5.

Airflow and Humidity

Many reptiles are terrestrial (ground-dwelling) or fossorial (burrowing); consequently, they require lower ventilation rates than mammals or birds. In fact, airflows appropriate for mammals can easily desiccate small reptiles. Most reptiles (with the exception of some desert-dwelling species) do well in a relative humidity range of 30-70%. Humidity requirements for selected species are listed in Table II. Low relative humidity can result in dysecdysis (difficult shedding), and humidity that is too high can result in "blister disease" (Rossi, 1996).

6.

Secondary Enclosures

Conventional animal rooms can be adapted for housing reptiles. Temperature and light cycles should be controlled for each room. Aquatic species can be kept in rooms with floor drains; tanks can be plumbed using PVC pipes to drain directly into floor drains. Sinks in animal rooms facilitate flushing and refilling aquatic housing units.

7.

Sanitation

Whenever possible, reptile cages should be sanitized in a cage washer. If this is not feasible, cages and tanks can be cleaned with quaternary ammonium compounds or a dilute sodium hypochlorite solution. A 3% sodium hypochlorite solution (1 part commercial bleach and 29 parts water) works well for sanitizing cages, hide boxes, water bowls, indoor-outdoor carpet, and equipment. Objects should be thoroughly rinsed and dried before returning to cages. Phenolic and cresolic compounds are very toxic to reptiles and must never be used around these animals, even to clean secondary enclosures (Page and Mautino, 1990; Anderson, 1991; Divers, 1996). Sanitation frequency of terrestrial reptile primary enclosures remains somewhat controversial. Although reptiles produce less waste than mammals, infrequent cleaning can result in buildup of pathogens such as Salmonella. Some individuals advocate prolonged intervals between cleaning, based on research demonstrating increased exploratory behavior (and presumably stress) following removal of feces and odor during cage cleaning (Conant, 1971; Pough, 1991). Other authors, however, have documented that the increased activity associated with being

subjected to a clean environment or other novel stimulus can result in positive behaviors (Huff, 1980; Radcliffe and Murphy, 1983). Chiszar et al. (1980) clearly demonstrated that the stimulus for increased exploratory behavior was the presence of a clean cage rather than the handling associated with the cleaning event, However, no information is currently available correlating this activity with acute or chronic stress in snakes (Chiszar et al., 1995). Until further studies clarify this issue, the most logical approach is to sanitize at intervals frequent enough to prevent pathogen buildup, while spot-cleaning as necessary between sanitizing. In general, changing cages at 1- to 2-week intervals works well for most species. Cages for garter snakes and other reptiles that generate large amounts of waste may need to be cleaned weekly. Many reptile cages and tanks are washed and sanitized by hand. Cleaning effectiveness should be monitored on a routine basis; cultures are one method of monitoring (Fig. 10). 8.

Handling

When handling any species of reptile, it is important to support the animal's body as much as possible. Reptiles should not be picked up or restrained by the tail. Many species of lizards have tail autotomy, and attempted tail restraint invariably ends in the appendage breaking off and the animal escaping. Lizards should have both the pectoral and pelvic girdles supported, with the tail gently held to prevent slapping. Very aggressive individuals may have to be restrained behind the head to prevent defensive biting. Lizards typically bite and hold on, and even relatively small animals can inflict a painful wound. Many lizards also have long, sharp claws and can scratch the handler. Small crocodilians can be held much the same as lizards, with more attention focused on head and tail restraint (crocodilians can administer a very powerful slap with the tail) (Fig. 11). Crocodilians may also roll when being held, in an effort to escape, and rough scales with dermal bones on the dorsal surface of the crocodilian can abrade hands. Large crocodilians should never be handled by one person alone. Tape can be wrapped around the animal's snout (taking care to avoid the nares) to prevent biting. Crocodilians have powerful jaws for crushing prey, yet relatively weak muscles for opening the mouth; therefore, muzzles work well. Many turtles can be restrained by holding the sides of the shell. However, species such as snapping turtles, softshell turtles, and mud and musk turtles have exceptionally long necks and can reach around to bite. These animals should be held by the back of the shell, taking care to avoid being scratched by claws on the hindfeet. When holding snakes, as much of the animal's body as possible should be supported. Many snakes are more comfortable if allowed to move about in the restrainer's hands. Snakes should never be held behind the head unless absolutely necessary. Grabbing a snake too tightly behind the head can damage tissues, restrict breathing, and elicit a much more


835

Fig. 10. Effectivenessof cage cleaning shouldbe monitored, particularly in cages that are washed by hand.

panicked escape response. Many snakes are quite docile if approached quietly and restrained gently (Fig. 12). 9.

Identification

Reptiles can be identified in a variety of ways. Shell notching, tail notching, and scale clipping have been used to identify turtles, crocodilians, and snakes, respectively. If the process of notching or clipping includes cutting dermal bone or extends into dermal tissues, anesthesia, analgesia, and appropriate asep-

tic technique should be used. Toe clipping is another means of marking that is controversial. Removal of too many toes will result in impaired locomotion; this can severely impact feeding, climbing, and other necessary survival functions. Anesthesia should be used for this painful procedure unless it clearly threatens the postrelease survival of the animals. Appropriate aseptic technique should be used, to prevent postamputation infections. Species with large, well-vascularized digits should not be subjected to toe amputation. Reptiles with skin pigmentation, such as some box turtles,

Fig. 11. Limbedreptiles shouldbe held by supportingboth the pectoral and pelvic girdles.


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Fig. 12. Snakesshouldbe held with as much of the body supportedas possible and not restrained behind the head.

snakes, and lizards, can be identified based on skin color and pattern description. Implantable transponders offer permanent identification with minimal impact on the animal. Temporary identification methods include toe web tags in young crocodilians (these normally last for a few months) and nontoxic dyes. One of the reptile facilities in the University of Tennessee program records the animal's number in permanent ink on small pieces of yellow electrical tape and then affixes the tape to the head of the snake (Layne, personal communication). The tape is shed with the animal's skin, and a new piece is replaced immediately after shedding. 10.

Quarantine

All new reptiles entering a facility should be quarantined. Many animals purchased from vendors are in fact wild-caught, and depending on the practices of the holding facility, could be more severely stressed and diseased than animals collected directly from the wild. Animals that are colony-bred are normally healthier; however, few reptiles arrive with complete health reports. Therefore, it is best to quarantine all animals prior to introduction into an existing colony. Jacobson (1993b) recommends a 90-day quarantine period for snakes. On arrival, all animals should be weighed and receive physical examinations and appropriate diagnostic tests. External and internal parasites should be tested for and treated. Infectious disease should be diagnosed and treated. Feeding records should be kept, and any relevant observations should be recorded. A reptile with clinical signs of disease should never be introduced into an existing collection. 11.

Zoonoses

Salmonellosis is the zoonotic disease most frequently associated with reptiles (Mermin et al., 1997; Austin and Wilkins, 1998). Turtles, snakes, and lizards can carry the organism in their gastrointestinal tracts, and shed only when stressed (Chio-

dini and Sundberg, 1981; Austin and Wilkins, 1998). Transmission to humans is through the fecal-oral route, and indirect transmission through handling contaminated objects is common. Signs of salmonellosis in humans include fever, vomiting, cramps, and diarrhea. Individuals who are immunosuppressed, elderly, very young (less than 1 year old), or taking medication to increase gastric pH are at greater risk of infection. The disease can progress and cause dehydration, meningitis, osteomyelitis, and sepsis (Anonymous, 1992, 1995; Austin and Wilkins, 1998). Treatment of reptiles to eliminate the carrier state has not been successful and can result in antibiotic-resistant strains of the organism (D'Aoust et al., 1990). Immunocompromised individuals, pregnant women, and children under 5 years of age should avoid contact with reptiles (Anonymous, 1992, 1995). A set of dedicated cleaning equipment and supplies should be used for reptiles, and hands must be washed thoroughly after contacting reptiles or the equipment (Austin and Wilkins, 1998). Aquatic species of reptiles have been implicated in cases of atypical mycobacteriosis (Mycobacterium chelonae) and Edwardsiella tarda infections. Although these infections are rare, the organisms may pose a health hazard, particularly for immunocompromised individuals or people with underlying disease (Miller et al., 1990; Darrow et al., 1993). Appropriate precautions, such as avoiding direct contact with contaminated water (particularly if fingers and hands have preexisting injuries), wearing gloves, and washing hands thoroughly after handling aquatic reptiles or their caging and equipment, should help prevent disease transmission. 12.

Venomous Species

Numerous snake species and two species of lizards are venomous. Venoms have been broadly classified as hemotoxic and neurotoxic; however, most are complex mixtures of digestive enzymes and have varying effects on tissues and organs. For

18. BIOLOGYAND DISEASES OF REPTILES this reason, it is essential to be thoroughly familiar with the biology, behavior, and toxicity of any venomous species housed in the laboratory animal facility. Essential considerations for management of venomous reptiles include housing in unbreakable, locked cages; provision of equipment such as snake hooks, long forceps, large plastic garbage cans, and acrylic tubes for handling, feeding, and restraint; protocols for routine cage changing, animal handling, and accidental envenomation; and thorough training of all personnel involved with these species. Special caging can be designed for highly aggressive or specialized species (Mason et al., 1991; Greene, 1997). One of the reptile facilities at the University of Tennessee houses and studies venomous snakes. The animals are housed in specially constructed, locked cages. Two trained individuals must be present when any venomous snake is handled. A detailed protocol for accidental envenomation is prominently posted on the animal room door (which is kept locked). Antivenom is kept on hand for transport to the hospital with the victim in the event of a bite. Procedures will vary among species, depending on relative toxicity and behavior of the venomous animal being housed.

II.

A.

BIOLOGY


1. Integumentary System Reptiles are covered primarily by scales, which are made of [3-keratin. The elastic form found in most vertebrates, a-keratin,

837

covers the skin between scales that do not overlap. Softshell and leatherback sea turtles have cx-keratin covering their shells, and snakes and lizards have an epidermal structure with a-keratin on the inside and [3-keratin on the outside (Zug, 1993). In crocodilians and turtles, epidermal growth is continuous, as is the shedding of pieces of skin. In contrast, lizards and snakes have a synchronized pattern of ecdysis (shedding). Germinal cells undergo a resting phase prior to synchronously beginning to divide. A second epithelial layer is formed beneath the original; this renewal phase takes 1-2 weeks. During this time, the skin is dull and the eyes appear cloudy (opaque) (Fig. 13). Lymph then diffuses between the layers and enzymatically cleaves them. The skin and eye opacities resolve, and shedding usually occurs 3 - 4 days following this clearing (Zug, 1993; Rossi, 1996). Crocodilians, some lizards, and turtles have osteoderms (bony plates in the dermis); these are usually found on the dorsal and lateral surfaces of the animals. Skin glands are present and have a variety of functions. Many turtles have musk glands in the inguinal and axillary regions. Male tortoises and both sexes of crocodilians have glands in the mandibular area; crocodilians also have cloacal glands. A linear array of secretory pores is evident on the inside of the thighs in male lizards of certain species. Snakes and some lizards have paired scent glands that empty through the cloaca and seem to function in defense and sexual recognition. Salt glands are found in a variety of locations (tongue, orbit, nasal passage) in several species of marine or desert reptiles (sea snakes, crocodiles, lizards, turtles). These glands excrete excess salt, because reptiles cannot concentrate urine above blood osmolality (Jacobson, 1984; Zug, 1993).

Fig. 13. The ocular scales of the snake become cloudy (opaque)prior to shedding(arrow).

838 2.

DORCAS P. O'ROURKE AND JUERGEN SCHUMACHER Musculoskeletal System

Reptiles have undergone a variety of musculoskeletal adaptations during evolution. Among the most notable is the bony and muscular arrangement that allows crocodilians to close their mouths with incredible crushing force. Another is the refinement of cranial bones in snakes that enables them to ingest large prey items. Flexible ligaments, rather than a mandibular symphysis, connect the independently moving mandibles. Each mandible also has a joint in the middle, which permits outward bowing and expansion. Additionally, the mandibles are independently attached to outward-slanted, free-swinging quadrate bones (Greene, 1997). These adaptations allow snakes to swallow prey items much larger than their own heads. Primitive snakes such as boas and pythons have remnants of pelvic girdles, which are visible radiographically. Boas and pythons also have spurs, clawlike hindlimb remnants, which are used during courtship. The vertebrae and ribs of turtles are fused to a bony shell. The upper part is referred to as the carapace, and the lower part of the shell is called the plastron. Box turtles have a hinge in the front part of the plastron, which closes to seal off access to the body when the legs and head are retracted.

valve, a fold of tissue at the caudal aspect of the tongue, which meets the palate to form a watertight seal and permits these animals to hold prey in their mouths while submerged (Schaeffer, 1997). The primary respiratory organ in reptiles is the lung. Most reptiles have paired lungs; many snakes have an elongate right lung and either no or a small vestigal left lung. The lungs of snakes and many lizards are saclike structures that may end in rather extensive air sacs (Fig. 15). Turtles and crocodilians have compartmentalized lungs. In turtles, movement of the head and limbs causes air to be forced in and out of the lungs. Crocodilians have a muscular septum that functions like a mammalian diaphragm (Stoakes, 1992; Zug, 1993). Many reptiles, particularly aquatic species, are tolerant of anoxia and can go for prolonged periods without breathing (Zug, 1993; Wasser et al., 1997; Hicks and Wang, 1998). This physiologic adaptation can significantly impact methods of anesthesia and euthanasia for these species. Most reptiles do not routinely vocalize. Obvious exceptions are crocodilians, some species of turtles and lizards, and the snake Pituophis. Pituophis is the first snake described to have a functional vocal cord (Young et al., 1995). 4.

3.

Respiratory System

Compared to that of mammals, the glottis of reptiles is easy to visualize and access (Fig. 14). It lies in the cranial part of the mouth in snakes and many lizards, which enables them to hold large prey items and still respire, and is behind the base of the tongue in turtles and crocodilians. Crocodilians have a basihyal

Cardiovascular System

With the exception of crocodilians, all reptiles have a threechambered heart, consisting of paired atria and a single ventricle. Reptiles also have paired aortas (Fig. 16). Crocodilians have essentially a four-chambered heart, with the foramen of Panizza being the sole intraventricular connection. The functioning of this septum, along with paired aortas arising from

Fig. 14. The glottis of snakes and otherreptiles is easyto visualize.


839

Fig. 15. The snake lung is elongate and ends in an air sac.

opposite separate ventricles, results in shunting of blood to cephalic and coronary circulation during anoxic events such as diving (Axelsson et al., 1991; Zug, 1993; Axelsson et al., 1996). Lizards have a midventral abdominal vein that lies just inside the abdominal wall. This vein should be avoided when making surgical incisions (Barten, 1996b). 5.

Gastrointestinal System

Snakes and a few lizard species demonstrate remarkable adaptation of the salivary glands. In these species, digestive enzymes have been modified into venoms. Vipers, elapids, and some other snakes have true venom glands, which secrete

venom through a duct to a single fang (generally fixed in elapids and erectile in viperids). Duvernoy's gland, composed of branched tubules, is present under the skin of the maxillary region, near the angle of the jaw. A duct connects Duvernoy's gland to a sometimes enlarged, grooved maxillary tooth. Duvernoy's gland is present in many species of colubrids (Greene, 1997). Venom glands are found in the lower jaw of Gila monsters and beaded lizards; these species chew to ensure envenomation. Crocodilians, snakes, and lizards have teeth; turtles have a horny beak that is used to bite off chunks of food. The esophagus of snakes is thin-walled and distensible, to accommodate large prey. Crocodilian stomachs are round,

Fig. 16. All reptiles except crocodilians have a three-chamberedheart, and all have paired aortas (arrows).

840


muscular, and thick-walled, and often contain gastroliths to aid in digestion (Lane, 1996). Crocodiles have a number of connective tissue septa that separate the body cavity into several separate components (Van Der Merwe and Kotze, 1993; Mushonga and Horowitz, 1996). Unlike that in mammals, the pancreas in snakes is discrete and compact (Moscona, 1990). The sequence of glucagon and insulin appearance and development of the alligator pancreas is identical to that of mammals and birds (Jackintell and Lance, 1994). The liver of snakes is elongate, in accordance with other anatomic features of this group (Fig. 17). Alligators have fibrous trabeculae that course through the liver (Beresford, 1993). Some turtle species have pigmented cell aggregations in the spleen and liver; these are macrophages, which increase in number as animals age (Christiansen et al., 1996). The small intestine and colon tend to have larger volumes in herbivorous species of reptiles (Jacobson, 1984; Barboza, 1995), as for other classes of vertebrates. In some snake species that eat infrequently, energy is conserved through atrophy of the small intestine and related organs between meals (Secor and Diamond, 1997). In all reptiles, products of the digestive, excretory, and reproductive tracts empty into the cloaca.

6.

Excretory System

Reptiles have paired, metanephric kidneys. Aquatic species tend to excrete ammonia, semiaquatic species excrete urea, and terrestrial species excrete primarily uric acid (Coulson and Hernandez, 1983; Jacobson, 1984). A renal portal system that drains venous blood from the caudal half of the reptile's body directly through the kidneys has been described. Recent evidence, however, suggests that at least in some species, venous blood may be diverted past the kidney directly to the liver in cer-

tain circumstances (Holz et al., 1997). Both situations should be considered when administering drugs that undergo renal or hepatic metabolism and excretion (Fig. 18). Many species of turtles and lizards have a urinary bladder; snakes and crocodilians do not.

7.

Nervous System/Special Senses

The reptilian brain has cerebral hemispheres and 12 cranial nerves. The spinal cord extends to the tip of the tail and contains locomotor control centers, thereby allowing animals to respond at the spinal level (Davies, 1981; Lawton, 1992). Most reptiles have lidded eyes and nictitating membranes. Snakes and some lizards (e.g., geckos) are the exception; the lidless eyes are protected by a transparent scale or spectacle. Snakes also lack nictitating membranes and scleral ossicles present in other reptilian species (Millichamp and Jacobson, 1983). Harderian glands are present in the orbits of many reptilian species and may function in vomerolfaction (Rehorek, 1997). Many reptiles have a vomeronasal organ, which has an accessory olfactory function (Zug, 1993). In snakes, particles of odor are picked up by the forked tongue and carried to the vomeronasal organ, located in the palate. Some species of snakes have pit organs that house infrared heat receptors. In pythons, a row of pits occurs in the labial (lip) scales; and in pit vipers, a single organ is located in the loreal scale between the eye and nostril. Many lizard species have a parietal or "third" eye, which contains photoreceptors that may permit enhanced detection of dawn and dusk (Solessio and Engbretson, 1993). Other lizards have dermal photoreceptors that may function in regulation of basking behavior (Tosini and Avery, 1996). Snakes lack external ears. Vibration is transmitted through

Fig. 17. The snake liver is large and elongate.

841


Fig. 18. Healthysnake kidney (A) and kidney affected with renal gout (B) from inappropriate antibiotic administration.

the body to the quadrate bone and then to the columella and inner ear (Greene, 1997). 8.

Normal Values

Reptilian red blood cells are nucleated and are lower in number than in mammals and birds. Lizards normally have a higher number of red blood cells than do snakes, who have a larger number than do turtles. Total erythrocyte counts can vary with sex, season, and nutritional status (Duguy, 1970; Sypek and Borysenko, 1988; Campbell, 1996). The white blood cells of reptiles include heterophils, eosinophils, basophils, thrombocytes, lymphocytes, monocytes, and in some species, azurophils. In most species, heterophils have fusiform, eosinophilic cytoplasmic granules; the cytoplasmic granules of squamates can be pleomorphic. Heterophils respond to inflammation or infection and function as phagocytic cells, similar to the neutrophils of mammals. Eosinophils resemble heterophils but have spherical eosinophilic cytoplasmic granules; the function of reptilian eosinophils is unclear. Basophils are small cells filled with basophilic granules, which frequently obscure the nucleus. Some species of reptiles have normally high basophil counts. These cells appear to function in reptiles as they do in mammals. Thrombocytes are the reptilian equivalent of platelets. Lymphocytes, the most numerous leukocytes in many species of reptiles, resemble and function like those of mammals. Monocytes tend to be large cells with vacuolated cytoplasm and are involved in granuloma formation and immune response. Squamates have a unique leukocyte, the azurophil, which appears to be a type of monocyte (Hawkey and Dennett, 1989). Like erythrocytes, many leukocytes in reptiles are af-

fected by temperature, season, sex, and nutritional status of the animal (Montali, 1988; Raphael et al., 1994; Campbell, 1996; Anderson et al., 1997). Additionally, factors such as site of venipuncture, type of anticoagulant, and method of analysis can affect blood cell counts in reptiles (Hawkey and Dennett, 1989; Bolten et al., 1992; Gottdenker and Jacobson, 1995; Muro et al., 1998). Representative CBC and plasma chemistry values are presented in Tables III and IV. B.

Nutrition

Reptiles fill a variety of ecological niches and range from carnivores to herbivores. Crocodilians begin life preying on insects and other invertebrates, small frogs, and fish. As crocodilians grow, prey size increases and may include other reptiles, birds, and mammals. The gharial is a specialized feeder, with fish being its mainstay. Other crocodilians, such as the Nile crocodile, eat prey as large as wildebeest. Large saltwater and Nile crocodiles will also prey on humans; most attacks on people are attributed to these two species. In captivity, small to medium crocodilians can be fed whole fish or rodents. Crocodilians can be adapted to dry chows; however, these should contain over 40% protein (Staton et al., 1990; Donoghue and Langenberg, 1996). In some areas, commercial "alligator chow" may be available. Young crocodilians will eat daily; older animals can have longer intervals between meals. Snakes are carnivorous and insectivorous, and several species have very specialized diets. Colubrids (king snakes, rat snakes) and boas feed readily on rodents; king snakes will also eat other snakes. Fish, frogs, and earthworms are common food items for

842

D O R C A S P. O ' R O U R K E A N D J U E R G E N S C H U M A C H E R T a b l e III

CBC Values for Selected Reptilian Species

Parameter

Iguana

Co ruc ia

C ro c ody lus

Te rrap e ne

Trac h e m y s

Te stuclo

Python

iguana a

ze b rata b

p o rosus r

carolina a

scripta a

he r m a n n i e

re gius f

0.84-1.43 24-60 7.4 - 11.6

0.6-1.3 17-41 4.7-12.2

0.27-0.83 20-38 5.0 -8.5

0.37-0.78 12-26 5.9-8.9

0.49 24.44 6.14

10.5-28.0 3.41-8.79

152-600

240-311

309-587

211-296

514.8

17-56

26.1-31.9

~

42-111 3.93-22.4 2-40 16-58 0-18 4-26 0-6 8-42

72-92 6.4-25.7 70-84 13-29 0-2.7 0-1.6 0-4.7 ~

79-131 7.5 56 11 ~ 8 9.4

Red Blood Cells (106/gl) 0.8-2.0 Hematocrit (gm%) 24-37 Hemoglobin (gm/dl) ~ Mean Corpuscular Volume (fl) ~ Mean Corpuscular Hemoglobin Concentration (gm/dl) ~ Mean Corpuscular Hemoglobin (pg) m White Blood Cells (103/gl) 4.5-10 Lymphocytes (%) 40-65 Heterophils (%) 30-45 Eosinophils (%) 0-2 Basophils (%) 1-4 Monocytes (%) 1-4 Azurophils (%) 15-25

m

26.32 96-118 9.7 39.5 34 ~ 1.5 1 ~

122.64 7.24 22.1 50.3 21.0 2.5 3.7

4.6-38.2 1-49 40-82 0-4 0-2 7-34

From Frye (1995). bFrom Wright and Skeba (1992). c From Millan et al. (1997). d From Wallach and Boever (1983). eFrom Muro et al. (1998). fFrom Johnson and Benson (1996). a

Table IV

Chemistry Values for Selected Reptilian Species

Parameter Total protein (gm/dl) Albumin (gm/dl) Glucose (mg/dl) Cholesterol (mg/dl) Alkaline phosphatase (U/liter) Aspartate Aminotransferase (U/liter) Lactate Dehydrogenase (U/liter) Creatine Phosphokinase (U/liter) Calcium (mg/dl) Phosphorus (mg/dl) Sodium (mEq/liter) Potassium (mEq/liter) Chloride (mEq/liter) Uric Acid (mg/dl) Total CO2 (mEq/liter) From Frye (1995). bFrom Wright and Skeba (1992). c From Millan et al. (1997). d From Marks and Citino (1990). e From Johnson and Benson (1996). ;From Drew (1994). a

Iguana iguana a

4.5-8.0 2.0 -3.5 65 - 155

240 - 450 < 3800 < 1000 10.5-13.6 5.3-6.8 152-164 2.5-3.8 0.5-3.2

Corucia zeb rata b

70 - 122 11-252 < 4 -76 27-940 11.0-21.2 2.8-6.7 145-167 1.4 -5.0 123-129 1000~ per min). The intracellular cryoprotectant is toxic and must be removed from the embryonic cells as quickly as possible before the cells become metabolically active. To accomplish the rapid removal of the cryoprotectant agents, the thawed embryos are placed in a series of relatively large volumes of room-temperature buffer solution (e.g., M2 or DPBS). Expected survival and recovery rates for 8cell-stage embryos (e.g., cryopreserved by a slow-freeze process using DMSO) should range from 60 to 90%. Between 5 and 50% of the thawed embryos should become live-born following ET, with the skills of the embryology laboratory personnel determining the actual rates of success. The genotype of frozen transgenic embryos may be confirmed by performing a rapid PCR amplification for the transgene on a small aliquot of embryos immediately following thawing.

1140

GLENN M. MONASTERSKY AND JAMES G. GEISTFELD

The ability to freeze and recover viable, motile, fertilizationcapable mouse sperm is an enticing alternative to embryo freezing. Although common for the majority of mammalian species, mouse sperm cryopreservation has been exceptionally challenging. Nevertheless, this technique is rapidly developing (Songsasen and Leibo, 1998; Wakayama and Yanagimachi, 1998; Mobraaten, 1999).

The result of this array of intellectual property claims is that a given transgenic mouse is covered by multiple patents so that several licensing agreements must be negotiated before a line can be distributed freely.

REFERENCES E.

Transgenic Mice and Intellectual Property Rights

Transgenic technology has introduced a novel, and sometimes uncomfortable, complication into the animal research community. This new element involves intellectual property rights and the patenting of transgenic life-forms. Intellectual property rights may range from very broad coverage of a technique or process (e.g., recombinant DNA technology or pronuclear microinjection) or may protect the ownership of a specific gene sequence. Transgenic mouse patents may result in licensing or follow-through royalty agreements and have generated significant controversy in the research and laboratory animal communities. The reality of intellectual property rights must be confronted whenever transgenic and knockout mice are created, produced, or distributed. Obviously, the production, acquisition, and use of transgenic mice have become more complex and more costly as a result of patent protection for individual lineages, genes, and techniques. Transgenic mice may be covered by any or all of the following areas of intellectual property. (1) The first stage of the transgenic mouse creation process involves DNA cloning (Cohen and Boyer; assigned to Stanford University). Until 1998, any institution using recombinant DNA technology to construct transgene or knockout expression vectors had to obtain a license from Stanford. (2) The second stage of patent protection involves general creation techniques for genetically engineered animals. For example, the patents covering creation of embryonic stem cell knockout mice, "Techniques for Selection of Homologous Recombination Events m Gene Targeting Using Positive Negative Selection" (Capecchi; assigned to University of Utah) and the creation of pronuclear microinjection transgenic animals, "The Method of Genetic Transformation Resulting from Microinjection" (Wagner and Hoppe; assigned to DNX Corporation) are techniques that must be licensed. (3) The third patent stage covers specific creation techniques. For example, "Gene Targeting in Animal Cells Using Isogenic DNA Constructs" (Berns; Netherlands Cancer Institute) describes the creation of an inbred knockout mouse. (4) The fourth patent stage covers specific genetically mediated phenotypes. An example of this type of intellectual property would be the renowned "Oncomouse" patent: "Oncogene Bearing Rats and Mice" (Stewart and Leder; assigned to DuPont, Inc.). This patent applies to any transgenic or knockout model that is predisposed to genetically mediated tumor formation. (5) The fifth stage of patent protections covers the outright ownership of specific genes. An example of this property would be the "p53" patent (Donehower and Bradley; Baylor University).

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28. TRANSGENIC AND KNOCKOUT MICE

son, D. L., Schauer, D., and Fink, E J. (1998). Diminished reproduction, failure to thrive, and altered immunologic function in a colony of T-cell receptor transgenic mice: Possible role of Citrobacter rodentium. Lab. Anim. Sci. 48(2), 145-155. Mobraaten, L. E. (1999). Cryopreservation in a transgenic program. Lab Anim. (January), pp. 15-18. Monastersky, G. M. (1995). Transgenic mouse strategies: Embryology and animal science. In "Strategies in Transgenic Animal Science" (G. M. Monastersky and J. M. Robl, eds.), pp. 3-36. ASM Press, Washington, D.C. Monastersky, G. M., and Geistfeld, J. G., (1997). The production and verification of transgenic mouse strains. Lab. Anim. (26), pp. 36-40. Nakagata, N. (1996). Use of cryopreservation techniques of embryos and spermatozoa for production of transgenic (TG) mice and for maintenance of TG mouse lines. Lab. Anim. Sci. 46, 236-238. National Research Council (1989). "Immunodeficient Rodents m A Guide to Their Immunobiology, Husbandry, and Use." National Academy Press, Washington, D.C. National Research Council (1991). "Infectious Diseases of Mice and Rats." National Academy Press, Washington, D.C. Overbeek, P. A. (1994). Factors affecting transgenic animal production. In "Transgenic Animal Technology, a Laboratory Handbook" (C. A. Pinkert, ed.), pp. 96-107. Academic Press, San Diego. Percy, D. H., and Barthold, S. W. (1993). "Pathology of Laboratory Rodents and Rabbits." Iowa State Univ. Press, Ames. Pinkert, C. A. (1997). The history and theory of transgenic animals. Lab. Anim. (26), pp. 29-34. Pinkert, C. A., ed. (1994). "Transgenic Animal TechnologymA Laboratory Handbook." Academic Press, New York. Pinkert, C. A., Ornitz, D. M., Brinster, R. L., and Brinster, R. D. (1987). An albumin enhancer located 10Kb upstream functions along with its promoter to direct efficient liver-specific expression in transgenic mice. Genes Dev. 1, 268-276. Polites, H. G., and Pinkert, C. A. (1994). DNA microinjection and transgenic animal production. In "Transgenic Animal TechnologymA Laboratory Handbook" (C. A. Pinkert, ed.), p. 23. Academic Press, New York. Reetz, I. C., Wullenweber-Schmidt, M., Kraft, V., and Hedrich, H. J. (1988). Rederivation of inbred strains of mice by means of embryo transfer. Lab. Anim. Sci. 38, 696-701. Robertson, E., Bradley, A., Kuehn, M., and Evans, M. (1986). Germ-line transmission of gene introduced into cultured pluripotential cells by retroviral vector. Nature 323, 445-448. Rguleau, A., Kovacs, P., Kunz, H. W., and Armstrong, D. T. (1992). Decontamination of rat embryos and transfer to SPF recipients for the production of a breeding colony. Theriogenology 37, 289. Rulicke, T., and Autenried, P. (1995). Potential of two-cell mouse embryos to develop to term despite partial damage after cryopreservation. Lab. Anim. 29, 320-326.

1141 Shomer, N. H., Foltz, C. J., Li, K., and Fox, J. G. (1997). Diagnostic exercise: Infertility in two chimeric mice. Lab. Anim. Sci. 47(3), 321-323. Simpson, E. M., Linder, C. C., Sargent, E. E., Davisson, M. T., Mobraaten, L. E., and Sharp, J. J. (1997). Genetic variation among 129 substrains and its importance for "targeted mutagenesis" in mice. Nat. Genet. 16, 19-27. Small, J. D. (1984). Rodent and lagomorph health surveillance-quality assurance. In "Laboratory Animal Medicine" (J. G. Fox, B. J. Cohen, and E M. Loew, eds.), pp. 709-723. Academic Press, San Diego. Songsasen, N., and Leibo, S. P. (1998). Live mice from cryopreserved embryos derived in vitro with cryopreserved ejaculated spermatozoa. Lab. Anim. Sci. 48, 275-281. Taketo, M. Schroeder, A. C., Mobraaten, L. E., Gunning, K. B., Hanten, G., Fox, R. R., Roderick, T. H., Stewart, C. L., Lilly, E, Hansen, C. T., and Overbeek, P. A. (1991). FVB/N: An inbred mouse strain preferable for transgenic analyses. Proc. Natl. Acad. Sci. U.S.A. 88, 2065-2069. Tinkle, B. T., Bieberich, C. J., and Jay, G. (1994). Molecular approaches involved in mammalian gene transfer: Analysis of transgene integration. In "Transgenic Animal Technology--A Laboratory Handbook" (C. A. Pinkert, ed.), p. 230. Academic Press, San Diego. Van Ruiven, R., Meijer, G. W., van Zutphen, L. E M., and Ritkes-Hoitinga, J. (1996). Adaptation period of laboratory animals after transportation: A review. Scand. J. Lab. Anim. Sci. 23(4), 185-190. Waggie, K., Kagiyama, N., Allen, A., and Nomura, T., eds. (1994). "Manual of Microbiologic Monitoring of Laboratory Animals, 2nd ed. NIH Publ. 942498. National Institutes of Health, Bethesda, Maryland. Wakayama, T., and Yanagimachi, R. (1998). Development of normal mice from oocytes injected with freeze-dried spermatozoa. Nat. Biotech. 16, 639641. Weisburd, S. (1987). Beyond the cutting edge of cold. Sci. News 132, 129-144. Whitmore, S. P., Gilliam, A. E, Hendren, R. W., Lewis, S. E., Rao, G. N., and Whisnant, C. C. (1996). Genetic monitoring of inbred rodents from controlled production colonies through biochemical markers and skin grafting procedures. Lab. Anim. Sci. 46(2), 585-588. Whittingham, D. G., Lyon, M. E, and Glenistor, P. H. (1977). Re-establishment of breeding stocks of mutant and inbred strains of mice from embryos stored at - 196~ for prolonged periods. Genet. Res. 30, 287-299. Wilkie, T. M., Brinster, R. L., and Palmiter, R. D. (1986). Germline and somatic mosaicism in transgenic mice. Dev. Biol. 118, 9-18. Wood, M. J., Whittingham, D. G., and Rail, W. E (1987). The low temperature preservation of mouse oocytes and embryos. In "Mammalian DevelopmentmA Practical Approach" (M. Monk, ed.), pp. 255-280. IRL Press, Oxford. Yokoyama, T., Copeland, N. G., Jenkins, N. A., Montgomery, C. A., Elder, E E B., and Overbeek, P. A. (1993). Reversal of left-right asymmetry: A situs inversus mutation. Science 260, 679-682.


Chapter 2 9 Factors That May Influence Animal Research Neil S. Lipman and Scott E. Perkins

I. II.

III.

Introduction .................................................

1143

Intrinsic Considerations

1143

.......................................

A.

Genetics ................................................

1144

B.

Age ....................................................

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C.

Sex ....................................................

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D.

I m m u n e and Nutritional Status

1146

E.

Circadian Rhythms

F.

Endocrine Factors

.......................................

1146

........................................

1146

Extrinsic Considerations .......................................

1147

A.

Physical Factors ..........................................

1147

B.

Chemical Factors .........................................

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C.

Microbial Agents .........................................

1159

D.

Stressors ................................................

1163

References ...............................

I.

INTRODUCTION

Animal research involves the collection of data from carefully designed experiments. The validity of the research and the conclusions drawn from the data are influenced by many factors. Some of these factors may confound experimental results and therefore must be carefully considered and controlled. Otherwise, distortions may result that lead to false observations or conclusions. Although disease is recognized to complicate in vivo research, there is a wide variety of more subtle factors that may alter experimental findings. This chapter will provide an overview of the multitude of complicating factors that have been described in the literature. The reader should understand that there are likely additional factors yet to be recognized, as well as interactions among fac-


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.9. . . . . . . . . . . . . . . . . .

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tors that may also influence experimental outcomes. This chapter has been divided into two principal sections: intrinsic considerations are those inherent to the animal itself, such as its genotype; and extrinsic considerations are those external to the animal that may influence its response. In order to obtain reliable, meaningful results, an attempt should be made to control or standardize all known biological, environmental, and social factors when conducting experiments involving animals.

II.

INTRINSIC CONSIDERATIONS

Experimental animals vary among one another based on their genetic constitution, age, sex, health, and nutritional and


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NEIL S. LIPMAN AND SCOTT E. PERKINS

immune status, as well as on other biological factors. Although animals with genetic uniformity are available and often used, other intrinsic factors must also be considered.

A.

Genetics

The genotype of an animal is an important consideration in designing an experimental protocol, as genetic differences clearly exist between species, breeds, and even strains. Both inbred and outbred animals are widely used in biomedical research, each having advantages and limitations. For outbred animals, the breeding colony from which they are obtained must be large enough to maintain heterogeneity, and management techniques must be employed to ensure genetic variability. Heterogeneity can be monitored by using computer models; analyzing biochemical, genetic, and immunologic markers; and examining physiologic variables (Groen and Lagerwerf, 1979; MacCleur et al., 1986; van Oorschot et al., 1992; WilliamsB langero, 1993). The development and availability of inbred strains permit researchers to address specific questions while generating reproducible and comparable data. However, genetic differences exist even when considering the same strain or even substrain, thus potentially altering experimental data (Heywood and Buist, 1983; Simpson et al., 1997). It is important to understand that the genetic integrity of an animal is not necessarily guaranteed by its nomenclature. Genetic divergence is common; thus, the genetic purity of inbred animals must be monitored and maintained (Festing, 1982; Hedrich, 1983; Threadgill et al., 1997). Genetic differences between individuals of the same inbred strain from the same colony may be the result of incomplete inbreeding, mismatings, inadvertent outcrossings with other strains, spontaneous mutations, chromosomal aberrations, or residual heterozygosity (Baily, 1982). H'Doubler et al. (1991) determined spontaneously hypertensive (SHR) and Wistar Kyoto (WKY) rats to be genetically disparate despite derivation from the same parental Wistar stock. In animal studies using SHR rats, WKY rats have been used almost exclusively as controls due to their presumed homozygosity. However, skin graft experiments and analyses of immunologic and biochemical markers determined SHR and WKY rat strains to be heterogeneic, and some sources of WKY rats to be outbred stocks (H'Doubler et al., 1991). In addition, Kurtz et al. (1989) demonstrated genetic heterogeneity among WKY rats distributed from different commercial sources and even among a single breeding facility. The genetic variability may have been the result of the National Institutes of Health (NIH) distributing breeding stocks of WKY rats as early as the F 6 generation (Kurtz et al., 1987). Thus, rats designated WKY do not constitute an inbred strain. St. Lezin et al. (1992) demonstrated that SHR rats and their WKY controls share only approximately 50% of their DNA fingerprint bands in common, while the hy-

pertensive inbred Dahl salt-sensitive rats (SS/Jr strain) and their normotensive controls, Dahl salt-resistant rats (SR/Jr strain), share only approximately 80% of their DNA fingerprint bands in common. These findings provide evidence of extensive genetic polymorphism between spontaneously hypertensive rat strains and their corresponding normotensive controls and suggest that the continued comparison of these strains may have limited value in interpreting the results on the pathogenesis of hypertension. Today, commercial rodent vendors diligently control and monitor genetic integrity and carefully manage breeding practices for inbred and F1 hybrid colonies, using immunologic and biochemical markers, as well as evaluate polymorphic alleles, using molecular techniques (Cramer 1983; Groen 1977; Russell et al., 1993; Whitmore et al., 1996). When using animals generated by gene targeting, additional details must be considered. Choosing the best genetic strain or stock of mice when developing targeted mutant mice requires an extensive knowledge of their endogenous traits (Crawley et al., 1997). In transgenic animals, the number of copies and site of gene integration may not be known. Additionally, in "knockout" or "knockin" animals the integrated or deleted gene(s) may interact with other genes and extrinsic factors, producing unexpected results that may compromise the interpretation of the mutant phenotype. Threadgill et al. (1995) demonstrated that the phenotype of epidermal growth factor receptor (EGFR) knockout mice was dependent on the genetic background. Development of homozygous EGFR-deficient mutants resulted in peri-implantation death due to degeneration of the inner cell mass in CF-1 mice; midgestational death due to placental defects in 129/Sv mice; and early neonatal death in CD1, 129/Sv X C57BL/6, and 129/Sv x C57BL/6 X MF1 mice due to organ abnormalities (Threadgill et al., 1995; Sibilia et al., 1995). In prostaglandin E2 EP4 receptor-deficient mice on a 129 background, remodeling of the ductus arteriosus (DA) does not occur after birth, resulting in death of the pups. However, 5% of EP 4 ( - / - ) mice on a mixed genetic background survive, and with selective breeding of these mice, there is a 21% survival rate, suggesting that alleles at other loci can provide an alternative mechanism for DA closure (Nguyen et al., 1997). Spearow et al. (1999) detected large differences in susceptibility to juvenile male reproductive development by 17[3-estradiol (E2) in different strains of mice. For example, spermatid maturation was eliminated by low doses of E2 in C57BL/6 and C17/Jls mice, while there was little or no inhibition of spermatid maturation in CD-1 mice, even in response to 16 times the dose of E2. Crawley et al. (1997) provided an extensive overview of behavioral phenotypes of inbred mouse strains and the implications and recommendations for molecular studies based on these behavioral traits. Importantly, the understanding of behavioral phenotypes of the strain in which a mutation will be analyzed can avoid overinterpretation of the mutant phenotype (Crawley et al., 1997). As with inbred animals, comprehensive genetic monitoring, including monitoring

29. FACTORSTHAT MAY INFLUENCEANIMAL RESEARCH

1145

the inserted gene and number of copies, should be performed to when evaluating defeat-induced learned submissiveness (Siegmaintain these unique resources and to verify the presence and fried et al., 1984). Therefore, species and strain selection of an zygosity of the genotype (National Research Council, 1996). animal may have profound effects on experimental results. Species and strains of animals may metabolize xenobiotics differently as a result of inherent quantitative and qualitative B. Age variations. Quantitative variations include differences in cytochrome P450 isoenzymes; cytochrome P450 concentrations; The age of an animal may affect research outcomes and has and competing isoenzyme reactions within the host. Qualitative variations generally involve differences in metabolic pathways. been shown to be a source of variability in rodent carcinogenicThey result from defective or absent enzymes or the ability of a ity studies (Hardisty, 1985; Haseman et al., 1989). Neonatal anspecies to conduct unique enzymatic reactions. Examples in- imals have immature body systems compared to those of adults, clude defects in mercapturic acid formation in guinea pigs; de- and depending on the genotype of the animal, the immunologic fects in glucuronidation in cats; deficient sulfate conjugation in competence of neonates is considerably more immature swine; the lack of N-acetyltransferase in dogs, preventing them (Williams and Weisburger, 1991). As an example, the total and from acetylating aromatic amines; and the singular ability of differential white blood cell count of dogs and cats less than dogs to biotransform 2,2 ',4,4 ',5,5 ' -hexachlorobiphenyl due to 6 months of age are higher than those of adults; the total leukoa unique cytochrome P450 isoenzyme present in their livers cyte, neutrophil, and lymphocyte counts in kittens are highly (Sipes and Gandolfi, 1991). Williams- Blangero et al. (1996) at- variable (Bounous et al., 1995); and neonatal dogs and cats tributed phenotypic variation in alanine transaminase (ALT) have lower blood pressures, stroke volumes, and peripheral vasactivity in chimpanzees to genetic factors. The authors provided cular resistance, and higher heart rates, cardiac output, plasma evidence that the genetic components causing variations in ALT volumes, and central venous pressures (Driscoll et al., 1979). levels can have significant effects on experimental parameters These differences reflect their immature sympathetic innervain hepatitis C research. Examples of intraspecies differences in- tion as compared to the more mature parasympathetic system. clude differences in rates of hexobarbital oxidation in Sprague- Other reported age differences include the demand for oxygen, Dawley and Wistar rats (Sipes and Gandolfi, 1991); differing respiratory rate, hepatic microsomal enzyme system developsensitivities to neurotoxicants such as diisopropyl fluorophos- ment, renal concentrating and diluting capability, and the abilphate (DFP) in Sprague-Dawley, Long-Evans, Fischer 344, and ity to thermoregulate (Hosgood, 1995). The ability to biotransWistar rats (Gordon and Watkinson, 1995); carcinogenic effects form xenobiotics is severely limited in fetuses and neonates of 7,12-dimethyl-benz[a] anthracene (DMBA) on different types due to the immaturity of their hepatic microsomal enzymes of mammary tissue in Wistar-Furth (WF) and Copenhagen (Sipes and Gandolfi, 1991). As an example, neonates are more (COP) rats (Kusunose et al., 1990); and differences in learning, sensitive to certain carcinogenic compounds (Williams and memory, and attention tasks among rat strains and even the same Weisburger, 1991). Conversely, enzyme activities decrease with strain obtained from different suppliers (Andrews, 1996; Craw- increasing age, which may lead to an increased toxicity of ley et al., 1997; Kacew et al., 1998). In addition, significant dif- xenobiotics that serve as their substrates. Older animals may ferences in acute cadmium intoxication have been demonstrated have decreased hepatic and renal blood flow, smaller livers, inin mice. Only C3H mice develop severe hepatocellular damage creased body fat, and decreased excretory capability (Sipes and and significantly lower metallothione induction at 6 hr post- Gandolfi, 1991). Han et al. (1998) demonstrated age-related cadmium injection compared to B ALB/c and DBA/2 strains changes in blood pressures of two strains of senescence(Hata et al., 1980). Strain differences are also observed with accelerated mice (SAM). The SAMP1 mice had a significantly chloroform toxicity in mice; the DBA/2J strain is sensitive, increased blood pressure with age, possibly due to progressive renal changes, and SAMP8 mice had a gradual decrease in while C57BL/6J is resistant (Hill et al., 1975; Vessel et al., 1976). Chloroform toxicity in DBA/2J mice is due to faster re- blood pressure after 5 - 7 months of age. All of these age-related nal conversion of chloroform to phosgene, a reactive intermedi- biochemical and physiologic factors may alter the response to ate, compared to that in C57BL/6J mice. Inbred strains of mice xenobiotics and confound experimental results. also differ with respect to the frequency, type, and age at which neoplasia develops (Fox and Witham, 1997) and also in sensoC. Sex rimotor gating as measured by differences in prepulse inhibition of auditory and tactile startle responses (Paylor and Crawley, The gender of the animal may also influence experimental 1997). A/J and C57BL/6J mice demonstrate differences in anxiety-related behavioral phenotypes and responses to di- outcome. Marked differences in pharmacologic and toxicologic azepam, methyl-[3-carboline 3-carboxylate, and benzodiazepine responses to a variety of xenobiotics have been demonstrated (Mathis et al., 1994, 1995). Additionally, DBA/2 mice have a in male and female rats (Sipes and Gandolfi, 1991). Female greater endogenous analgesic response than C57BL/6 mice rats have a reduced capacity to biotransform certain chemicals,

NEIL S. LIPMANAND SCOTT E. PERKINS

1146

including hexobarbital and parathion. Thus, they sleep significantly longer than males when given an equivalent dose of hexobarbital, and parathion is approximately twice as toxic to female rats than male rats (Sipes and Gandolfi, 1991). Genderspecific forms of cytochrome P450 have been discovered in rats (Sipes and Gandolfi, 1991). The renal conversion of chloroform to phosgene occurs 10 times faster in male mice than in female mice, making male mice susceptible and female mice resistant to chloroform toxicity (Sipes and Gandolfi, 1991; Vessel et al., 1976). Additional examples of sex-related differences are observed when cognitive testing rats and examining the development of DMBA-induced mammary tumors in Wistar-Furth rats, as the incidence of tumors is 100% in females and only 19% in males (Andrews, 1996). However, in Copenhagen rats, the neoplastic response to DMBA-induced mammary tumors is not sex-dependent (Kusunose et al., 1990).

D.

Immune and Nutritional Status

The immune and nutritional status of an experimental animal may have profound effects on the experimental outcome, depending on the nature of the research. The immune system has many functions, including homeostasis of leukocyte differentiation and maturation, immunoglobulin production, and immune surveillance, providing the host a defense system against microbes, neoplastic cells, and environmental agents (Dean and Murray, 1991). Resistance to infectious agents may be dependent on the type of immune response that the host generates. For example, CBA/J mice are highly resistant to Leishmania major, as they develop a predominantly cell-mediated or THl-type response; whereas BALB/c mice are not resistant, as they develop a predominantly humoral or TH2-type response (McEachron et al., 1995). The response to murine leukemia virus (E-55 +) is different in B ALB.K mice and in B 10.BR mice, which express the same H-2 haplotype but differ in genetic background. One hundred percent of BALB.K mice develop leukemia and generate a predominantly TH2 response to infection, while B 10.BR mice generate a predominantly TH1 response and do not develop the final leukemic phase (McEachron et al., 1995). In contrast, it is the TH2 response that confers resistance to helminth infections in BALB.K mice but the TH1 response in B 10.BR mice (McEachron et al., 1995). Immunologic dysfunction, including hypersensitivity and allergy, autoimmunity, and immunodeficiency may influence experimental outcome. There is a wide variety of agents that alter immune function, including chemicals, drugs, food additives, metals, and microbes (Dean and Murray, 1991). The increased sensitivity to chemical carcinogens in neonatal animals may be related to immunologic mechanisms. Penn (1988) demonstrated an alteration in the rate and extent of tumor development in humans with different types of immunodeficiencies.

An animal's nutritional status is dependent on the type(s) of food provided, method and amount of feeding, appetite, and age (Sipes and Gandolfi, 1991). Various dietary conditions, such as mineral, vitamin, and protein deficiencies; lipid composition; and the composition of the diet, alter the biotransformation of xenobiotics (Sipes and Gandolfi, 1991). For example, vitamins C, E, and B complex are involved in cytochrome P450 regulation, and their deficiency reduces the rate of xenobiotic biotransformation (Sipes and Gandolfi, 1991). E.

Circadian Rhythms

Circadian rhythms are endogenous rhythms of physiologic functions and are partially influenced by the time of day, intensity of light, and the suprachiasmatic nuclei (Hastings, 1970; Scheving et al., 1983). Many behavioral, biochemical, and physiologic parameters display rhythmic minima and maxima daily, occurring at specific times of the day and night (Hastings, 1970; Mock et al., 1978). These include blood counts; blood coagulation times; concentrations of CD-3 molecules on the surface of T cells; T-cell response to mitogens; plasma steroid concentrations; body temperature; thermal preference; sensitivity to audiogenic seizure induction; susceptibility to endotoxin and pneumococcus exposure; infectivity pattern following murine leukemia virus inoculation; drug metabolism and toxicity, including barbiturate sleep times and response to ethanol administration; susceptibility to neoplasia; DNA synthesis; protein synthesis; and release of urinary chemosignals that accelerate puberty (Alder and Friedman, 1968; Berger, 1980, 1981; Bowman et al., 1970; Cayen et al., 1972; Chedid and Nair, 1972; Deimling and Schnell, 1980; Drickamer, 1982; Feigin et al., 1969; Gordon et al., 1998; Halberg et al., 1973; Haus et al., 1983; Izquierdo and Gibbs, 1972; Jori et al., 1971; LeBouton and Handler, 1971; Levi et al., 1991; McEachron et al., 1995; Mitropoulos et al., 1972; Nair and Casper, 1969; Nash and Llanos, 1971; Radzialowski and Bousquet, 1968; Ramaley, 1972; Romero, 1976; Scheving and Pauly, 1967; Scheving et al., 1968, 1978; Tavadia et al., 1972; Wada and Asakura, 1970; Wongwiwat et al., 1972; Zbiesieni, 1980). Caution should be exercised when comparing data that have been collected at different times. F.

Endocrine Factors

The endocrine system is one of the principal control systems of the body, producing hormones that regulate both cellular and metabolic functions (Williams and Weisburger, 1991). The interrelationship of the endocrine and nervous systems is essential to integrate a variety of factors that are important in an animal's response to general arousal, physiologic processes, and aversive stimuli (Clark et al., 1997).

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29. FACTORS THAT MAY INFLUENCE ANIMAL RESEARCH

Sex hormones are important determinants of cytochrome P450 enzyme activity (Sipes and Gandolfi, 1991). Testosterone administered to female rats increases their ability to biotransform xenobiotics. The opposite effect is observed when male rats are castrated (Sipes and Gandolfi, 1991). Certain chemicals have been shown to disrupt the endocrine system, leading to cancer induction in specific target organs, such as the adrenals, uterus, or thyroid (Williams and Weisburger, 1991). Gonadectomy, hypophysectomy, or adrenalectomy alters hormonal level(s), potentially resulting in a variety of biological changes (Williams and Weisburger, 1991). Neonatal gonadectomy of CE and DBA strains of mice leads to a high incidence of estrogen-secreting adrenal tumors (Fekete et al., 1941; Murthy et al., 1970). Lipman et al. (1993) postulated that hypersecretion of trophic pituitary hormones in gonadectomized ferrets may result in stimulation of the adrenal gland, producing adrenal gland tumors. Induced ovulators, restricted from normal reproductive activity, may develop hormonal alterations and subsequent biological responses. Prolactin-secreting pituitary adenomas may develop in nulliparous rabbits because of exposure of pituitary acidophils to high concentrations of plasma estrogens secreted by ovarian follicles (Lipman et al., 1994). This hypothesis is supported by the development of prolactin-secreting tumors in rats following prolonged administration of natural or synthetic estrogens (Ito, 1976). Interspecies variations at the hypothalamic-pituitary axis appear to have an important bearing on the differential activities of estrogens and antiestrogens (Hart, 1990). Toxicity of estrogens is species-dependent. Carnivores are more susceptible than rodents (Hart, 1990). Female ferrets may develop estrogen-induced bone marrow suppression due to prolonged estrus or an estrogen-secreting ovarian remnant resulting in pancytopenia and death (Bernard et al., 1983; Sherrill and Gorham, 1985).

Ill.

EXTRINSIC CONSIDERATIONS

A.

Physical Factors

The provision of a stable environment for the conduct of animal research is essential to ensure the integrity of both the animals and the results obtained. The environment to which the animals are exposed must be considered from two perspectives, the m a c r o e n v i r o n m e n t of the room in which the animals are housed, and the cage m i c r o e n v i r o n m e n t with which the animals have direct and prolonged contact. Dependent on the species housed and the caging system utilized, macro- and microenvi-

ronmental conditions may be vastly dissimilar. The principal factors influencing the microenvironment include the macroenvironment, the caging system, and the animals. Extensive effort is devoted to maintaining the temperature, relative humidity, and air quality within the macroenvironment. Guidelines (the "Guide") have been established for these parameters to ensure the well-being of the animals, as well as to provide a stable environment (National Research Council, 1996). Alterations and fluctuations of environmental conditions are well recognized to influence results in a variety of disciplines. For example, the effect of environment on mouse behavior was recently examined in a variety of inbred strains and a targeted mutant mouse line. Despite using animals of the exact genotype, the same experimental setup and procedures, and the same caging, and controlling for the effects of shipment, differences in results were observed and attributed to environmental influences, including differences in air handling and humidity (Crabbe et al., 1999).

1. Temperature The thermoneutral zone (TNZ) of an animal is the temperature range in which homeotherms exert minimal energy to maintain core body temperature. When an animal is exposed to temperatures above or below the TNZ, both behavioral and physiologic adjustments are made to ensure homeostasis, including postural adjustments, huddling, piloerection, peripheral vascular dilatation or constriction, alterations in the respiratory rate and pattern, and food consumption. If alterations in ambient temperature persist or the adaptive adjustments are inadequate, changes in the animal's basal metabolic rate result (Clough, 1982; Newton, 1978). However, the TNZ is not necessarily identical to the temperature range providing optimal development, comfort, reactivity, and adaptability, or for particular species such as the rat, even to the animal's thermal preference (Gordon et al., 1991; Weihe, 1965). Gwosdow and Besch (1985) have proposed that the temperatures to which rats are acclimated can alter their TNZ and set-point temperatures. Importantly, metabolic adaptation can occur within minutes of temperature change (Hart, 1963). Alterations in ambient temperature have greater impact on small mammals, such as rodents, because of their large surface area-body weight ratios. The confounding influence of environmental temperature on research has been recognized for over 50 years. However, most reports reflect the effects of temperature extremes; few describe more subtle changes expected to occur in modern animal holding facilities. In mice and rabbits, both the onset and severity of hypoglycemia and associated seizures were observed to differ with ambient temperature when the biological effects of insulin were assayed (Chen, 1943; Johlin, 1944). The influence of temperature on studies of drug-induced toxicity is well recognized. The toxicity of sympathomimetic amines such as amphetamine

NElL S. LIPMAN AND SCOTT E. PERKINS

1148

increases with a rise in ambient temperature, while the toxicity of others, e.g., ephedrine, decreases (Chance, 1957). Malberg and Seiden (1998) demonstrated that an environmental temperature change of only 2 ~C, from 20 ~ to 22 ~C, resulted in a drop in core body temperature in 3,4-methylenedioxymethamphetamine (MDMA)-treated rats. Not only is the temperature at which a study is conducted important, but the thermal history of the animals has also been demonstrated to play an important role. Mice exposed to cold (4 ~C) for 7 days prior to evaluation of the [3-agonist isoproterenol at 24~ demonstrated enhanced toxicity (up to 10,000 times) when compared to mice maintained at 24~ for the entire test period (Balazs et al., 1962). Weihe (1973) has suggested that differences in drug toxicity attributed to high cage-population density may actually have resulted from difficulty in thermoregulation. Reduction in temperature, as well as its elevation, is also of importance. Hypothermia resulting from housing rats in isolation has been attributed to a decrease in systemic clearance of antipyrine, a marker compound for hepatic oxidative metabolism and for the estimation of total body water (Brunner et al., 1994). Three drug toxicity curves have been described with respect to environmental temperature: (1) A V- or U-shaped curve with minimal toxicity observed at or near the TNZ, with increasing toxicity observed above or below thermal neutrality, characterizes many centrally acting drugs affecting the thermoregulatory system; (2) a linear relationship between toxicity and increasing temperature, as exemplified by sympathomimetic amines, which stimulate heat production; and (3) a skewed relationship in which no change in toxicity is obtained until the TNZ is exceeded, after which a linear relationship is observed (Fuhrman and Fuhrman, 1961). Effects of temperature are not limited to drug toxicity. Macroenvironmental temperature elevation in a rat production colony for 2 days at 31.6~ resulted in death of 33% of the animals. Twenty-five percent of the surviving males, 4 - 6 6 days of age at time of exposure, were sterile as a result of bilateral testicular atrophy (Pucak et al., 1977). Rabbits whose dams were exposed to elevated temperatures (33~ 14 days prepartum responded differently to cold exposure, endotoxin administration, and noradrenalin infusion, than those whose dams were maintained in a stable environment (Cooper et al., 1980). Reported effects on lactation include reduced latency to first milk ejection and reduction in the quantity of milk produced by directly impacting the activity of the mammary alveolae in rats (Jans and Woodside, 1987; Yagil et al., 1976). Exposure to elevated environmental temperatures for extended periods during rearing can influence morphological features, including tail, ear, paw, and salivary gland size in rodents (Caputa and Demicka, 1995; Clough, 1982). Reductions in growth rate, litter size, and neonate viability, which may extend into subsequent generations, have been reported in rodents exposed to low temperatures (Barnett, 1965, 1973).

2.

Relative Humidity (RH)

Both high and low RH have the potential to affect research. Elevation in RH directly impacts the ability of animals to thermoregulate, as evaporative heat loss is essential for core body temperature control in homeotherms. Rats consume approximately 5% more food when maintained at the same temperature but at 35 % as compared to 75 % RH (Weihe et al., 1961). These findings may be of relevance to studies in which test agents or chemicals are administered in the food or in which the amount of food consumed is critical. Mice are more active at low RH (Clough, 1982). Low humidity has also been shown to delay sexual maturity (Drickamer, 1990a). Environmental moisture impacts the viability and transmission of infectious microorganisms. Transmission of Sendai virus is enhanced by high RH (60-70% as compared to 40-45%), whereas transmission of influenza virus is reduced, as the virus survives best at 17-25% RH (Hemmes et al., 1960; Schulman and Kilbourne, 1962; van der Veen, et al., 1972). In general, the viability of microorganisms appears to be lowest when RH is 50% (Anderson and Cox, 1967). RH has a dramatic impact on the generation of intracage ammonia, especially in static rodent isolator cages (Corning and Lipman, 1991; Gamble and Clough, 1976). In these cages, microenvironmental RH may be as much as 38% higher than macroenvii'onmental RH. RH has implications for skin-absorption studies, particularly when animals devoid of fur are used. Variation in RH alters absorption of the topically applied substances by altering evaporative rates, the animals' peripheral circulation, and the material's viscosity (Clough, 1982). Variation would also be expected when test substances are administered as aerosols. Ringtail in rats, which in part is genetically determined and influenced by both caging and nutrition, is characterized by annular constrictions on the tail skin, which may lead to tail sloughing. The incidence of ringtail increases when the RH falls below 40% and the environmental temperature falls outside the animal's TNZ (Flynn, 1959; Njaa et al., 1957). The syndrome is observed most frequently in young animals. Ringtail has also been observed in mice and M y s t r o m y s albicaudatus (Nelson, 1960; Stuhlman and Wagner, 1971). 3. Air Exchange and Composition

Small laboratory animals breathe considerable quantities of air. It is estimated that a mouse breathes approximately 35 liters of air per day under normal conditions (Clough, 1982). The composition of the air is important not only to the animal, but also to the researcher because of the potential implications for research. The quality of air to which an animal is exposed is dependent on a multitude of factors, including but not limited to geographic location, particularly with respect to proximity to

29. FACTORS THAT

MAY INFLUENCEANIMAL RESEARCH

heavy industry and urban populations; the scope of its treatment by the HVAC (heating, ventilation, air conditioning) system; and the location of air intake(s), especially in consideration to exhaust locations of the building and to automobile traffic. Air may contain particulates and/or volatile substances that may be injurious to the respiratory system, skin, and mucous membranes, or may be absorbed and cause systemic effects. Microenvironmental pollutants, principally the metabolic waste gases ammonia and carbon dioxide, may be of major importance. In addition, cage components, feed, and bedding may potentially off-gas undesirable substances (Perkins and Lipman, 1995; Vessel et al., 1976; Wade et al., 1968). The quality of microenvironmental air is influenced by the caging system employed, the strain or stock of animal housed, bedding utilized, and housing density, as well as macroenvironmental conditions (Choi et al., 1994; Corning and Lipman, 1991; Hasenau et al., 1993; Perkins and Lipman, 1995). Although of concern for all laboratory-housed species, poor microenvironmental air quality is of particular concern for rodents maintained in isolator caging. Static isolator caging systems used to house rodents significantly impede air exchange, leading to accumulation of gaseous pollutants, notably ammonia (NH3) and carbon dioxide (CO2) (Corning and Lipman, 1991; Gamble and Clough, 1976; Keller et al., 1989; Murakami, 1971; Serrano, 1971; Simmons et al., 1968). Considerable intercage variability in microenvironmental conditions is frequently observed when housing animals of the same strain and biomass in identical caging (Corning and Lipman, 1991). Ammonia is formed by urease-producing bacteria or bedding containing heat-labile urealytic and urease-activating enzymes, which convert urea in urine and feces into ammonia. Microenvironmental NH 3 may reach 350 ppm within 7 days when housing the maximum number of mice prescribed in the "Guide" (Perkins and Lipman, 1996). This concentration exceeds, by as much as 14-fold, limits of 25 ppm established as an 8 hr timeweighted average (TWA) by the American Conference of Governmental Industrial Hygienists (ACGIH) for human exposure in the workplace (ACGIH, 1995). Physiologic alterations and interference with research may occur at NH 3 concentrations that can be observed in static isolator cages. Ammonia is a potent respiratory irritant that can induce morphologic changes, including reduction in the number of cilia of the respiratory epithelium, hyperplasia of epithelial cells, and formation of glandular crypts in respiratory and olfactory epithelium (Broderson et al., 1976; Gamble and Clough, 1976). In addition, exposure of M y c o p l a s m a p u l m o n i s - i n f e c t e d rats to NH3 concentrations observed in isolator cages (2-250 ppm) enhances M. p u l m o n i s isolation and severity of infection (Broderson et al., 1976; Schoeb et al., 1982). Pneumocystosis and a variety of immunosuppressive effects have also been attributed to elevations in NH3 (Gordon et al., 1980; Targowski et al., 1984; Walzer et al., 1989). It has also been suggested that NH 3 inhibits select com-

1149

ponents of the hepatic microsomal enzyme (HME) system and is a contributing factor in the development of corneal opacities in a variety of inbred and F1 hybrid mouse strains (Van Winkle and Balk, 1986; Vessel et al., 1976). Concentrations of CO2 may also be significantly elevated in static isolator cages (Corning and Lipman, 1991; Huerkamp and Lehner, 1994; Serrano, 1971). Concentrations up to 4000 ppm higher than those observed in the macroenvironment, when housing the maximum biomass permissible in the "Guide," have been reported (Perkins and Lipman, 1995). However, maximum reported concentrations do not exceed ACGIH exposure limits of 5000 ppm established as an 8 hr TWA (ACGIH, 1995). As a result, minimal concern has been raised with its elevation. However, CO2 is a respiratory and cardiovascular stimulant and has the potential to act as an asphyxiant by displacing oxygen. Physiologic alterations that could influence research investigations are clearly possible. The potential of other microenvironmental contaminants must also be considered. Bolon et al. (1991) questioned the presence of an uncharacterized pollutant in isolator cages while evaluating the inhalational effects of methyl bromide on F344 rats. Although elevated ammonia concentrations were contributory to the nasal lesions induced by methyl bromide, the olfactory sensory-cell loss observed could not be attributed to either agent. In another study, acetic acid (0.86 ppm) was determined to have been off-gassed from corncob bedding in isolator cages, although no adverse effects are known to occur at the concentrations observed (Perkins and Lipman, 1995). Volatile chemicals used for research purposes, such as gas anesthetics, and agents used for sanitization and environmental control (e.g., disinfectants and pesticides), have the potential to influence the air the animals breathe. The effects of aromatic hydrocarbons released from cedar and pine bedding on induction of hepatic microsomal enzymes have been well documented (Vessel et al., 1976; Wade et al., 1968). Similarly, pesticides used intentionally or introduced inadvertently and a variety of room-deodorizer constituents may similarly alter hepatic microsomal enzyme function (Cinti et al., 1976; Conney and Burns, 1972; Hodgson et al., 1980; Jori et al., 1969; Robacker et al., 1981). Modern pesticides frequently contain organophosphates or carbamates that may alter mammalian cholinergic transmission, even at low levels of exposure. Pesticides, frequently applied as a spray in an oil base or an aqueous carrier, are likely to result in concentrations in ambient air that increase the risk of direct animal contact (Pakes et al., 1984). Pesticides are known to induce immune cell dysfunction, act as tumor promoters, be toxicants, and act as antiandrogens (Kelce and Earl Grey, 1999; Rought et al., t999). Odorants, such as citrus fragrance used in cleaning products, have been observed to restore stress-induced immunosuppression, decrease locomotor activity during open field testing, and reduce total immobility time in a forced swimming test (Komori et al., 1995a,b).

1150

Because of the high volatility of odorants or other volatile chemicals, sufficient amounts of their vapor at concentrations sufficient to affect the animals may drift into animal holding rooms from corridors or storage sites (Lindsey et al., 1978). Clough (1982) has raised concerns regarding the possible effects that electrically charged airborne molecules generated by the treatment of air by the HVAC system may have on research. Air-conditioned buildings with metal duct distribution systems have a negative ion concentration that is typically 5% of the outdoor value. Rats exposed to a positively charged atmosphere do not do as well in select behavioral tests (Clough, 1982).

4.

Noise

The impact of intense noise of frequencies detectable by humans on both the physiology and behavior of laboratory animals has been recognized for many decades (Peterson, 1980). However, the effects of noise of lower intensity and ultrasound have received considerably less attention. Noise can induce auditory effects, principally destruction of auditory structures and degradation of hearing. Of considerable importance are the nonauditory effects attributed to exposure to sounds of particular intensities and frequencies. The hearing range of species used in the laboratory overlaps only partially with that of humans. Rodents, cats, dogs, and small primates can detect ultrasounds (> 20 kHz) that are outside a human's hearing range. While the human ear is most sensitive to sounds at 2 kHz, many rodents have peak auditory sensitivities in the range of 30 to 60 kHz (Bell, 1974; Heffner and Heffner, 1980). Ultrasounds are used for communication in rodents, small primates, and possibly cats (Bell et al., 1972; Brown, 1976; Milligan et al., 1993; Sales and Pye, 1974). Ultrasound communication is important to a number of intraspecies interactions in rodents, including those be' tween dam and pup, in establishing dominance hierarchies, and during mating (Hofer and Shair, 1978; Sales and Pye, 1974; Sales and Smith, 1980; White and Barfield, 1987). Low-frequency sounds (< 20 kHz) have been demonstrated to alter water consumption; blood pressure; blood corticosteroid, glucose, and insulin concentrations; reproductive performance; body weight; eosinophil counts; immune responsiveness; tumor resistance; histology of the pituitary gland; and learning ability, as well as to induce adrenal and cardiac hypertrophy and hypertension in rodents (Anthony, 1962; Anthony and Harclerode, 1959; Armario et al., 1985; Buckley and Smookler, 1970; Fay, 1988; Fink and Iturrian, 1970; Geber, 1970; Jensen and Rasmussen, 1970; Lockett, 1970; Morseth et al., 1985; Wolstenholme and O'Connor, 1967; Zondek and Tamari, 1967). Noise can induce effects that remain long after it is removed. Barlow (1972) observed that pups born to mice stressed by sound during pregnancy had reduced learning ability. Ultrasound has been observed to reduce fertility and productivity, cause diuresis and increased urinary sodium secretion, in-

NEIL S. LIPMANAND SCOTT E. PERKINS duce audiogenic seizures, reduce locomotor activity, and destroy auditory structures in rodents (Fink and Iturrian, 1970; Lockett, 1970; Peterson, 1968; Pye, 1973; Sales, 1991; Zondek and Tamari, 1967). Structural damage to the auditory system can occur even though subjective (conditioned) responses to sound stimuli remain normal (Catlin, 1986). The direct application of ultrasound to the body wall of the rat, at power similar to that used for human echography, resulted in a significant decrease of fetuses detectable at embryonic day 15 (Bologne et al., 1983). Ultrasound has been shown to alter behavior in cattle, horses, poultry, sheep, and swine (Algers, 1984). Sound has also been demonstrated to entrain circadian rhythms in several species (Menaker and Eskin, 1966; Mrosovsky, 1988; Richter, 1968). Ultrasonic irradiation (> 1 MHz) has been shown to interfere with prenatal development in both the mouse and hamster (Shoji et al., 1975; Weinland, 1963). As this frequency is above the hearing range of any species, the mechanism is presumed to be nonauditory. The ability of sound to induce audiogenic seizures, a model for human epilepsy, has been well studied. Audiogenic seizure activity has been studied principally in mice and rats, although a variety of other species are susceptible (Pierson and Liebmann, 1992; Clough, 1982). Genetically susceptible strains of mice include the AKR, BALB/c, CBA, C57, and DBA/2, although weanlings of any murine strain can be made susceptible if they are audio-conditioned between 15 and 21 days of age, when they are most sensitive (Clough, 1982). Some mouse strains remain susceptible to seizure activity for several months after conditioning (Clough, 1982). Both Sprague-Dawley and Wistar rats can be made epileptogenic by noise exposure as neonates, although the severity of seizures and the age dependence of maximum severity differ among stocks and epilepsyprone substrains (Pierson and Liebmann, 1992). Anthony (1962) recommended that noise levels in animal facilities not exceed 85 dB, a level equivalent to the current 8 hr time-weighted average established for human occupational exposure to noise (ACGIH, 1995). The noise generated by the daily activity of animal facility personnel, by the use of equipment routinely employed to meet animal husbandry and research needs, as well as by particular species such as rabbits and dogs, may be of sufficient intensity to result in behavioral and physiologic alterations. Milligan et al. (1993) examined soundpressure levels in animal holding rooms at five institutions housing a variety of species at both low (0.01-12.5 kHz) and high (12.5-70 kHz) frequencies for periods up to 24 hr. The authors concluded that high sound levels were most frequently generated by human activity at frequencies within the audible range of animals, but often outside the human audible range. Another study undertaken to determine the presence and source of ultrasound in laboratories and animal holding facilities demonstrated that 24 of 39 sources monitored, including cage washers, oscilloscopes, and video display terminals, emitted ultrasonic sound, as did running water taps and rotating glass

29. FACTORSTHATMAYINFLUENCEANIMALRESEARCH

stoppers (Sales et al., 1988). Of particular concern was the finding that some equipment produced only ultrasound that was inaudible to humans. Concerns regarding the activation of fire alarm annunciators and the effect on animals of the noise generated by them led to the development of an annunciator that produces noise below the auditory frequency threshold of mice and rats (Clough and Fasham, 1975). The increased use of ventilated caging systems and laminar flow and biological safety cabinets within animal holding rooms heightens concerns regarding noise generation in both the macroenvironment, where it may impact personnel, and the microenvironment, where it may affect animals (Perkins and Lipman, 1996). Construction noise ranging between 70 and 100 dB at 50 to 2000 Hz has been demonstrated to alter changes in 3,-aminobutyric acid (GABA) release and uptake in rat amygdaloid and hippocampal slices (Fernandes and File, 1993).

5.

Light

The impact of light, including its periodicity, intensity, and wavelength, on the reproduction, behavior, and physiology of mammalian species is well documented. Light is an important synchronizer of circadian ~rhythm. In the absence of light, the cycle length of diurnal rhythms deviates more than an hour from the usual 24 hr period (Weihe, 1976). The time during which the nadir or peak of a specific parameter occurs shifts with an alteration of the light-dark (L: D) cycle. The synchronizing effect of light on the 24 hr clock and the amplitude of the cycle are closely associated with its intensity. The direction of amplitude shift frequently depends on whether the species is nocturnal or diurnal. Aschoff (1960) demonstrated that the nocturnal hamster's wheel-running activity decreased as light intensity increased. Intensity of lighting also affects reproductive physiology. The incidence of anestrus increases significantly when hamsters are exposed to light at an intensity less than 15 lux, and fecundity rates are highest when rats are housed at 250 lux as compared to 6 other ambient light intensities (Weihe et al., 1969). Additionally, light intensity has been reported to affect the mean vaginal opening time, ovarian and uterine weights, estrous cycle length, preweaning mortality, and defecation rates in rodents (Donnelly and Saibaba, 1993; Hautzinger and Piacsek, 1973; Porter et al., 1963; Weihe et al., 1969; Williams, 1971). Phototoxic retinopathy occurs in humans and a variety of animal species. It is most commonly reported in rodents, especially albino rat stocks and strains. Light intensity well below that which causes thermal epidermal burns leads to retinal damage. In addition to light intensity and albinism, other variables, including photoperiod duration, body temperature, nocturnality, the light level under which the animals were raised, age, hormone status, and time of day during light exposure, all affect the extent of photoreceptor damage (Duncan and O'Steen, 1985;

1151

Lanum, 1978; Semple-Rowland and Dawson, 1987a,b; Weihe, 1976). Continuous illumination as low as 110 lux for 7 to 10 days can damage photoreceptor cells in rats (Noell and Albrecht, 1971). Numerous studies indicate that retinal degeneration may occur at illumination levels observed in animal holding rooms (Bellhorn, 1980; ILAR, 1978; LaVail, 1976; O'Steen and Anderson, 1972; O'Steen et aL, 1973; Shear et al., 1973). Current recommendations for illumination at the cage level are light levels between 130 and 325 lux to prevent photoreceptor degeneration in sensitive species, unless animals were raised at extremely low (6 lux) light levels, which may require lower than the recommended light levels (National Research Council, 1996; Semple-Rowland and Dawson, 1987a,b). The location of cages on a rack is important, as light intensity decreases with the square of the distance from its source. The intracage light intensity may differ by as much as 80-fold in transparent plastic cages from the top to the bottom of a rack, while differences up to 20-fold have been observed within a single cage (Weihe et al., 1969). Greenman et al. (1982) concluded that differences in retinal morphology observed in BALB/c mice used in a chronic toxicity study were due to cage position. The caging system utilized, as well as the shelf, rack, and room positions in which the cages are located, should be rotated to reduce complications induced by light (Weihe et al., 1969). Photoperiodicity is an important stimulant and regulator of reproduction. Complex neuroendocrine pathways initiated at the retinal photoreceptors result in the release of hypothalamic hormones, including the gonadotropins (Brainard et al., 1997). Many, but not all, commonly used laboratory species remain highly sensitive to changes in photoperiod. The decline in the reproductive performance of hamsters when the light phase is shortened, accompanied by a regression of the size and activity of their gonads, is well established (Nelson and Zucker, 1987). The marked increases in body weight associated with increased testicular size, elevations in plasma testosterone, and spermatogenesis in the male squirrel monkey are also photoperioddependent (Baldwin, 1968; DuMond and Hutchinson, 1967; Mendoza et al., 1978). When species are maintained in the laboratory under constant photoperiod, many of these seasonal reproductive changes disappear; however, seasonal fluctuations in fecundity, sex ratios of litters, body weight at weaning, and age of sexual maturation are observed in rodents (Drickamer, 1977, 1984, 1990b; Lee and McClintock, 1986). The optimal photoperiod is unknown for most species. A 12L: 12D cycle is used for most species; however, longer photoperiods (14L) are used by some laboratories for rodent breeding (Mulder, 1971). Estrous cycle length has been shown to increase in SpragueDawley rats from a 4- to - 5-day cycle when photoperiod is increased from 12L to 16L (Hoffman, 1973). Photoperiod has also been observed to affect body weight gain and feed intake in livestock (Tucker et al., 1984). Alternating light-dark cycles weekly has been used to induce mild chronic stress in Brown

1152

Norway rats, resulting in a decrease in the cellular immune response while the spontaneous tumor incidence remain unchanged (Kort and Weijma, 1982; Kort et al., 1986). Contamination with as little as 0.2 lux light exposure during the dark phase of the light cycle can reset the circadian rhythm in rodents (Brainard et al., 1983; Minneman et al., 1974). Dauchy et al. (1997) determined that light contamination (0.2 lux) during the dark phase of the light cycle altered growth, lipid uptake, and metabolism of the transplantable Morris hepatoma in rats by inhibiting melatonin secretion. The function of time-controlled lighting systems should be carefully monitored. Although continuous dark cycles are highly unlikely to go undetected, the provision of continuous illumination for varying periods may not be noticed unless monitored. Continuous lighting has an overstimulating effect on reproduction, leading to cessation of cycling, permanent vaginal cornification, and development of excess ovarian follicles in rodents (Hoffmann, 1973; Weihe, 1976). Constant light also enhances the growth and metabolism of tumors (Dauchy et al., 1997). Although few laboratory species, except cats and many nonhuman primate species, have color vision, the wavelength and hence, color of light, have been shown to alter both behavioral and physiologic parameters in many species. Light wavelength, including the color generated by fluorescent lights, has been shown to alter voluntary wheel-running activity, the time of vaginal opening, reproductive organ weights, body weight, submandibular gland development, sexual maturity, and the development of dental caries in various rodent species (Hautzinger and Piacsek, 1973; Saltarelli and Coppola, 1979; Sharon et al., 1971; Spalding et al., 1969a,b; Wurtman, 1975). 6.

Radiation

Electromagnetic and ionizing are the two principal forms of radiation. Electromagnetic radiation consists of oscillating electric and magnetic fields composed of different wavelengths or frequencies. Ultraviolet, visible, and infrared light, and microwaves and radio waves are forms of electromagnetic radiation (ER). Electromagnetic radiation can alter biological responses and thus possibly influence the actions of drugs and the outcomes of disease (Izmerov, 1985; Wilkening and Sutton, 1990). An increased level of ultraviolet B (UVB) radiation in the environment due to ozone depletion has increased the risks of ocular damage, immunosuppression, immune modulation, and cancer (Goettsch et al., 1994; Longstreth et al., 1998; Selgrade et al., 1997). In animal studies, UVB-related immunosuppression has had a negative effect on the outcome of some infectious diseases and cancers (Goettsch et al., 1994; Longstreth et al., 1998). Infrared light diminishes diabetes induced by alloxan in rats. Hapke (1983) proposed that infrared radiation alters blood glucose regulation and is antidiabetogenic. Exposure of laboratory animals to electromagnetic fields has


demonstrated a variety of biOlogical effects, including lymphomas, pulmonary sarcomas, hepatomas, and mammary and skin tumors. However, other animal studies have found no carcinogenic effects (Harris et al., 1998; Repacholi, 1997; Repacholi et al., 1997). Although further studies are required, human exposure to high field strength and low-level, extremely lowfrequency electric and magnetic fields, and chronic exposure to static magnetic fields have been associated with biological effects such as pediatric leukemias and other cancers, adverse pregnancy outcomes, and changes in eye structure and function (Repacholi, 1998; Repacholi and Greenebaum, 1999). Exposure to sinusoidal, bipolar oscillating magnetic fields has been reported to cause malformations in chicken embryos (Bryan and Gildersleeve, 1988). Ionizing radiation is the result of rays and particles producing enough energy to release free electrons from atoms, leaving the atom electrically charged or ionized. Gamma rays, X rays, and atomic particles (a and [3) are types of ionizing radiation. They can cause harmful biological effects, such as damage of DNA resulting in genetic, teratogenic, and somatic effects, including death; and increases in the incidence of neoplasms (Burkart et al., 1999; Report to the Congress, 1981; U.S. Regulatory Commission, 1981, 1987). 7.

Caging and Housing-Related Issues

Modern caging is generally manufactured from stainless steel or synthetic polymers. These materials differ dramatically in their thermal conductivity. Metabolic rate, evaporative water loss, and colon temperatures are altered when rats are housed in cages with floors made from metal or plastic for periods as short as 60 min, as were the animals' responses to MDMA, a psychoactive drug (Gordon and Fogelson, 1994). Cage design is also recognized to influence animal health. Pododermatitis can occur in rats housed for extended periods on wire caging (Anver and Cohen, 1979). Wire caging has also been associated with an increased incidence and severity of urologic syndrome in AKR mice (Everitt et al., 1988). The provision of a complex cage environment for rodents, in lieu of housing in standard shoe-box cages, enhances cell proliferation and improves response in behavioral tests following implantation of intracerebral grafts and lesions of the hippocampus and cortex (Galani et al., 1997; Kelche et al., 1988; Kempermann et al., 1998). However, cage enrichment, especially when providing nest boxes or modifications that allow spatial and visual separation, may result in the loss of stable dominance hierarchies, leading to increased aggression and neuroendocrine alterations dependent on the individual animal's social position (Haemisch and Gartner, 1994, 1997; Haemsich et al., 1994). Cage enrichment has been shown to decrease immune function, reducing resistance following challenge with Babesia microti in CFLP mice (Barnard et al., 1996). The influence of cage size has also been reported. The febrile

29. FACTORS THAT MAY INFLUENCEANIMAL RESEARCH

response to lipopolysaccharide (LPS) is less when hamsters are housed in cages providing 200 cm 2 of floor space as compared to cages providing 1815 cm 2. The author theorized that the diminished response was a result of the stress from housing in a small cage, as glucocorticoids and other stress hormones alter the response to LPS (Kuhnen, 1997, 1998). In examining play activity in young rats, Siegel and Jensen (1986) determined that animals housed in smaller cages exhibited greater social play as defined by pinning behavior. When housed in groups of 3 in shoe-box caging providing 32.2, 64.5, 96.8, or 129 cm 2 per mouse, C57BL/6 mice provided the least floor area consumed or wasted more water and responded more vigorously to a T-cell mitogen than mice provided greater space (Fullwood et al., 1998). However, an increase in aggressive behavior as well as adrenal gland weight and plasma glucocorticoid concentration was observed as progressively more space was provided. Provision of greater floor space to male mice alters the animals' dominance rank (Poole and Morgan, 1976). Cage size was not observed to significantly influence the behavior or physiology of dogs and rhesus monkeys (Hite et al., 1977; Line et aL, 1989). The use of ventilated caging for rodent housing may potentially alter research findings. Although intracage ventilation improves microenvironmental conditions, excessive intracage ventilation, especially when air is supplied at the level of the cage, may lead to chilling and dehydration, with neonates and hairless mutants being particularly sensitive. Intracage air velocities as high as 100 linear feet per minute (lfpm) have been measured in a commonly used caging system (Tu et al., 1997). As 20~ air moving at 60 lfpm has a cooling effect of 7~ exposure of animals to a ventilated cage may alter behavioral and physiologic responses (Weihe, 1971). Ventilated caging systems may cause pheromone dilution and alter breeding. A negative synergistic effect has been reported between ventilated cages and the use of automatic watering systems, resulting in increased mouse pup mortality (Huerkamp et al., 1994). The generation of noise and vibration by these systems is an additional consideration that may influence experimental findings (Lipman, 1999). 8.

Miscellaneous

The effect of vibration on animals has been subject to minimal evaluation. However, occupational exposure of humans to vibration has been associated with a variety of physiologic and biochemical alterations (Tzvetkov et al., 1992). Changes in lipid metabolism, electrolyte and trace element concentration, acid-base balance, and reproductive function are altered in animals exposed to vibration (Shenaeva, 1990; Tzvetkov, 1993). With the increasing use of ventilated caging systems worldwide, many of which contain blower systems mounted directly on the rack, the possibility of continuous long-term exposure to low levels of vibration should be considered. Sanz et al. (1988)

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attributed anomalies detected while evaluating the molecular mechanism of toxic substances to meteorological (storms), geological (earthquakes), and astronomical (lunar phase) events.

B. 1.

Chemical Factors

Xenobiotics (Other than Pharmaceuticals)

Xenobiotics are any chemicals or compounds that are foreign to a biological system. Exposure to xenobiotics may occur via the air, water, diet, bedding, caging, and/or equipment, or may be intentionally introduced pharmacologic agents as part of the routine conditioning or experimental procedure. The effect or toxicity of a xenobiotic is based on its dose and disposition. Absorption, distribution, biotransformation, and excretion all affect the disposition of a xenobiotic (Sipes and Gandolfi, 1991). In addition, host barriers, i.e., the skin, lungs, and alimentary tract, and the physical and chemical composition of a xenobiotic also affect its toxicity. A xenobiotic or its metabolites may cause physiologic alterations in the animal and thus affect the outcome of the experiment by altering immune function, and by acting as a mutagen and/or a teratogen (Dean and Murray, 1991; Sipes and Gandolfi, 1991). Examples include aflatoxins; heavy metals such as lead, mercury, and cadmium; organochlorine insecticides; and commonly administered anesthetic agents (Degraeve, 1981; Elis and DiPaolo, 1967; Gerber et al., 1980; Ito and Ingalls, 1981). a.

Biotransformation

Compounds are generally altered chemically by enzymatic activity before being excreted. This process yields metabolites that are more hydrophilic and thus more readily eliminated. Xenobiotic biotransformation is affected by the physiochemical properties of the compound as well as by its protein-binding ability, the dose, and route of administration, Biotransformation is also affected by a variety of host factors including species, strain, age, sex, time of exposure, state of its biotransforming enzymes, nutritional and disease status, and environmental factors (Sipes and Gandolfi, 1991). For example, cadmium accumulation from diet, water, and ambient air is greater in the tissues of hypertension-sensitive Dahl rats than of hypertensionresistant rat strains (Ohanian and Iwai, 1980). b.

Diet

The diet is usually the principal source of xenobiotic compound exposure (Torronen et al., 1994). Ideally, laboratory animal diets should not contain compounds that can alter experimental response. However, many animal diets contain natural and synthetic chemical compounds that may have significant effects on physiologic processes and thus alter the experimental

1154


outcome, especially in pharmacologic and toxicologic studies had decreased blood levels of cholesterol and phospholipid. The (Schecter et al., 1996; Torronen et al., 1994). Diets may contain differences were attributed to the level and composition of the inducers, suppressors, activators, inhibitors, and substrates that fiber fraction of the cereal-based diet (Rutten and de Groot, influence P450 levels (Torronen et al., 1994; Yang et al., 1992). 1992). Additionally, transgenic mice expressing a human breast Feed contaminants include chlorinated hydrocarbons, organo- cancer oncogene, c-neu, fed a diet containing fiber from nonphosphates, polychlorinated biphenyls, heavy metals, aflatox- purified cereal ingredients, had delayed development of mamins, nitrates, nitrosamines, and estrogenic compounds (Edwards mary cancer (Rao et al., 1997). The amount of dietary fat may et al., 1979; Newberne and McConnell, 1980; Silverman and influence the biotransformation and response of mice to barbiAdams, 1983). Many contaminants are found naturally in plant turates. The differences in response result from hepatic fat acmaterials or are agricultural residues. Diets may also be con- cumulation and a redistribution of the lipophilic barbiturate into taminated during storage or formulation. Examples include adipose tissue (Hapke, 1983). In chimpanzees, certain diets inaflatoxin contamination of corn, wheat, and other cereals during crease the urinary excretion of isoflavonoids, phytoestrogens, storage; the presence of phytoestrogens in dietary constituents; daidzein, and enterolactone, which may be partially responsible and contamination of diets with estrogenic compounds during for resistance to the development of estrogen-induced cancers formulation (Hadlow et al., 1955; Wogan, 1968; Wright and (Musey et al., 1995). Additional human and animal studies sugSeibold, 1958). Thigpen et al. (1987a) developed a standardized gest that dietary phytoestrogens help prevent cancer, heart dismouse bioassay for detecting estrogenic activity in rodent diets. ease, and osteoporosis through a variety of mechanisms (Kurzer Female mice, weaned at 15 days, receiving diet containing di- and Xu, 1997). Variations in the quantity or availability of essential vitamins ethylstilbestrol (DES) or negative control diet were sacrificed at 22 days, and uterine weights and uterine:body weight (U:BW) and minerals may alter drug-metabolizing systems, affect memratios were evaluated. Mice receiving the DES diet had sig- brane integrity, or predispose to the effects of carcinogens nificant increases in both uterine weights and U:BW ratios, (Newberne and McConnell, 1980). Deficiencies in calcium, thus providing a standardized bioassay for evaluating rodent di- copper, iron, magnesium, and zinc have been shown to decrease ets. Using this bioassay, Thigpen et al. (1987b) evaluated com- cytochrome P450 enzyme levels and redox reactions (Sipes and mercially available rodent diets and determined two diets to Gandolfi, 1991). Hypervitaminosis A has been associated with have estrogenic activity. However, additional studies concluded teratogenic effects in rabbits, including fetal resorptions, aborthat fats and carbohydrates in the food caused increases in uter- tions, and stillbirths (DiGiacomo et al., 1992). Miller et al. ine weights and in U :BW ratios, thus partially accounting for (1997) demonstrated that the amount of dietary iron intake in the estrogen-like uterine growth-promoting activity (Thigpen common marmosets can affect liver iron content and health. Diet al., 1987c). Variations in the constituents and formulation ets high in iron, 350-500 ppm, can lead to hepatic hemosideroof the diet result in a wide variety, type, and concentration of sis with subsequent effects, including death. Excessive caloric intake may have a serious impact, as caloric chemical contaminants as well as nutrient quality (Newberne and McConnell, 1980; Wise, 1982). Variations in dietary con- restriction has been documented to be beneficial to the host for stituents may alter the toxicity of chemical contaminants and resisting effects of aging, degeneration and infectious diseases, potentially affect the animal's response to specific drugs or neoplasia, and the toxicity of chemical agents (Anderson et al., 1985; Masoro, 1992; Newberne and McConnell, 1980; Pickerchemicals (Sipes and Gandolfi, 1991). The protein and fat content may have profound effects on ing and Pickering, 1984; Ross and Bras, 1973). Restriction of physiologic processes and on the toxicity of certain xenobiotics. dietary energy maintains most physiologic systems in a youthOlovson (1986) demonstrated dramatically increased breeding ful state, retarding a broad spectrum of disease processes, and efficiency, decreased mortality, and increased body-weight gains delays death due to neoplasms (Masaro, 1992). Further, in aniin both male and female cats when dietary fat content was in- mals and humans, dietary restriction has a marked effect on the creased from 15 to 27% in a conventional cat-breeding colony. endocrine system and the cell-cycling rate in various organs In inbred rats, both diet and strain strongly influence the num- (Williams and Weisburger, 1991). Laboratory diets may be contaminated by carcinogenic niber, size, and hemoglobin content of red blood cells (Hackbarth et al., 1983). Rats maintained on a low-protein diet have de- trosamines (Edwards et al., 1979; Walker et al., 1979) and nicreased lethality and hepatotoxicity produced by dimethylni- trates (Newberne and McConnell, 1980), which can be controsamine, while rats fed diets with a high polyunsaturated fat verted to nitrosamines in the gastrointestinal tract. In rodents, content have decreased concentrations of cytochrome P450 exposure to heavy metals, such as lead and cadmium, has been isoenzymes (Sipes and Gandolfi, 1991). In rats, aflatoxin B1- shown to suppress disease resistance (Cook et al., 1975; induced hepatic neoplasms were more significant when corn oil Hemphill et al., 1971); suppress the effects of endotoxin (Cook was the source of dietary fat compared to beef fat as the source et al., 1974); cause immunosuppression (Blakley et al., 1980; (Newberne et al., 1979). In comparing a cereal-based and a Koller, 1979; Loose et al., 1978); and influence reproductive purified diet, rats, mice, and hamsters fed the cereal-based diet performance (Degraeve, 1981; Gerber et al., 1980), and may be

29.

FACTORSTHATMAY INFLUENCEANIMAL RESEARCH

carcinogenic (Goyer, 1991). The type and quantity of feed provided to rats may cause differences in central nervous system responsiveness and function (Kacew et al., 1998). In cats, increased dietary cysteine promotes higher methionine, homocysteine, glutathione, and oxidized glutathione concentrations in blood (Fettman et al., 1999). Aged female beagle dogs fed a diet with n-6 to n-3 fatty acid ratio of 1:4 had increased total lymphocyte and CD4 § T-cell counts and a decrease in CD4 § to CD8 + ratio after vaccination with a keyhole limpet hemocyanin suspension (Hall et al., 1999). Finally, food deprivation can have a dramatic effect on the outcome of toxicology studies, as deprivation can induce hepatic microsomal enzymes and reduce the concentration of cofactors and conjugating agents (Sipes and Gandolfi, 1991). Good Laboratory Practice (GLP) Regulations published by the Food and Drug Administration require compounds be excluded that could act synergistically or antagonistically with test compounds, or that could be expected to produce a result similar to that of the test compound (McSheehy, 1983). c.

Water

Animal drinking water is generally supplied from a local potable water source that meets standards applied to human consumption. Depending on geographic location; area geology; the use of surface or well water; proximity to industrial, agricultural, or urban centers; and the type of water treatment used, the water consumed is subject to considerable variation. Drinking water may be contaminated by pesticides, heavy metals, radionuclides, and other compounds that may produce biological effects (Cantor, 1997; Surbeck, 1995). In Europe, a variety of pharmaceuticals, including antibiotics, analgesics, antiseptics, beta-blockers, and cholesterol-lowering drugs, were detected in the drinking-water supply (Garcia, 1998). There is epidemiologic evidence of an association between drinking-water contaminants, including pesticides, arsenic, volatile organics, asbestiform fibers, and radionuclides, and the formation of one or more types of cancer in humans (Cantor, 1997). Nitrogen fertilizers and pesticides have been used worldwide since the 1960s, and pesticide contamination of drinkingwater supplies has increased over the past decade (Morales et al., 1993; Shapiro, 1980; Taets et al., 1998). In Spain, contamination of drinking water by nitrates has been associated with a significant increase in bladder cancer in humans (Morales et al., 1993). Raszyk et al. (1995) demonstrated increased mutagenicity, via the Ames test, in pesticide and polychlorinated biphenyl (PCB)-contaminated drinking water collected on swine farms, establishing that mutagens present in drinking water may jeopardize animal health. Kligerman et al. (1993) demonstrated cytogenetic damage in splenocytes of male F344 rats and female B6C3F1 mice following exposure to simulated California groundwater contaminated with a mixture of pesticides and the fertilizer ammonia nitrate. Herbicides have also

1155

been shown to cause chromosomal damage to Chinese hamster ovary (CHO) cells in vitro (Taets et al., 1998). Heavy metals, such as lead, copper, cadmium, nickel, and silver, may contaminate drinking water; and lead contamination in drinking water has been associated with adverse pregnancy outcomes in humans including cardiovascular defects and stillbirths (Aschengrau et al., 1993). Ronis et al. (1998a,b) evaluated chronic lead exposure in pregnant SD rats. Pregnant rats exposed at embryonic day 5 to lead acetate, 0.05-0.45% (w/v), in drinking water had a dose-responsive decrease in neonatal birth weights and crown-to-rump lengths and a subsequent delay in sexual maturity. The disruptions in reproductive physiology were accompanied by a significant decrease in neonatal sex steroids and suppression of sex hormones during puberty. These results suggest the reproductive axis of the rat is sensitive to lead during development. Zheng et al. (1996) exposed weanling male SD rats to lead acetate in drinking water, 0, 50, or 250 ktg/ml, and demonstrated a dose-responsive decrease in production of choroid plexus transthyretin, a major cerebrospinal fluid (CSF) protein manufactured by the choroid plexus that is responsible for the transport of thyroid hormones to the developing brain. Water treatment can be used to minimize microbial contamination; however, many forms of treatment result in physiologic alterations that may affect experimental data (Fidler, 1977; Hall et al., 1980; Hermann et al., 1982; Homberger et al., 1993). Chlorine may be introduced into the water supply as a component of water treatment. Chlorine may cause alterations in the immune response (Exon et al., 1987; Hermann et al., 1982), and chlorination by-products may be carcinogenic and mutagenic (Koivusalo and Vartiainen, 1997; Morris et al., 1992; Pilotto, 1995). Trihalomethanes are formed as a result of interactions between chlorine or bromine with methane groups from natural organic materials. Chloroform, a trihalomethane, is found in relatively high concentrations and has demonstrated biological impact, including cytotoxicity, increased DNA synthesis, and carcinogenesis (Lee et al., 1998; Lipsky et al., 1993; Vessel et al., 1976). For GLP studies, the quality of the water must be analyzed and documented to ensure that the water does not contain chemical pollutants (McSheehy, 1983). The processing and delivery system of water in a lab animal facility may affect water constituents. Hall et al. (1980) evaluated the effects of acidified drinking water on select biological phenomena of normal and immunosuppressed male mice. Depending on the pH of the water and the acid (hydrochloric vs. sulfuric) used for acidification, there may be a decrease in weight gain, water consumption, and number of bacteria species isolated from the terminal ileum, with more pronounced changes noted in immunosuppressed mice. The authors concluded that the acidification of drinking water was not innocuous and it should be evaluated as an environmental variable. Lohmiller and Lipman (1998) documented increases in silicon concentration and formation of silicon crystals from autoclaving glass water bottles. The increase in silicon and variations

1156


from bottle to bottle in silicon concentration could cause alterations in experimental variables. Renal lesions have been induced in guinea pigs following the experimental administration of silica-containing compounds (Dobbie and Smith, 1982). Kennedy and Beal (1991) evaluated rubber water bottle stoppers for mineral content and mineral-leaching ability into both deionized and acidified-deionized drinking water. Minerals were present in all three types of stoppers evaluated. Acidifieddeionized drinking water typically leaches more minerals from the stoppers. The authors concluded that certain types of stoppers may be more suitable for particular nutritional and toxicologic studies. 2.

Pharmaceuticals

Pharmaceutical agents are administered to laboratory animals for a host of reasons. For example, pharmaceuticals are administered to induce and maintain anesthesia, to relieve pain, to prevent or treat microbial disease, or to activate an inducible promoter that turns on or off specific genes. In general, their administration is necessary but may be ancillary to the primary experimental goal. Pharmaceuticals may result in physiologic changes, distinct from those expected from their principal mechanism of action, or they may alter the metabolism of other chemicals and therefore alter experimental results. Importantly, effects induced by pharmaceutical agents may frequently be dose- and species-dependent. a.

Anesthetics, Tranquilizers, and Analgesics

The induction and maintenance of general anesthesia lead to significant physiologic alterations, principally of the cardiovascular, pulmonary, and nervous systems. Anesthetics, tranquilizers, and analgesic combinations that minimize physiologic disturbance of the system under study are usually selected. This choice is most critical when experimentation is conducted while the animal is under anesthesia, as most significant cardiopulmonary alterations return to normal following recovery. However, some anesthetics may induce physiologic and behavioral changes distinct from their cardiopulmonary effects, which may persist after the animal has awakened from anesthesia. Anesthetics may be directly toxic. Commonly used anesthetics such as avertin, xylazine, and the combination ketamine and xylazine have been shown to induce tissue injury. When administered at clinically relevant doses, and depending on the species and route of administration, examples of tissue injury include pulmonary parenchymal damage, muscle necrosis, peritonitis, corneal calcium deposition, and keratoconjunctivitis sicca (Celly et al., 1999; Gaertner et al., 1987; Guillet et al., 1988; Kufoy et al., 1989; Smiler et al., 1990; Zeller et al., 1998). Lesions associated with the use of avertin in mice are speculated to have resulted from improper anesthetic prepara-

tion or the concentration administered rather than from any inherent toxicity of the anesthetic (Papaioannou and Fox, 1993; Weiss and Zimmerman, 1999). Methoxyflurane is nephrotoxic in F344 rats, causing a dose-related diabetes insipidus syndrome (Clifford, 1984; Mazze et al., 1973). Reactive intermediates formed during reductive metabolism of halothane and isoflurane under hypoxic conditions lead to hepatic toxicity (Eger et al., 1987; Harper et al., 1982). Anesthetics may enhance or inhibit the toxicity of other agents. Barbiturates and xylazine induce hepatic cytochrome P450-metabolizing enzymes that may influence the metabolism of other chemicals (Nossaman et al., 1990). Enflurane, halothane, and methoxyflurane have been shown to inhibit cytochrome P450-dependent type I substrates (Rice and Fish, 1987). The authors speculate that methoxyflurane, because of its high lipid solubility, would have long-lasting effects. The effects of anesthetics on the immune system are well recognized. Halothane reduces the responsiveness of lymphocytes to mitogensi decreases their chemotactic, phagocytic, and transforming capabilities, as well as their ability to synthesize RNA and protein; and inhibits cell-mediated cytotoxicity, neutrophil and monocyte chemotaxis, and neutrophil phagocytosis (Bruce, 1972, 1975; Cullen, 1974; Cullen et al., 1972, 1976; Mougdil, 1986). Decreased neutrophil and monocyte chemotaxis has been associated with administration of isoflurane and methoxyflurane (Mougdil, 1986). A variety of anesthetics and opioid analgesics, including avertin, ether, fentanyl, halothane, isoflurane, ketamine-xylazine, morphine, sufentanil, and sevoflurane, reduce the cytotoxic activity of natural killer (NK) cells, a lymphocyte subtype involved in nonspecific immune response to tumors, viruses, and select bacteria in the postoperative period (Beilin et al., 1989; Markovic and Murasko, 1990, 1991, 1993; Markovic et al., 1993). Hyporesponsiveness of NK cells lasts for at least 11 days after anesthesia (Markovic et al., 1993). Markovic and Murasko (1993) hypothesized that the anesthetic effects on NK cell activity results from the induction of CD8 § cells that suppress stimulation of NK cells by interferon. Following anesthesia, NK cells fail to respond to interferon or poly I:C, an inducer of endogenous interferon synthesis, as they do normally (Markovic and Murasko, 1990, 1991; Markovic et al., 1993). Anesthetic-induced NK cell depression strongly accelerated progression of spontaneous lung metastasis produced by the 3LL Lewis lung carcinoma and B16 melanoma (Katzav et al., 1986; Shapiro et al., 1981). The administration of local anesthetics inhibits lymphocyte capping; depresses phagocytosis in neutrophils; reduces both the number and function of CD4 § and CD 19 § cells; alters lymphocyte secretion of interferon, tumor necrosis factor, interleukin (IL)-I, and soluble IL-2 receptor following stimulation by a variety of mitogens; and increases plasma endothelin-like immunoreactivity (Brand et al., 1998; Corsi et al., 1995; Kutza et al., 1997; Sato et al., 1996; Shirakami et al., 1995). Local anesthetics, including procaine, lidocaine, butacaine, tetracaine,

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FACTORSTHAT MAYINFLUENCEANIMALRESEARCH

and dibucaine, have been shown to enhance the toxicity of the bleomycin derivative, peplomycin (Mizuno and Ishida, 1982). The effects of anesthetics on cardiovascular function are well recognized. At concentrations used clinically, most volatile anesthetics depress the contractile force of the heart. The mechanisms underlying the negative inotropic effects of these agents, although partially involving the effect of Ca 2§ on the myofibrillar apparatus, are not completely understood (Bosnjak, 1991). Volatile anesthetics, including halothane, isoflurane, and sevoflurane, have an inhibitory effect upon both vascular and tracheal smooth muscle, leading to both vascular and airway dilatation (Kai et al., 1998; Mercier and Denjean, 1996; Zhang and List, 1996). These agents attenuate and prevent airway smooth muscle constriction when exposed to allergen and leukotriene D4 (Tudoric et al., 1995). These effects are mediated by influencing Ca 2§ sensitivity (Kaiet al., 1998; Zhang and List, 1996). Effects have been described following the use of select anesthetic agents, which can be of critical importance to experimental outcome although they may be clinically inapparent. The application of the topical anesthetic benzocaine is associated with methemoglobinemia in a variety of species (Davis et al., 1993). A 2 sec burst of anesthetic spray or direct application of 56 mg of benzocaine increased methemoglobin concentrations sufficiently to substantially alter cardiovascular and pulmonary function (Davis et al., 1993; Lagutchik et al., 1992). The widely used a2-adrenergic agonist xylazine lowers basal plasma insulin concentrations and abolishes the rise in insulin following glucose administration, resulting in elevations in fasting glucose and glucose intolerance in multiple species (Brockman, 1981; Goldfine and Arieff, 1979; Hsu, 1988; Koppel et al., 1982). Select anesthetics have been shown to exert effects on the neuroendocrine system. Reported alterations include both increased and decreased cortisol and catecholamine secretion; increased concentrations of serum growth hormone, thyroxine, antidiuretic hormone, and renin; and decreased secretion of aldosterone and testosterone (Bardin and Peterson, 1967; Fariss et al., 1969; Oyama, 1973; Pettinger et al., 1975). Anesthetics may also influence behavior. The analgesic buprenorphine was associated with pica behavior in the rat. Rats administered this agent ingested hardwood bedding on which they were maintained (Clark et al., 1997b). b.

Euthanasia Agents

There are limited studies examining the effects of euthanasia technique on experimental results. Butler et al. (1990) observed differences in prostacyclin production in aortic tissue and response of aortic and colonic smooth muscle to acetylcholine when rabbits and rats were sacrificed using methoxyflurane, carbon dioxide, and pentobarbital. Cervical dislocation with or without methoxyflurane or pentobarbital anesthesia, and CO2 and halothane overdose were observed to alter both mitogen-

1157

induced lymphoproliferation and the induction of alloantigenspecific cytolytic T lymphocytes in mice (Howard et al., 1990). Decapitation has been associated with dramatic increases in plasma catecholamine concentrations in rats, presumably caused by environmentally induced changes in sympathoadrenal medullary activity (Popper et al., 1977). Carbon dioxide inhalation, dependent on concentration, has varying effects on brain excitability, causes acidosis, and decreases the cerebral concentrations of both sodium and potassium (Granholm and Siesjo, 1969; Pincus, 1969). It is also reported to cause tissue petechiation, particularly in the lungs. The euthanasia agent T-61 causes intravascular hemolysis, which interferes with serum hexosaminidase measurements; artifactual damage of the pulmonary parenchyma, characterized by congestion, edema, and endothelial necrosis; and endothelial swelling of the renal glomerular tufts (Doughty and Stuart, 1995; Port et al., 1978; Prien et al., 1988). The pulmonary architecture is extremely sensitive to effects of a variety of euthanasia techniques (Feldman and Gupta, 1976). Most euthanasia techniques are unsuitable to maintain the integrity of enzymatically labile neurochemicals; therefore, microwave irradiation is utilized by neuroscientists to fix brain neurochemicals and metabolites in vivo while maintaining the brain's anatomic integrity (Stavinoha, 1993). c.

Antimicrobials

The potential of antibiotics to influence physiologic response is well founded. As they are commonly administered to animals used in biomedical research, it is imperative to recognize the nature and scope of their potential effects. Antibiotics may be toxic to a variety of laboratory animal species. Guinea pigs, hamsters, gerbils, and rabbits may develop fatal enterotoxemias, as a variety of antibiotics alter the bacterial microflora in the gut, permitting colonization and proliferation of either toxin-producing Clostridium difficile or C. spiroforme, dependent on the animal species, the route of administration, and the dose (Borriello, 1995; DeLong and Manning, 1994; Frisk, 1987). Although a host of antibiotics may induce the disease, penicillin and clindamycin are most commonly associated. Dihydrostreptomycin toxicity occurs in the gerbil (Wightman et al., 1980). Procaine, used in some penicillin formulations, is toxic to guinea pigs, mice, and rabbits (Galloway, 1968). The adverse effects of the aminoglycosides on renal function and hearing, as well as the potential of fluoroquinolones to damage cartilage, are well recognized by clinicians (Krasula and Pernet, 1991; Riviere and Spoo, 1995). Strain differences in the sensitivity to aminoglycoside (tobramycin) nephrotoxicity have been reported in Fischer 344 and Sprague-Dawley rats (Reinhard et al., 1991). Toxicity also fluctuates temporally in association with diurnal cycles (Linet al., 1996). Cardiovascular dysfunction has been associated with the administration of particular classes of antibiotics. Aminoglycosides produce negative inotropic effects in both cardiac and

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arterial muscle (Adams, 1976). Additionally, they alter the positive inotropism of other agents, including the catecholamines and calcium (Adams, 1976). These effects result from the interference with calcium-dependent membrane phenomena and inhibition of ganglionic neurotransmission (Adams et al., 1976). Lincomycin at high doses disrupts myocardial conductance and is arrhythmogenic in dogs (Daubeck et al., 1974). Aminoglycosides also inhibit synaptic transmission at the somatic myoneural junction (Adams, 1973). The clinical significance of neurotransmission blockade relates to the potential for respiratory arrest when antibiotics are administered concurrently with anesthetics or neuromuscular blocking agents. Antibiotics can also influence immune responsiveness. The tetracyclines have been extensively evaluated. They reduce delayed-type hypersensitivity (DTH) responses; inhibit mitogeninduced lymphocyte proliferation and NK cell activity; and alter granulocyte adherence, migration, and phagocytic ability (Belsheim et al., 1983; Goh and Ferrante, 1984; Ingham et al., 1991; Potts et al., 1983a,b; Thong and Ferrante, 1979, 1980). Similarly, the aminoglycosides have been shown to selectively suppress lymphocyte response to mitogens, depress chemotaxis, and inhibit the microbiocidal activity of phagocytes (Hauser and Remington, 1982; Metcalf and Wilson, 1987). Ciprofloxacin also causes immune perturbation (Jimenez-Valera et al., 1995). When administered to mice prior to immunization with sheep red blood cells, the IgG response is suppressed; however, the IgM response remains unchanged. When the antibiotic is administered 3 days after immunization, the IgM response is enhanced. These authors also demonstrated that ciprofloxacin suppressed the DTH response, inhibited mitogen stimulation of lymphocytes, caused leukopenia, and increased the number of granulocyte-macrophage colony-forming cells in the bone marrow. Both trimethoprim-sulfamethoxazole and chloramphenicol have been shown to depress the anamnestic antibody response following immunization (Hauser and Remington, 1982). Antibiotics may influence the pharmacokinetics and metabolism of other agents. Chloramphenicol inhibits hepatic microsomal enzymes. The concurrent administration of chloramphenicol and ketamine-xylazine or pentobarbital prolongs the duration of anesthesia (Adams and Dixit, 1970; Nossaman et al., 1990). The fiuoroquinolones compete with GABA receptors and therefore may interfere with studies involving the central nervous system (Green and Halliwell, 1997). Bacitracin, gentamicin, and nystatin alter cecocolonic motility and increase fecal excretion of dry matter and water in rats, and amoxicillinclavulanate alters intestinal motility in humans (Caron et al., 1991; Cherbut et al., 1991). Therefore, the concurrent administration of antibiotics with test compounds should be carefully considered. d.

Miscellaneous

There are countless other pharmaceuticals and biologics that would be expected to alter physiologic responses and there-

fore complicate the research utilization of the animals receiving them. Careful consideration must be given to the use and selection of any agent administered. Parasiticides and multivalent vaccines, both frequently administered to laboratory animals, are provided as examples. The anthelmintic levamisole has a variety of immune-potentiating effects, including stimulating cell-mediated immune responses, enhancing the rate of T-lymphocyte differentiation, and increasing the activity of effector lymphocytes (Brunner and Muscoplat, 1980). Ivermectin, commonly used both as an anthelmintic and acaricide, is toxic to certain strains of young mice as well as some genetically altered mice (Schinkel et al., 1994; Skopets et al., 1996). The lack of drug-transporting P-glucoproteins and other developmentally sensitive features of the blood-brain barrier are factors in its toxicity. Ivermectin also has immunostimulatory properties associated with the altered function of T-helper cells (Blakley and Rousseaux, 1991). Vaccination of dogs with modified live vaccines may be associated with thrombopathia and may alter immune function. Vaccination with mixed vaccines, routinely used for disease prevention in puppies and adult dogs, altered DTH responses for at least 2 months postvaccination (Miyamoto et al., 1992). In addition, vaccination of dogs reversed immune suppression in puppies resulting from halothane anesthesia (Taura et al., 1995). 3. Pheromones Pheromone signals are an important form of communication in many species and are involved in social and sexual behaviors in mice and rats (McClintock, 1998; Monahan and Maxson, 1998; Mucignat-Caretta et al., 1998; Vagell and McGinnis, 1998). In mammals, pheromones are detected by the vomeronasal organ, an olfactory sensory structure in the brain (Matsunami and Buck, 1997). The endocrine system of vertebrates and higher invertebrates has been shown to produce a variety of cyclic nucleotides, peptides, fatty acids, prostaglandins, and sterols with endocrine-altering effects (Brown, 1998). Male reproductive behavior in Syrian hamsters is dependent on pheromones from the female and presence of gonadal steroid hormones (Romeo et al., 1998). In mice, pheromones may have an inhibitory influence on action-potential generation and on cAMP levels in receptor cells of the vomeronasal organ, where olfactory receptor cells are thought to detect pheromone-like molecules important for reproductive physiology (Guo et al., 1997; Moss et al., 1998; Zhou and Moss, 1997). Animal odors from conspecifics or a differing species may lead to behavioral or physiologic alterations. Odors emanating from ferrets housed in an adjacent animal holding room have been observed to negatively impact the breeding performance of hamsters (G. E. Schneider, personal communication, 1988). Cat odor resulted in an increased release and decreased uptake of GAB A in hippocampal and cortical brain slices derived from exposed rats as compared to those derived from rats exposed to a neutral odor (File et al., 1993). When stressed, rats can release

29. FACTORSTHAT MAYINFLUENCEANIMALRESEARCH

odors (alarm chemosignals) from their body surface into the air as well as into their urine. These odors act as inhibitory or stimulatory activity signals on conspecifics (Mackay-Sim and Laing, 1981a,b). It was erroneously believed that normal blood (rat) exerted similar effects; however, it has been subsequently determined that the effects exerted by blood are dependent on the stress to which the rats were subject prior to blood collection and not simply due to a factor present in normal rat blood (Mackay-Sim and Laing, 1981a). BALB/c mice exposed for 24 hr to odors from donor mice that were foot-shocked had suppressed cellular and humoral immune function (Cocke et al., 1993). Odors from unfamiliar male mice cause primary developmental defects displayed by embryos developing in a deteriorated uterine environment (Chung et al., 1997).

C.

Microbial Agents

Microbes frequently confound research findings. As a result, there is a tremendous effort to produce, distribute, maintain, and use animals free of microbial pathogens. The effects that microbes may exert on biomedical research are multifaceted. Clinical disease, with its attendant morbidity and mortality, can devastate a research project, especially when disease strikes well into a study of prolonged course. More commonly however, the effects are more insidious. Microbes may induce histologic or biochemical alterations that increase the difficulty of interpreting results, or of greater concern, may lead to their misinterpretation. Diseases causing hepatic injury or renal damage may alter the biotransformation and excretion of experimentally administered compounds (Sipes and Gandolfi, 1991). Subtle immunomodulatory effects and the subsequent impact of microbial pathogens may go unrecognized. The routine use of genetically altered animal models, many of which have phenotypes that are influenced by the microbial flora with which they are associated, requires an improved understanding of the interaction between flora and host to fully understand and exploit these models. As the scope of this section is limited, the reader is referred to more extensive reviews on the impact of infectious agents on research by Bhatt et al. (1986), Institute of Laboratory Animal Resources (ILAR) (1991), and Baker (1998). The agents included in this section have the potential to cause pathologic lesions in a variety of organs. As descriptions of these can be found elsewhere in this book, they are not included in the discussion below. I.

Viruses a.

M o u s e Hepatitis Virus ( M H V )

Mouse hepatis virus is considered to be the most important and common viral pathogen of mice. The virus is distributed worldwide, is highly contagious, contaminates transplantable

1159

tumors and cell lines, and has repeatedly demonstrated its ability to alter host physiology and impact research. Having the potential to induce both immune stimulation and suppression, MHV may significantly alter the immune system. The effects of MHV in immunocompetent mice, following natural or experimental infection, include inducing involution and apoptosis in the thymus (Cray et al., 1993; Godfraind et al., 1995; Lee et al., 1994); affecting the number and tumoricidal activity of macrophages (Bo0rman et al., 1982); decreasing the proliferative responses of lymphocytes to mitogens (Cray et al., 1993; de Souza et al., 1991; Even et aL, 1995; Kryzstyniak and Dupuy, 1983); triggering polyclonal immunoglobulin production (Lardans et al., 1996); stimulating or depressing antibody response following immunization, dependent on on the timing of the immunization with respect to infection (Lahmy and Virelizier, 1981; Leray et al., 1982; Virelizier et aL, 1976); altering mucosal immune responsiveness (Casebolt et al., 1987); reducing the number of lactate dehydrogenase-elevating virus (LDV)permissive macrophages, delaying plasma lactate dehydrogenase elevation after infection (Dillberger et al., 1987; Even et al., 1995); altering allograft rejection (Cray et al., 1993); depressing phagocytic activity (Gledhill et al., 1965; Williams and DiLuzzio, 1980); inducing macrophage procoagulant activity (Levy et al., 1981); increasing production of interferon (IFN), IL-1, IL-2, IL- 6, IL-12, tumor necrosis factor (TNF), afetoprotein, and antiretinal antibodies (Coutelier et al., 1995; Even et al., 1995; Hooks et al., 1993; Mallucci, 1964; Pearce et al., 1994; Piazza et al., 1965; Schindler et al., 1982); depressing production of IL-2 and IL-4 (de Souza et al., 1991); altering the behavior of ascitic tumors and expression of cell surface markers on T cells (Fox et al., 1977; Nelson, 1959); activating NK cells (Schindler et al., 1982; Tardieu et al., 1980); inducing anemia, leukopenia, and thrombocytopenia (Hunstein et al., 1969; Namiki et al., 1977; Piazza et al., 1965); altering the course or susceptibility of mice to Sendai virus, pneumonia virus of mice (PVM), K virus, and leukoviruses as well as E p e r y t h r o z o o n coccoides, Salmonella typhimurium, and Schistosoma m a n s o n i (Braunsteiner and Friend, 1954; Carrano et al., 1984; Fallon et al., 1991; Gledhill, 1961; Gledhill et al., 1965; Lavelle and Bang, 1973; Manaker, 1961; Nelson, 1952a,b; Niven et al., 1952; Tisdale, 1963; Warren et al., 1969); altering hepatic fermokinetics (Tiensiwakul and Husain, 1979; Vacha et al., 1994); inducing immune-mediated demyelinization (Houtman and Fleming, 1996); reducing the incidence of diabetes in NOD mice (Wilberz et al., 1991); and altering hepatic regeneration, the number of hepatic sinusoidal fenestrae, and the proliferative activity of the bowel (Carthew, 1981; Barthold et al., 1982; Steffan et al., 1995). Mouse hepatitis virus also modulates a variety of enzyme systems. Hepatic isocitrate dehydrogenase, glucose-6-phosphate dehydrogenase, and aspartate transaminase are all markedly increased during infection, while cytochrome P450 microsomal enzymes, including those induced by phenobarbital, NADPH oxidase, aniline hydroxylase, and succinate hydrogenase, are significantly decreased

1160


(Budillon et al., 1972, 1973; Cacciatore and Antoniello, 1971; Paradisi et al., 1972; Ruebner and Hirano, 1965). Observations have shown that MHV also induces a number of important changes in immunocompromised mouse strains. Importantly, the extent and severity of lesions induced are considerably more severe in immunosuppressed animals (Huang et al., 1996; Ishida et al., 1978; Sebesteny and Hill, 1974; Ward et al., 1977). Mouse hepatitis virus also affects the immune system, causing spontaneous differentiation of lymphocytes bearing T-cell markers (Scheid et al., 1975; Tamura et al., 1978); enhances both the IgM and IgG antibody responses to sheep erythrocytes (Tamura et al., 1978; Tamura and Fujiwara, 1979); increases the number and phagocytic activity of macrophages (Tamura et al., 1980); enhances NK cell activity (Tamura et al., 1981); leads to xenograft rejection (Akimaru et al., 1981; Kyriazis et al., 1979); results in hepatosplenic myelopoiesis (Ishida et al., 1978); induces production of IL-1, IL-2, IL-6, and tumor necrosis factor (TNF) (Pearce et al., 1994); inhibits hepatic regeneration following partial hepatectomy (Carthew, 1981); and leads to the increase of a variety of hepatic enzymes and bilirubin (Huang et al., 1996). b.

Sendai Virus

Infection with Sendai virus (SV), an important pathogen of rats and mice, not only affects the respiratory system for which it has tropism, but also alters both humoral and cell-mediated immunity. Sendai virus depresses pulmonary bacterial clearance by altering pulmonary macrophage function (Degre and Glasgow, 1968; Degre and Solberg, 1971). The effects of SV on pulmonary macrophages include altering phagocytosis; inhibiting of phagosome-lysosome fusion; decreasing lysosomal enzymes; altering Fc and non-Fc receptor-mediated attachment; and altering the ability to degrade ingested bacteria (Jakab, 1981; Jakab and Warr, 1981). The effects of SV on the immune system are well characterized. The virus inhibits the response of T lymphocytes to mitogens (Garlinghouse and Van Hoosier, 1982; Roberts, 1982; Wainberg and Israel, 1980); causes a lifelong increase in cytotoxic T-cell precursors (Doherty et al., 1994); increases NK cell cytolytic activity and both the IgM and IgG splenic primary plaque-forming cell responses to sheep erythrocytes (Brownstein and Weir; 1987; Clark et al., 1979); stimulates TNF and interferon (INF) expression (Milone and Fitzgerald-Bocarsly, 1998; Payvandi et al., 1998; Uhl et al., 1998); enhances rejection of skin isografts (Streilein et al., 1981); reduces the severity of adjuvant-induced arthritis (Garlinghouse and Van Hoosier, 1978); inhibits the development of leukemia following inoculation of mice with Friend leukemia virus (Wheelock, 1966); and decreases the tumorigenicity of transplantable tumors (Matsuya et al., 1978; Takeyama et al., 1979). The effects of SV on immunity have been reported to be lifelong and include an increased prevalence of spontaneous autoimmune diseases (Kay, 1978, 1979; Kay et al., 1979). Prior or concurrent infection with SV alters the neoplastic response

of the respiratory system to carcinogens (Hall et al., 1985; Nettesheim et al., 1974, 1981; Parker, 1980; Peck et al., 1983), and the virus has also been shown to alter wound healing and interfere with early embryonic development and fetal growth (Kenyon, 1983; Lavilla-Apelo et al., 1992). c.

Lactate D e h y d r o g e n a s e - E l e v a t i n g

Virus (LDV)

Infection with LDV results in a lifelong viremia, which is the principal reason that LDV is one of the most common contaminants of transplantable tumors, infectious inocula, and biological materials originating from or passaged in mice. Elevation of one isozyme of lactate dehydrogenase, resulting from reduced clearance, is the hallmark of the disease, although other serum enzymes may also be elevated (Brinton, 1982). Carbon particle and asparaginase clearance is also reduced (Mahy et al., 1965; Notkins and Scheele, 1964; Riley et al., 1970). The immune system is also adversely affected by LDV. Infection with LDV results in elevation of serum gamma globulin concentrations, impairs the antigen-presenting capacity of macrophages, and alters the humoral immune response, dependent on chronicity of infection (Isakov et al., 1982a,b,c; Mergenhagen et aL, 1967; Michaelides and Simms, 1980; Notkins et al., 1966; Riley et al., 1975); causes polyclonal lymphocyte activation (Michaelides and Simms, 1980); delays allograft rejection (Howard et al., 1969); inhibits contact sensitivity to 2,4-dinitro fiuorbenzene (DNFB) (Hayashi et al., 1991); prevents experimental allergic encephalomyelitis (Inada and Mims, 1986); reduces autoimmune disease, adhesion molecule expression, and antinuclear antibody formation in NZB x NZWF1 mice (Hayashi et al., 1993; Kameyama and Hayashi, 1994; Oldstone and Dixon, 1972); depresses graft versus host disease (Notkins, 1971); stimulates NK cell activity and the production of IFN (Koi et al., 1981; Nicklas et al., 1988); alters macrophage superoxide anion production (Hayashi et al., 1992a,b); inhibits binding of asparaginase to monocytes (Mori et al., 1992); suppresses streptozotocin-induced insulitis (Hayashi et al., 1994); and potentiates the severity of Eperythrozoon coccoides, Plasm o d i u m yoelii, and Listeria m o n o c y t o g e n e s infections (Bonventre et al., 1980; Fitzmaurice et al., 1974; Henderson et al., 1978; Riley, 1964). Lactate dehydrogenase-elevating virus also enhances or suppresses the growth of transplantable and inducible tumors. Tumors transplanted shortly after infection demonstrate enhanced growth because of cellular immune suppression (Bailey et al., 1965; Michaelides and Schlesinger, 1974; Riley et al., 1978). Bittner agent-induced mammary tumors and the development of pulmonary adenomas following treatment with urethane are both suppressed by LDV (Riley, 1966; Theiss et al., 1980). d.

Lymphocytic Choriomeningitis Virus ( L C M )

Lymphocytic choriomeningitis virus is an important zoonotic pathogen and a common contaminant of transplantable tumors,

29. FACTORSTHATMAYINFLUENCEANIMALRESEARCH cell lines, and biological materials. The virus induces a persistent infection of T cells, resulting in lifelong viremia and viral shedding. The effects of LCM on research, following natural and experimental infection, include developing severe cellular and humoral immune suppression (Bro-Jorgensen and Volkert, 1974; Bro-Jorgensen et al., 1975; Guttler et al., 1975; LehmannGrube et al., 1972; Mims and Wainwright, 1968; Thomsen et al., 1982; Wu-Hsieh et al., 1988); enhancing NK cell activity, interferon production, macrophage function, and virus-specific cytotoxic T-cell proliferation (Blanden and Mims, 1973; Pfau et al., 1982; Ronco et al., 1981; Welsh, 1978; Welsh and Doe, 1980; Zinkernagel and Doherty, 1975, 1979); increasing endothelial adhesion molecules in serum (Christensen et al., 1995; Marker et al., 1995); altering cytokine gene expression (Colle et al., 1993); delaying skin allograft and tumor rejection (Guttier et al., 1975; Lehmann-Grube et al., 1972); increasing susceptibility of mice to ectromelia virus, E. coccoides, bacterial endotoxin, and irradiation (Barlow, 1964; Bro-Jorgensen and Volkert, 1972; Hotchin, 1962; Mims and Wainwright, 1968; Seamer et al., 1961); inhibiting the tumorigenic potential of polyoma virus, the Bittner agent, and Rauscher leukemia virus (Hotchin, 1962; Padnos and Molomut, 1973; Yuon and Barski, 1966); inactivating experimental hepatitis B infection (Guidotti et al., 1996); preventing the development of insulin-dependent diabetes mellitus in the BB rat and NOD mouse (Dyrberg et al., 1988; Oldstone, 1990; Schwimmbeck et al., 1988); and altering murine behavior, synaptic plasticity, and cognitive functions (de la Torre et al., 1996; Gold et al., 1994). Mouse Parvovirus and Rat Parvovirus Type 1 (MPV-1; RPV-1)

Mouse parvovirus and rat parvovirus type 1 have been identified as viruses that infect the mouse and rat, respectively. Although recognized serologically for several years, MPV-1 was first isolated after infecting and interfering with cultures of CD8 +, CD4 +, and y8 T-cell clones used for studying lymphocyte activation and immunoregulatory mechanisms (McKisic et al., 1993). In addition to causing lyric infection and inhibiting proliferation of T cells, MPV-1 was shown to inhibit T-cell proliferation following exposure to IL-2 and antigen; depress the proliferative response of spleen and lymph node from antigen-primed mice; and accelerate tumor allograft rejection, not by directly infecting the graft, but by inducing a "bystander help" effect (McKisic et al., 1993, 1996). RPV-1 is not as well characterized. It may suppress the development of lymphoid tumors (Jacoby and Ball-Goodrich, 1995). f.

Kilham Rat Virus (KRV) and H-1 Virus

Kilham rat virus contaminates transplantable tumors and cell lines. The virus alters in vitro responses of lymphocytes to mitogens and allogeneic lymphoid cells (Campbell et al., 1977); affects cytotoxic T-cell activity (Darrigrand et al., 1984); and

1161

induces interferon production (Kilham et al., 1968). Kilham rat virus also suppresses the development of leukemia induced by the Moloney murine leukemia virus (Bergs, 1969); induces diabetes mellitus in the diabetes-resistant BB/Wor strain (Brown et al., 1993); alters lipid metabolism (Schuster et aL, 1991); alters leukocyte adhesion to aortic endothelium (Gabaldon et al., 1992); and may reduce the incidence of Yersinia-associated arthritis (Gripenberg-Lerche and Toivanen, 1993, 1994). H-1 virus inhibits tumor induction by DMBA and adenovirus in hamsters (Toolan and Ledinko, 1968; Toolan et al., 1982); causes hepatic necrosis in rats exposed to other pathogens and hepatotoxic chemicals (Kilham and Margolis, 1970; Ruffolo et al., 1966); and may reduce the incidence of arthritis associated with Yersinia (Gripenberg-Lerche and Toivanen, 1993, 1994). As both viruses replicate in and are cytolytic to dividing cells, they also have the potential to alter fetal development or to be teratogenic. g.

Minute Virus o f Mice (MVM)

Minute virus of mice is a common contaminant of transplantable tumors, cell lines, and virus stocks. Evidence that MVM may interfere with research is based principally on studies of the immunosuppressive variant, MVM(i), although experimental inoculation of the prototype strain, MVM(p), inhibits the growth of intraperitoneally administered Ehrlich ascites tumor cells (Guetta et al., 1986). It is unclear whether MVM(i) occurs as a natural pathogen of mice; however, MVM(i) is immunosuppressive (Segovia et al., 1995); cytolytic to T-cell clones (Tattersall and Cotmore, 1986); inhibits cytotoxic T lymphocytes (Bonnard et al., 1976; Herbermann et al., 1977); suppresses the response of T cells to mitogens (McMaster et al., 1981); and suppresses T helper-B cell responses (Engers et al., 1981). h.

Sialodacryoadenitis Virus (SDA)

Sialodacryoadenitis virus may potentially interfere with research involving the lacrimal and salivary glands, the respiratory system, the eyes, or fetal and neonatal development. Effects associated with SDA infection include alteration of estrous cycles (Utsumi et al., 1991); embryonic and neonatal mortality (Utsumi et al., 1991); impairment of olfaction and chemoreception for up to 2 weeks postexposure (Bihun and Percy, 1995); alteration of growth rates in young rats (Utsumi et al., 1980); reduction in food consumption and weight gain (Nunoya et al., 1977); depletion of epidermal growth factor in salivary glands (Percy et al., 1988); and reduction of IL-1 production by alveolar macrophages (Boschert et al., 1988). 2.

Bacteria

a.

Citrobacter rodentium

The etiologic agent of transmissible murine colonic hyperplasia significantly alters the cytokinetics of the colonic mucosal

1162


epithelium (Barthold, 1979). Both the susceptibility to colonic neoplasia and the latent period for induction are increased in mice infected with C. rodentium and exposed to the carcinogen, 1,2-dimethylhydrazine (Barthold and Beck, 1980; Barthold and Jonas, 1977). The hyperplastic colonic lesions may also be misinterpreted as preneoplastic, as they resemble focal atypia. Citrobacter rodentium may also cause immunologic alterations, including inhibition of antigen-specific cytotoxic T-cell activity (Maggio-Price et al., 1998). b.

Clostridium piliforme

Frequently a subclinical infection, Clostridium piliforme causes overt Tyzzer's disease when animals are subject to experimental manipulations, such as whole body X-irradiation (Taffs, 1974; Takagaki et al., 1966); treatment with cortisone or adrenocorticotropic hormone (ACTH) (Takagaki et al., 1966; Yamada et al., 1969); feeding with a high-protein diet (Maejima et al., 1965); exposure to carbon tetrachloride (Takenaka and Fujiwara, 1975); and intraperitoneal passage of ascitic tumors (Craigie, 1966). Tyzzer's disease increases hepatic transaminases and alters the pharmacokinetics of trimethoprim and warfarin (Fries and Ladefoged, 1979; Naiki et al., 1965). c.

Helicobacter spp.

The importance of this expanding genus on research is becoming well recognized. Helicobacter hepaticus has considerable potential to influence research studies, as the bacterium is associated with progressive hepatitis, proliferative typhlitis, and/or colitis in immunocompetent and immunodeficient mouse strains (Foltz et al., 1998; Li et al., 1998; Ward et al., 1994a,b); causes hepatocellular and hepatocholangiolar adenomas and carcinomas in A/JCr and B6C3F1 mice (Fox et al., 1996a; Hailey et al., 1998; Ward et al., 1994a,b); elevates hepatic transaminase levels in serum (Fox et al., 1996b); and serves as an immunologic target promoting the development of inflammatory bowel disease in gene-targeted mice and in an adoptively transferred severe combined immunodeficient (SCID) mouse model (Cahill et al., 1997; Kullberg et al., 1998). The proneoplastic effects of H. hepaticus on the liver are mediated by increasing the levels of select cytochrome P450 isoforms involved in carcinogen bioactivation (Chomarat et al., 1997; Sipowicz et al., 1997); enhancing glutathione S-transferase activities while decreasing the activity of glutathione peroxidase and the content of glutathione (Chomarat et al., 1997; Sipowicz et al., 1997); increasing the production of superoxide (Chomarat et al., 1997); altering cytokinetics (Nyska et al., 1997); stimulating cyclin D expression, accelerating the development and progression of hepatic tumors in carcinogen-treated mice (Diwan et al., 1997); up-regulating a variety of growth factors, cell cycle proteins, and transcription factors (Ramljak et al., 1998); and producing a cytotoxin (Taylor et al., 1995). A1-

though not as well characterized, H. bilis induces similar lesions in immunocompromised mice and rats, but no association with hepatic neoplasia has been reported (Franklin et al., 1998; Haines et al., 1998; Shomer et al., 1997). d.

Mycoplasma pulmonis

Naturally occurring mycoplasmosis of rodents is an insidious disease that may significantly impact research in a variety of disciplines, especially long-term studies, as the organism may disseminate widely in the host. Mycoplasma pulmonis infection is likely to impact studies of the respiratory tract, as it alters mucociliary clearance (Cassell et al., 1981); ciliary function (Irvani and van As, 1972; Westerberg et al., 1972); cell kinetics (Wells, 1970); and immune function, as it alters both the number and subpopulation distribution of lymphocytes in the lung (Cole et al., 1975; Davis et al., 1982; Naot et al., 1979a,b). Mycoplasma pulmonis has been demonstrated to enhance the pulmonary response to carcinogens (Schreiber et al., 1972). Infection of the genital tract alters fecundity, as M. pulmonis affects embryo implantation, spermatozoan motility, and fertilization; skeletal development and ossification are also altered (Cassell, 1982; Fraser and Taylor-Robinson, 1977; Goeth and Appel, 1974; Lal et al., 1980; Leader et al., 1970). The effects of M. pulmonis on the immune system include increasing NK cell activity (Lai et al., 1987); suppressing humoral antibody response (Aguila et al., 1988b); stimulating the production of mitogenic substances for B and T cells (Proust et al., 1985; Ross et al., 1992); enhancing production of TNF, IL-1, IL-6, and interferon (Faulkner et al., 1995); and reducing the incidence and severity of collagen and adjuvant-induced arthritides (Taurog et al., 1984). Subclinical mycoplasmosis exacerbates vitamin A and E deficiency (Lindsey et al., 1986). e.

Pasteurella multocida

Pasteurella multocida causes a variety of clinical syndromes in rabbits, all of which can seriously impact research. More subtle effects of pasteurellosis have also been described. Nasal instillation of P. m u l t o c i d a - f r e e rabbits with a suspension of the organism resulted in the expression of the adhesion molecule VCAM-1 (Richardson et al., 1997a). Richardson et al. (1997b) also demonstrated the ability of P. multocida to enhance atherosclerosis, as naturally and experimentally infected rabbits fed a high-lipid diet had increased VCAM-1 expression as well as enhanced aortic sudanophilia. f

Pseudomonas aeruginosa

A common commensal in many species as well as a ubiquitous environmental contaminant, Pseudomonas aeruginosa is of major importance to immunocompromised subjects and studies employing the use of indwelling catheters and other percuta-

29. FACTORSTHATMAYINFLUENCEANIMALRESEARCH

neously implanted devices. Effects associated with infection include high mortality following administration of immunosuppressive drugs (Hazlett et al., 1977; Millican, 1963; Pierson et al., 1976; Rosen and Berk, 1977, Schook et al., 1977; Urano and Maejima, 1978); premature death after exposure to lethal radiation, cytomegalovirus infection, or cold stress (Flynn 1963a,b,c; Halkett et al., 1968; Hamilton and Overall, 1978; Hammond et al., 1954, 1955; Hightower et al., 1966; Vincent et al., 1955); increased fibrosis following airway infection (Mclntosh et al., 1992); depressed contact sensitivity to oxazolone (Campa et al., 1975, 1976, 1977); altered pulmonary epithelium fluid transport (Pittet et al., 1996); stimulation of the release of immunosuppressive factors from macrophages (Marshall et al., 1993); thymic atrophy and apoptosis (Wang et al., 1994); proliferation of immature splenic T cells in nude mice (Dixon and Misfeldt, 1994); T-cell dependent immune suppression (Haslov et al., 1992); enhanced cardiac automaticity and depression from hypoxia (Kwiatkowska-Patzer et al., 1993); impaired wound healing (Heggers et al., 1992); and altered behavioral and clinical pathologic parameters following experimental wound infection (Bradfield et al., 1992). 3.

Fungi

Infections caused by fungi, with the exception of Pneumocystis carinii in immunocompromised hosts, are relatively uncommon in laboratory-bred animals. Pneumocystis carinii alters alveolar capillary permeability (Yoneda and Waltzer, 1980); induces activating and inhibitory innate cellular immune-response mechanisms (Warschkau et al., 1998); increases levels of TNF, IL-1, IL-6, IL-8, and arachidonic acid and its metabolites (Castro et al., 1993; Chen et al., 1992, 1993; Kolls et al., 1993; Lipschik et al., 1996; Pottratz et aL, 1998); induces expression of the adhesion molecule ICAM-1 and inhibits cyclin-dependent kinase activity and fibrinogen expression in pulmonary epithelium (Limper et al., 1998; Simpson-Haidaris et al., 1998; Yu and Limper, 1997); alters pulmonary GTP-binding proteins and the amount and type of surfactant produced (Kernbaum et al., 1983; Oz and Hughes, 1997; Sheehan et al., 1986); and modifies the uptake of intratracheally administered compounds (Mordelet-Dambrine et al., 1992). 4.

Parasites

Parasites can confound the experimental use of animals in a variety of ways, including inducing an immune response, altering immune responsiveness, competing with the host for nutrients, consuming body fluids, altering cytokinetics, stimulating or depressing tissue growth, causing mechanical obstructions, altering host physiology and biochemistry, and affecting behavior. They may also transmit or enhance the pathogenicity of other infectious agents. In addition, anthelmintic and acaricide administration may significantly alter the biology of the host.

1163

Some examples of the effects of parasites on experimental results are well known. The carcinogenicity of saccharin was questioned, as the rats used to evaluate the compound were infected with Trichosomoides crassicauda, which causes hyperplasia or papillomatosis of the urinary bladder (Homburger, 1978). Gongylonema neoplasticum is associated with esophageal tumors in the rat. However, the effects of parasites are likely to be more subtle. Pinworms are commonly found in rodents and can alter a variety of biological processes. Oxyuriasis is reported to reduce the development of adjuvant-induced arthritis (Pearson and Taylor, 1975); induce splenic T- and B-cell proliferation and occasional germinal center formation (Beattie et al., 1981); increase the antibody response to sheep erythrocytes (Sato et al., 1995); retard growth (Wagner, 1988); impede colonic water and electrolyte absorption (Lubcke et al., 1992); accelerate the development of the hepatic monooxygenase system (Mohn and Philipp, 1981); be associated with the development of lymphomas in nude mice (Baird et al., 1982); and cause a significant reduction of activity in behavioral studies (McNair and Timmons, 1977). Fur mites, also commonly found in rodents, may significantly alter the histologic features of the skin, activate the immune system, alter animal behavior, and inhibit reproduction. Specifically, mites cause secondary amyloidosis (Galton, 1963; Weisbroth, 1982); alter the production of both IL- 2 and IL-4 and the immunoglobulin isotype profile in serum (Jungmann et al., 1996a,b); cause lymphocytopenia and granulocytosis (Jungmann et al., 1996a,b); and reduce the contact sensitivity reaction to oxazolone (Laltoo and Kind, 1979). The response to mites in mice is strain-specific. The inbred mouse strain NC develops an exaggerated clinical and immunologic response to infestation with Myocoptes musculinus (Morita et al., 1999). The NC strain develops severe pruritis, skin lesions, and elevated serum IgE levels as compared to similarly infested BALB/c and C57BL/6 strains. Encephalitozoon cuniculi infects a broad host range; however, infections are uncommon in most species bred for the laboratory with the exception of the rabbit. Parasite-induced lesions in the brain and kidney may make interpretation of experimental evaluation of these organs difficult. Encephalitozoon cuniculi may also alter immune responsiveness. The protozoan is reported to increase NK cell activity (Niederkorn et al., 1983; Niederkorn, 1985); reduce cellular and humoral responses to a variety of immunogens (Cox, 1977; Cox and Gallichio, 1978; Didier and Shadduck, 1988; Niederkorn et al., 1981; Waller et al., 1978); and alter host responsiveness to transplantable tumors (Arison et al., 1966; Meiser et al., 1971; Petrie, 1966). D.

Stressors

Stress is the effect produced by external or internal factors that induce an alteration in an animal's biological equilibrium

1164

(National Research Council, 1992). The alterations that result, dependent on the severity, nature, and duration of the stressor, may significantly impact experimental findings. The response to stress is influenced by a variety of factors not limited to host sex, age, and genetics. Stress results in stimulation of the hypothalamic-pituitary axis, resulting in release of corticosteroids from the adrenal cortex and catecholamines from the adrenal medulla. Opioids, released centrally and peripherally, also mediate stress effects (Madden et al., 1977; Plotnikoff et al., 1985; Wybran, 1985). Stress elicits changes in numerous immunologic processes. A variety of murine tumor-host model systems have been used to demonstrate the effects of stress on tumor development (Fitzmaurice, 1988; Riley, 1975; Sklar and Anisman, 1979; Visintainer et al., 1982). Stress-induced alterations in tumor model systems include tumor rejection inhibition, decreased tumor survival, and altered tumor latency. Riley (1975) demonstrated that the incidence of mammary tumors in C3H/He mice carrying the Bittner agent could be altered from 7 to 92% at 400 days of age by increasing the degree of environmental stress to which the mice were subject. Stress may be a direct result of experimental manipulation or may be caused by environmental or psychosocial factors. Many stressors are discussed in this chapter. Handling, acute and chronic restraint, social interaction with humans, and activity of personnel in animal holding rooms are additional factors that have been reported to result in physiologic and/or behavioral changes, that, dependent on the design and nature of the study, could influence results (Long et al., 1991; Misslin, 1982; Nerem et al., 1980; Saibaba et al., 1996; Tuli et al., 1995a,b). One stress to which almost all animals are subject is that which occurs during transport between institutions. Numerous studies have documented the impact of transportation in a variety of laboratory species. Alterations attributable to shipment include elevation of plasma glucocorticoid concentrations, neutrophilia, lymphopenia, hyperglycemia, changes in serum biochemical indices, reduced splenic NK cell activity, depressed humoral and cell-mediated responses to sheep red blood cells, thymic atrophy, depressed food consumption, weight loss, depressed reproductive performance, behavioral alterations, increased susceptibility to disease, and decreased latency and incidence of tumors (Aguila et al., 1988a; Bean-Knudsen and Wagner, 1987; Dymsza et al., 1963; Hayssen, 1998; Landi et al., 1982; Peters and Kelly, 1977; Riley, 1975; Toth and January, 1990; Tuli et al., 1995b; Wallace, 1976). In general, altered immunologic parameters return to baseline within 48 hr after arrival, although corticosterone levels remained elevated in mice when measured at 48 hr (Aguila et al., 1988; Landi et al., 1982; Toth and January, 1990). Behavioral and reproductive function may require greater acclimatization before returning to normal. A 96 hr acclimatization period was insufficient for mouse behavior to return to normal after transport (Tuli


et al., 1995b). Reproductive performance was depressed in transatlantic-shipped deer mice for 110 days after arrival (Hayssen, 1998). Alterations attributed to transportation are detectable even when animals were shipped by environmentally controlled ground transportation (Aguila et al., 1988; Toth and January, 1990). When comparing air to ground transport, investigators found either no differences or greater physiologic change induced by air transport (Aguila et al., 1988; Landi et al., 1981; Toth and January, 1990). Transportation-induced changes could be detected following transport for as little as 4 hr (Toth and January, 1990). Interinstitutional shipment of animals is not the only source of transport stress. Drozdowicz et al. (1990) examined the effects of in-house transport in mice and found that moving animals on a cart for 12 min led to an increase in plasma corticosterone, a decrease in circulating white blood cells and lymphocytes, and reduction in thymic weight. Corticosterone, WBC, and lymphocyte counts returned to normal within 4 hr of transport; however, the circadian release of corticosterone remained abnormal for 1 day. Social interaction is an important experimental consideration. An isolation stress p h e n o m e n o n is well recognized in rodents. Housing animals in isolation influences a variety of behavioral and physiologic parameters, including creatinine clearance; urine flow rates; fractional reabsorption of sodium; antipyrine clearance; plasma glucose, triglyceride, and corticosterone levels; the incidence of spontaneous tumors; the prevalence of degenerative joint disease; humoral and cell-mediated immune response; body weight; behavioral reactivity; ethanol consumption, drug toxicity, and susceptibility to challenge with infectious agents; and the response of tumor allografts to chemotherapy (Benton and Brain, 1981; Brown and Grunberg, 1995, 1996; Brunner et al., 1994; Capitanio and Lerche, 1998; Consolo et al., 1965; Dairman and Balazs, 1970; Everitt et al., 1988; Haseman et al., 1994; Hilakivi-Clarke and Dickson, 1995; Jessop and Bayer, 1989; Karp et al., 1993; Kerr et al., 1997; Meisel et al., 1990; Nyska et al., 1998; Parker and Radow, 1974; Perez et al., 1997; Rabin and Salvin, 1987; Rao and Lindsey, 1988; Shanks et al., 1994; Steplewski et al., 1987; Vadiei et al., 1990; Vargas-Rivera et al., 1990; Weinreich et al., 1996). Temporary maternal separation may cause sufficient stress and nutritional deprivation to delay development. For example, neonatal rat pups separated from their dam for 6 hr a day resulted in delayed developmental landmarks, including righting ability, negative geotaxis, and eye opening. Body and brain weights were also decreased in comparison to those of controls (Vitarella et al., 1998). The effects of housing density and group size have been long recognized and must be distinguished from each other. Housing density reflects the number of animals housed per unit space. Group size can be increased without altering housing density by increasing cage space proportionally as the population is increased. When the same floor space per animal is provided, dif-


ferences in behavioral and physiologic parameters are more easily detected when comparing housing in isolation to that in groups, as differences between group sizes are difficult to distinguish (Bell et al., 1972; Davis, 1978). Increased housing density has been associated with a variety of biological effects, including declines in reproductive performance and increases in aggressive behavior (Christian and LeMunyan, 1958; Davis, 1978; Welch and Welch, 1969). In general, increasing housing density has an immunosuppressive effect. Increases in plasma corticosterone, reduction in the number of peripheral granulocytes and lymphocytes, and reductions in superoxide production and neutrophil phagocytic activity have been reported with increasing housing density (Peng et al., 1989; Tsukamoto et al., 1994). It has also been shown to increase the susceptibility of mice to coxsackievirus and Plasmodium berghei infection, enhance the diabetogenic response of streptozotocin, decrease antibody synthesis, depress homograft rejection, enhance the development of autoimmune disease, and alter the size and constituents of various regions of the brain (Johnson et al., 1963; Mazelis et al., 1987; Plaut et al., 1969; Solomon, 1969; Vessey, 1964; Welch et al., 1974). However, select studies on housing density have reported immune enhancement that may have resuited from differences in gender, strain, and housing condition (Joasoo and McKenzie, 1976; MacManus et al., 1971).

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Chapter 30 Animal Models in Biomedical Research Fred W. Quimby

I.

II.

III.

an Animal Model? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Types of Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Transgenesis and Mutagenesis: Creating Models for the Future . . . . . C. Sources of Animal Models . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Nature of Research . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Hypothesis Testing and Serendipity . . . . . . . . . . . . . . . . . . . . . . . . . . B. Breakthroughs in Technology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. T a k i n g Advantage of Interspecies Similarities: Research on Obesity. History of Animal Use in Biomedical Research . . . . . . . . . . . . . . . . . . . . . What Is

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Early History

............................................

From

Animal models have been used to understand the laws of nature throughout the millennium; however, since the scientific method was conceived, their use has accelerated and our knowledge has greatly expanded. Animal models include prokaryotes, invertebrates, and vertebrates, and each has played a significant role in defining principles in the biological sciences. Due to the high conservation of DNA between homologous genes and similar physiologic characteristics, vertebrate animals have frequently been the choice of investigators developing new drugs and medical devices to improve human health. For exactly the same reasons, vertebrate animals themselves have often been beneficiaries of such research. This chapter explores facets of the history of biomedical science and hypothesizes a scientific revolution in biologyma revolution where the principles of physics, chemistry, mathematics, and computer science will merge with biology to create advances unthinkable before. In LABORATORY ANIMAL MEDICINE,

2nd edition

1197 1200 1200 1201 1204 1206 1206

Pasteur to Prusiner . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Animals as Recipients of Animal Research . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B.

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this revolution the lowly mouse will reign as the principal animal model for investigations.

I.

WHAT IS AN ANIMAL MODEL?

A.

Types of Models

1. Introduction There are many types of models used in biomedical research, e.g., in vitro assay, computer simulation, mathematical models,

and animal models, and although living vertebrate animals represent only a fraction of the models used, they have been responsible for many important advances in biology and medicine Copyright2002, ElsevierScience (USA). All rightsreserved. ISBN 0-12-263951-0

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(National Research Council [NRC], 1985). Invertebrate models have made a profound impact in the areas of neurobiology, genetics, and development and include the nematode Caenorhabditis elegans, protozoa, cockroaches, sea urchins, the fruit fly Drosophila melanogaster, Aplysia, and squid, among others. The sea urchin, for instance, contributed to the discovery of meiosis, events associated with fertilization, discovery of cell sorting by differential adhesion, basic control of cell cycling, and cytokinesis (NRC, 1985). Similar lists can be prepared for insects, squid, and other marine invertebrates. These nonvertebrate models have been previously reviewed and are not the major focus of this discussion (NRC, 1985; Jasny and Koshland, 1990; Huber et al., 1990; Schulhof and Miller, 1990; Woodhead, 1989). A model, whether animal or nonanimal, is meant to be a mimic or surrogate and not necessarily identical to the subject being modeled (NRC, 1998; Scarpelli, 1997). In this chapter it is assumed that the human biological system is the subject being modeled; however, it is noteworthy that many of the advances made through studies of animal models have been applicable to animals other than humans (see Section III,C). Conceptually, animals may model analogous processes (e.g., relating one structure or process to another) or homologous processes (e.g., reflecting counterpart genetic sequences). Homology-based modeling appears to be uniquely biological, whereas finding corresponding features between two animals (or physical processes) is commonplace in physics, mathematics, and engineering (NRC, 1985). Prior to the current interest in genomics, many animal models were selected as analogs of human processes or conditions (they appeared similar); today we recognize many to be true homologies. However, caution should be exercised in interpreting the benefit of an animal homolog of humans; the physiologic adaptations occurring during the evolution of certain species may make their homologs poor analogs. Similarly, convergent evolution may result in good analogs from genetically distant structures and processes. Another useful concept in modeling concerns one-to-one modeling versus many-to-many modeling. In one-to-one modeling of a normal (or abnormal) human process, a species is sought that demonstrates analogous behavior for several features of the process and no negative features. This species is then considered a good model for this process, and when used in investigation, fruitful information is expected (consider an infectious disease like salmonellosis where the signs and symptoms in a monkey may be identical to those in humans; or a genetic disease such as X-linked muscular dystrophy where the signs in dogs resemble those in humans). Many-to-many modeling results from analysis of a process in an organism where each component feature of that process is evaluated at several hierarchical levels, e.g., system, organ, tissue, cell, and subcellular levels. Then, at each level all the taxa (species) are noted in which analogous features appear. Using this approach, a new kind of epistemic structure emerges, and the matrix of biologi-

FRED W. QUIMBY

cal knowledge replaces the one-to-one model for reaching an understanding, particularly of complex processes (NRC, 1985; Office of Technology Assessment [OTA], 1986). An attempt has been made to illustrate the advantages of the many-to-many model in the discussion of models for aging (see Section I,B, 2,b). Studies comparing analogies (and homologies) between many taxa have also contributed greatly in the understanding of central nervous system function, embryogenesis, and endocrinology. The current interest in comparative genomics promises for the first time to bring new order to the study of biology, where comparing the structure of genes to the function of their products may give new insight into the evolutionary history of species and what it means to be human. Animal models are used to elucidate basic physiologic, biochemical, and genetic processes found in health and disease. Rarely will the model share all the clinical, pathologic, and functional characteristics of the disease with humans, and while desirable, this is not necessary for the model to make substantial contributions on disease pathogenesis, prevention, and treatment. The differences between animal models and humans afflicted with type 1 (juvenile) insulin-dependent diabetes mellitus, Duchenne muscular dystrophy, and pancreatic ductal adenocarcinoma have been recorded, yet these models have contributed greatly to the understanding of the molecular basis of, and treatments for, these disorders (NRC, 1998; Scarpelli, 1997). In this context it is important to note that despite the many different factors modifying the evolutionary history of humans and that of the mouse, resulting in numerous differences in morphology and metabolism, there remains an impressive degree of genetic conservation between these species, including high sequence homology for both genes and their translated proteins, which appear to have similar, if not identical, functions in the two species (Davisson et al., 1998). Syntenic maps that document the order of genes along chromosomes between these species also demonstrate impressive similarities. Such mapping has aided the identification of new genetic loci in both species and in part explains the organization of complex processes that are often shared by both species (Copeland et al., 1993; Nadeau et al., 1989; Davisson et al., 1998). The characterization of murine models of various dwarfing syndromes has lead to an understanding of dwarfing in humans and suggested potential therapies (NRC, 1998). 2.

Spontaneous and Induced Animal Models

Animal models can be classified as spontaneous or induced. Spontaneous models may be represented by normal animals with physiologic mechanisms similar to those of humans or by abnormal members of a species that arise naturally through mutation(s). In contrast, normal animals submitted to surgical or another manipulation resulting in an abnormal physiologic state are induced models. The single largest category of induced models is that arising through genetic manipulation. Currently,

30. ANIMAL MODELS IN BIOMEDICAL RESEARCH

one new such animal model is being described each week. Occasionally, investigators will refer to another category of animal model, the so-called negative model. This is an animal that fails to develop a particular disease, e.g., infectious disease, or fails to respond to specific physiologic stimuli. Often insights into the genetic constitution of these animals lead to the discovery of genes (and proteins) that impart resistance to disease or an induced disorder, e.g., autoimmunity. Some of the best-characterized models are those with naturally occurring mutations that lead to disorders similar to those in man. Among the best-known spontaneous models are the Gunn rat (hereditary hyperbilirubinemia), piebald lethal and lethal spotting strains of mice (aganglionic megacolon), nonobese diabetic mouse and BB Wistar rats (type 1 diabetes mellitus), New Zealand Black and New Zealand White mice and their hybrids (autoimmune disease), nude mice (DiGeorge syndrome), SCID mice (severe combined immunodeficiency), Watanabe rabbits (hypercholesterolemia), Brattleboro rats (neurogenic diabetes insipidus), obese chickens (autoimmune thyroiditis), spontaneously hypertensive rats (SHR-primary hypertension), dogs and mice with Duchenne X-linked muscular dystrophy, dogs with hemophilia A and B, swine with hyper-low-density lipoproteinemia and malignant hyperthermia, mink with Chediak-Higashi syndrome, cats with achalasia, gerbils with epilepsy, cattle with ichthyosis congenita and hyperkeratosis, and sheep with Dubin-Johnson syndrome (Andrews et al., 1979; also see Section I,C). It is clear even from this very brief list that the types of diseases and the breadth of species involved are great. Induced models have been used to unravel some of the most important concepts in physiology and medicine. Whereas surgical models in nonhuman primates have contributed greatly to the understanding of brain plasticity following sensory-input deprivation and the largely separate cognitive domain with its own perception, learning, and memory experiences found in each cerebral hemisphere (Merzenich, 1998; Florence et al., 1998; Jones and Pons, 1998), surgical models in dogs led to breakthroughs in organ transplantation; coronary bypass surgery; balloon angioplasty; replacement of heart valves; development of cardiac pacemakers; the discovery of insulin and the treatment of diabetes mellitus; fluid therapy and other treatments for shock, liver failure, and gallstones; and surgical resection of the intestines, including the technique of colostomy (Council on Scientific Affairs, 1989; Quimby, 1994a; Bay et al., 1995; Quimby et al., 1995). Additional useful models have been induced by diet or administration of drugs or chemicals. Combining the chemicals alloxan and streptozotocin was found to selectively destroy the beta cells of the islets of Langerhans, rendering rats, rabbits, and other animal models susceptible to insulin-dependent diabetes mellitus (Sisson and Plotz, 1967; Golob et al., 1970). This model in mice has recently demonstrated NAD depletion by poly(ADP-ribose) polymerase as a dominant metabolic event in islet cell destruction, which may

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be useful in developing therapeutic strategies for type 1 diabetes (Burkhart et al., 1999), However, for studies of disease pathogenesis, the spontaneous mutant NOD mouse and B B rat are more analogous (and perhaps homologs) to human type 1 diabetes (Atkinson and Leiter, 1999). In 1981, investigators discovered that the drug 1-methyl-4 phenyl-l,2,3,6-tetrahydropyridine (MPTP) produced clinical signs of Parkinson's disease when injected into nonhuman primates. Further studies confirmed that this chemical destroyed the dopamine-producing cells of the brain-re-creating the pathologic lesion in the human disease (Lewin, 1984). The model system has contributed greatly to our knowledge of the structure and function of the basal ganglia as well as the mechanism of neurodegeneration (Grtinewald and Beal, 1999) and to the evaluation of new therapies such as transplantation and pallidotomy (Tolwani et al., 1999). Diet-induced models have been responsible for discovery of most vitamins and the necessity for trace minerals as nutrients, as well as for exploration of the pathogenesis of many diseases, e.g, atheroslcerosis. Eijkman's observations of chickens with beriberi (thiamin deficiency) resulted in a cure for humans (and animals) and led to the discovery of vitamins (Eijkman, 1965; see Section II,A,2). In fact, dietary manipulations in the chicken alone have contributed to our knowledge of rickets (Kwan et al., 1989), vitamin A deficiency (Band et al., 1972), vitamin B 6 deficiency (Masse et al., 1989), zinc deficiency (O'Dell et al., 1990), Friedreich's ataxia (van Gelder and Belanger, 1988), fetal alcohol syndrome (Means et al., 1988), and atherosclerosis (Dauber, 1944; Pick et al., 1952). Often complex induced models are used by combining drugs, surgery, diet, and infectious agents. An excellent example is the hu-SCID mouse, where a natural mutation in the RAG1 gene prevents T- or B-cell antigen receptor rearrangements, resulting in a severe combined immunodeficiency. When this mouse is injected with human lymphocytes or stem cells, it adopts the immune system of humans (Carballido et al., 2000). Finally, injection of this reconstituted mouse with HIV- 1 virus leads to viral propagation and a small-animal model for the assessment of anti-HIV drugs (Mosier et al., 1991). a. From past advances in amphibian developmental biology to the current global decline in frogs. An example illustrating the importance of previous basic science discoveries using normal animals to the contemporary problems facing people today relates developmental biology in frogs to the current problem of associating frog deformities and population declines with environmental agents. The African clawed frog, Xenopus laevis, has been an important model for elucidating various aspects of embryogenesis by acting at the interface of molecular and developmental biology. The Xenopus system was responsible for the first isolation of a eukaryotic gene, the initial studies on gene amplification, the first demonstration of accurate transcription from a cloned gene, and the first isolation and characterization of a eukaryotic transcription factor (Dawid and Sargent, 1988).

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Together, these discoveries have led to a frog oocyte expression least one report demonstrated embryotoxicity and deformities system that allows for efficient translation of proteins from in- (skeletal) in larvae hatching from eggs exposed to unshielded jected mRNA, as well as a functional transcription factor assay. ambient UVB radiation (Blaustein et al., 1997), and synergism The original Spermann-Mangold experiment conducted in between UVB radiation and the fungal parasite Saprolegnia 1924 demonstrated that the earliest visible sign of gastrulation, ferax was shown to increase embryo mortality for several spedevelopment of the dorsal lip, served as a central organizer ca- cies of frogs (Kiesecker and B laustein, 1995). Large-scale frog pable of directing mesodermal and neural derivatives in the em- die-offs have been associated with infections, including by the bryo. Later it was discovered that members of the transforming fungus Batrachochytrium dendrobatidis (Berger et al., 1998; growth factor (TGF)-[~2 family, located in the egg and dispersed Morell, 1999) and iridoviruses (Daszak et al., 1999); however, by cytoplasmic flow before the first cleavage stage, bestow only infestation by the digenetic trematode Ribeiroia has been distinct properties on vegetal blastomeres that allow them to associated with frog deformities. The trematode infestations are induce directed differentiation, embryo polarity, and ordered associated with extra or missing hindlimbs, but they cannot exglobal structure during embryogenesis (Dawid and Sargent, plain the full range of deformities seen in Minnesota (Johnson 1988). This organized sequence of development was abrogated et al., 1999; Sessions et al., 1999). An ichthyophonus-like inby exposure of eggs to ultraviolet light. Amphibian, especially fection has been described in frogs from areas characterized frog, development has been widely studied because of the di- by high deformity rate (Mikaelian et al, 2000; Babbit, Bowser, versity of strategies used by anurans to accommodate larval and and Sower, personal communication, 2000). A causal relationadult forms in aquatic and terrestrial environments. In fact, ship between this disease and limb deformity remains to be changes in developmental patterns, especially those concerned demonstrated. One interesting study of frog deformities in Quebec linked with the timing of developmental events in larvae (heterochrony), have been responsible for numerous species, many hindlimb malformations to the use of agricultural pesticides captured from the wild, to be included in investigations (Han- (Ouellet et al., 1997). Since the 1920s, investigators have linked ken, 1999). The dramatic developmental changes associated various chemicals with deformities in adult or larval frogs; with metamorphosis, which include remodeling of virtually these include hyperactivity and hindlimb deformity in Rana every larval organ system in certain families, e.g., Rana, have temporaria caused by DDT (Cooke, 1973); curvature of the attracted enormous interest and resulted in excellent reviews digits and abnormal limb articulations in Rana sylvatica by (Rose, 1999; Duellman and Trueb, 1994; Fritzsch, 1990; Mc- thiosemicarbazide (Riley and Weil, 1987); spinal curvature, Diarmid and Altig, 1999). Even with this body of information, blister formation, and abnormal behavior in Microhyla ornata scientists and government agencies were unprepared for the by malathion (Pawar et al., 1983); stunted growth, microcephaly, and curved spine in Rana pipiens exposed to paraquat events observed in 1995. On August 8, 1995, a group of middle school students on a na- (Dial and Bauer, 1984); spinal deformities in Rana pipiens, ture trip in central Minnesota discovered a high percentage of Rana catesbeiana, and Xenopus laevis by dieldrin (Schuytema deformed northern leopard frogs, Rana pipiens, at a local farm et al., 1991); and spinal deformity accompanied by convulsive pond. The frogs, all recent metamorphs, had a variety of hind- and twitching behavior in Rana pipiens by the pyrethroid inseclimb deformities, from missing legs and legs of different sizes ticide esfenvalerate (Materna et al., 1995). Again, none of these to legs that failed to bend properly (Souder, 2000). Within days synthetic chemicals mimicked the deformities seen in frogs the Minnesota Pollution Control Agency began investigations, across North America. In the 1980s, developmental biologists and toxicologists toand within a month biologists from the University of Minnesota were involved. What began as a middle school outing evolved gether developed an assay using Xenopus laevis embryos and into one of the most puzzling episodes in the annals of field bi- larvae to document developmental abnormalities associated ology, scores of independent observations (ranging from Cali- with environmental chemicals (Fort et al., 1988; Bantle et al., fornia to Maine and southern Canada) involving multiple spe- 1991). The test is called frog embryo teratogenesis assay: Xenocies of frogs with deformities of forelimbs and hindlimbs, eyes, pus (FETAX). Preliminary studies of water and sediment from jaws, and skin coloration. Missing digits (ectrodactyly) and Minnesota ponds identified with frog deformities demonstrated whole limbs (ectromelia), as well as supernumerary hands and a component in both that was associated with embryo mortality entire limbs were observed. Over the following 5 years investi- and malformations in a dose-dependent fashion. This compogators documented decreased numbers of frogs, as well as local nent(s) was removed by activated carbon filters (but not by boilextinctions, and many believed the deformities were linked to the ing) and was responsible for a wide range of malformations generalized population decline of amphibians seen worldwide. involving the skeletal system, brain, eyes, tail, gut, jaw, and kidBy 1996, the Environmental Protection Agency (EPA) had ney. Malformations were not seen in water or sediments colalso become involved, and amphibian developmental biologists lected from Minnesota reference ponds lacking frog deformities began searching for a cause. Results of early investigations ex- (Burkhart et al., 1998). Two subsequent papers extend these posing frog eggs to ultraviolet light were mixed; however, at findings to include additional affected ponds from Minnesota

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and Vermont and pond sediment-specific developmental alterations, including hindlimb deformities in which Xenopus tadpoles mimicked the deformities seen in Rana sp. (Fort et al., 1999a). In addition, Chemicals causing differing abnormalities in developing frogs were isolated from affected pond sediments. Among these were substances shown to inhibit thyroxin activity, which delayed tail resorption (Fort et al., 1999b). Addition of thyroxin to FETAX assays employing these chemicals abolished the chemical-induced deformity in Xenopus larvae. It is becoming apparent that multiple environmental insults, acting singularly or together, are responsible for the numerous deformities seen in North American frogs. It is also obvious that while years of previous research in amphibian developmental biology greatly accelerated the search for environmental chemicals associated with deformities, very little is known about infectious agents and how they impact frog populations. Finally, only through a multidisciplinary approach to this problem, involving developmental biologists, infectious disease experts, field biologists, toxicologists, aquatic ecologists, geneticists, and population biologists, will the true significance of these myriad causative agents on local extinctions and global population decline be understood. b. From equine infectious anemia to AIDS. Knowledge gained from spontaneous and induced models of retroviral infection led to the early recognition of human T-cell leukemia virus (HTLV) and human immunodeficiency virus (HIV) in humans. At the time of this writing, HIV virus has infected 30 million people worldwide with over 250,000 new infections per month and more than 10 million deaths (Nathanson, 1998). While trivalent therapy (HAART) has greatly delayed the progression of disease in many individuals and dramatically reduced maternal transfer to the fetus and newborn, its widespread use has yet to be adopted. Many believe only vaccination will stop the current epidemic among humans. New hope for a safe and effective vaccine has arisen, due to the new understanding of the infection cycle of HIV, the protection of viral proteins that bind cell receptors by sugar residues, the role of chemokine receptors, and effectiveness of cytotoxic T lymphocytes (Robinson et al., 1999; Cafaro et al., 1999; Wyatt and Sodroski, 1998; Emerman and Malim, 1998). Nonhuman primate models were essential in these studies. Likewise, nonhuman primate models have documented the efficacy of monoclonal antibodies to prevent mucosal transmission to the newborn (Mascola et al., 2000; Baba et al., 2000). However, the current knowledge of HIV was constructed from a long history of animal research, much of it conducted by veterinarians. One of the earliest studies demonstrating that particles smaller than bacteria were responsible for disease was the discovery by Loftier and Frosch of "filterable agents" that caused foot-and-mouth disease in cattle. They, along with Sigurdsson, who was the first to demonstrate long latency between infection and disease in retroviruses, were veterinarians (Leader, 1975).

Retroviruses were initially grouped together because of their ability to reverse-transcribe their viral RNA into host DNA through the action of the enzyme reverse transcriptase, but they are now classified into three subfamilies: Oncornavirinae, Lentivirinae, and Spumavirinae. Oncornaviruses and lentiviruses cause both human and animal disease and have been the subject of many investigations using animal models. Oncornaviruses are known to infect and cause cancer in sheep, cattle, chickens, cats, monkeys, humans, and laboratory rodents (Salzman, 1986). Despite a long history of veterinary research leading to their isolation, composition, replication, and mechanisms of resistance, human oncornaviruses were only documented in 1979 with the discovery of human T-cell leukemia virus (HTLV) (Poiesz et al., 1980). Lentiviruses are known to infect sheep, goats, cattle, horses, cats, and monkeys, and many were well documented in the literature before HIV was discovered in 1984 (Barre-Sinoussi et al., 1983; Popovic et al., 1984; Gallo et al., 1984). However, the pattern of disease among animals infected with lentiviruses is quite different among species. While sheep develop pneumonia, goats develop arthritis and encephalitis, and horses develop anemia (Mosier, 1996; Tenorio et al., 1992; Gardner, 1997; Tashijan and Crusberg, 1989); cats and nonhuman primates share the immunodeficiency seen in humans (Gardner, 1989; Sparger et al., 1989; Theilen, 1988). The pioneering work by Robert Gallo (National Institutes of Health) and Luc Montagnier (Pasteur Institute) led to the isolation and description of the first human lentivirus (HIV). Since then several different strains and many variants of this virus have been described (Nielsen et al., 1993; Baiter, 1998). In his book "Virus Hunting: AIDS, Cancer, and the Human Retrovirus," Gallo states: "The work that influenced me came from William and Oswald Jarrett of Scotland and later Essex, Bill Hardy, Ed Hoover and others (each of whom did work on the cat leukemia virus); Peter Biggs in England and Creighton in the U.S. (chicken leukemia virus), Janice Miller, Carl Olson (U.S.) and M. van Der Marten (U.S.) and later Arsene Burney of Brussels (cow leukemia virus) and Tom Kawakami (Gibbon ape leukemia virus) as well as from some of the earlier writings of Ludwik Gross and others regarding 'mouse leukemia virus'" (1991). The work that so influenced Gallo was, in large part, the work of veterinarians using a host of animal models. This body of research has led to our present-day understanding of lentiviruses, the development of therapeutic drugs, and the evaluation of genetically engineered vaccines (McMichael and Hanke, 1999). 3.

Validation and the "Ideal" A n i m a l Model

Various authors have attempted to defne the "ideal" animal model. Features such as (a) similarity to the process being mimicked, (b) ease of handling, (c) ability to produce large litters, (d) economy of maintenance, (e) ability to sample blood and tissues sequentially in the same individual, (f) defined

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genetic composition, and (g) defined disease status are commonly mentioned (Migaki and Capen, 1984; Leader and Padgett, 1980; Dodds and Abelseth, 1980). Perhaps the most important single feature of the model is how closely it resembles the original human condition or process. As previously mentioned, a model is never identical to a human but should be analogous to the targeted condition, i.e., share similarities that are important in elucidating new information relevant to the human condition or process, with the exception of negative models (see Section I,A). Shapiro uses the term "validation" as a formal testing of the hypothesis that significant similarities exist between the model and the modeled (Shapiro, 1998). He argues that to be valid, the animal model should be productive of new insights into and effective treatments for the human condition being modeled. While this goal is ideal, it may be impossible to attain in the preliminary stages of model characterization. In addition, many models are used to investigate nonpathologic processes, and models that simulate the target process most closely or are most likely to yield useful information are preferred (see Section II). The National Research Council (NRC) has advised the National Center for Research Resources (NCRR) on some criteria to be used in establishing priorities for fields and models to support (NRC, 1998). Among the criteria listed are that the model (1) is appropriate for its intended use(s) (a specific disease model faithfully mimics the human disease and a model system is appropriate for the human system being modeled); (2) can be developed, maintained, and provided at reasonable cost in relation to the perceived or potential scientific values that will accrue from it; (3) is of value for more than one limited kind of research; (4) is reproducible and reliable, so results can be confirmed; and (5) is reasonably available and accessible. These seem to be prudent criteria to follow when a funding organization seeks the greatest benefit within the confines of a finite budget. These recommendations also fulfill most of the criteria of an "ideal" model. There are instances where the only model (or the best model) cannot be maintained and provided at a reasonable cost (e.g., chimpanzees, which are invaluable for several vaccine efficacy trials) or are directed at one limited kind of research (e.g., murine knockout model of cystic fibrosis or murine transgenic model of amyotrophic lateral sclerosis). These exceptions are recognized by the NRC committee.

B.

Transgenesis and Mutagenesis: Creating Models for the Future

1. Methods

a. Techniques affecting the function of genes. Prior to the 1980s, most animal models were discovered by screening large breeding colonies or depending on the chance observations of

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veterinary clinicians (see Section I,C). However, several techniques, generally applied to small rodents, led to a large increase in available animal models. When the drug N-nitroso-N-ethylurea (ENU) is injected into male mice, single base pair mutations are created in the germ cells. By breeding progeny and backcrossing mice, homozygotes for the mutated allele are obtained. Genes in mouse embryonic stem cells (ES) can be mutated by use of ENU. This process is random, and in many earlier investigations the mutated gene had not been cloned or identified; however, since the 1980s, many useful models of human disease have been so created in mice, including models for phenylketonuria (mutated phenylalanine hydroxylase gene), c~-thalassemia (a-globin), [3thalassemia (~-globin), osteopetrosis (carbonic anhydrase II), glucose-6-phosphate deficiency, tetrahydrobiopterin-deficient hyperphenylalaninemia (GTP-cyclohydrolase I), Duchenne muscular dystrophy (dystrophin), triose-phosphate isomerase deficiency, adenomatous intestinal polyposis coli, hypersarcosinemia (sarcosine dehydrogenase), erythropoietic protoporphyria (ferrochelatase), and glutathionuria (y-glutamyltranspeptidase) (Herweijer et al., 1997). Zebrafish, Danio rerio, have been used extensively for studies in development because their embryos are transparent, each clutch contains 50-100 embryos, and the fish are amenable to large-scale mutagenesis using compounds like ENU (Driever and Fishman, 1996). Distinct genes have different mutability rates; however, ENU is reported to induce genetic mutations at average induction rates of 1 in 1000. This estimate serves as the basis for large-scale genomic screens (Ntisslein-Volhard, 1994). Because earlier work provided much of the outline for early embryonic cell fate, lineage, and patterning and for nervous system development (Eisen, 1994); and methods for production of homozygous fish, using genetically impotent sperm to induce the maternal chromosomes of the egg to complete meiosis II and pressure to prevent the first cell division (Steisinger et al., 1981), mutant phenotypes can be rapidly screened by visual inspection under a dissecting microscope, with mutants bred to homozygosity in three generations. Irradiation was used as a germline mutagen dating back to the early 1920s. X-rays have been shown to cause small chromosomal deletions in mouse spermatogonia, postmeiotic germ cells, and oocytes (Takahashi et al., 1994). Examples of radiationinduced models in wide use include the beige mouse (bg), dominant cataract (Cat-2t), and cleidocranial dysplasia (Roths et al., 1999). Chlorambucil has also been used to produce deletions in postmeiotic germ cells; however, because chlorambucil and Xrays often produce large deletions, in practice it is difficult to recover large numbers of mutant mice. Since the 1980s, the most popular and productive method for altering gene function is via transgenesis. The first method, described by Gordon and Ruddle (1981), involved direct insertion of cloned genetic material into the pronucleus of a fertilized mouse egg. While this method is simple, can be performed in many species, and does not appear to have limits on the size of


DNA that can be injected, it is limited in that the site of integration is random. Pronucleus injection has been successfully used to create transgenic rabbits, sheep, and goats, in addition to mice and rats (Pinkert, 1997; Mullins and Mullins, 1996). Earlier it was discovered that infection of preimplantation mouse embryos with Moloney murine leukemia virus led to germline integration of the proviral DNA in offspring (Jaenisch, 1976), and by the early 1980s, construction of retrovirus packaging mutants allowed for the integration of foreign genes in the mouse germline (Mann et al., 1983). This method generally results in low gene expression of the inserted element, the insertion takes place with minimal disruption of host genetic sequences, and insertion is nonrandom (the sequences flanking retroviral insertion sites contain chromatin that is DNase I hypersensitive). Around the same time, mouse embryonic stem cell (ES) lines were first produced and maintained in culture (Martin, 1981). This discovery allowed investigators to insert genes by homologous recombination, using vectors that contain both positive and negative selectable markers. As a result, ES cells with a targeted mutation can be combined with a developing mouse embryo that is implanted into pseudopregnant surrogate mothers. Offspring are chimeras for the insertion and after selection and appropriate matings, result in founders that have germline expression of the mutation (see Chapter 28 for details). Mice submitted to this form of targeted mutagenesis may have a nonfunctional gene (knockout), have a gene with altered expression, or gain a new functional gene (knockin). Gene expression can also be altered in a tissue-specific fashion by modifying a coding sequence such that critical components of the targeted gene are flanked by loxP sequences, which are targets for bacteriophage Cre recombinase. Crossing mice with loxP-modified loci to transgenic-expressing Cre recombinase under control of a linease-specific promoter results in tissue-specific exon excision and ablation of gene function (Gordon, 1997; Nagy and Rossant, 1996). The list of mouse mutations generated by targeted mutagenesis is staggering (see ). When alterations to the germline occur in specific genes, the result may be a gain of function (as when an extra copy of a human gene is introduced into the mouse genome) or a loss of function (as when a point mutation is induced by homologous recombination, a deletion produced by irradiation or antisense/ ribozyme constructs targeted against specific genes introduced via transgenesis) (Cameron and Jennings, 1989; Katsuki et al., 1988). Because ENU induces intragenic point mutations, it is likely that both gain-of-function and loss-of-function mutations can be produced. b. Controlling gene expression in transgenic mice. A variety of clever techniques have been developed to control the expression of transferred genes in addition to lineage-specific expression described above. Transgenic animals made by pro-

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nuclear injection may have the DNA construct designed with tissue-specific regulatory sequences (i.e., promoters) or with ubiquitous promoters that lead to widespread gene expression. Mice and rats have been made using shuttle vectors in which a bacterial gene inserted into the mouse genome is susceptible to mutation. Once the mouse is exposed to a mutagen, the high molecular weight genomic DNA is isolated and a bacteriophage packaging extract is used to isolate the bacterial sequence. Analysis of bacterial sequences in phage particles can establish the mutation frequency of the original mutagen (Roths et al., 1999). Another modification of the standard microinjection method for producing transgenic mice is one in which large multilocus segments of human DNA were transferred into the mouse pronucleus in the form of yeast artificial chromosomes (YACs). The entire [3-globin multigene locus (248 kb) was cloned into yeast, and once integrated, this locus could be mutated at precise points by homologous recombination. After transferring YACs and mutated YACs into mice, the full developmental expression of epsilon, gamma, beta, and delta genes was observed since the YAC also contained the human locus control region that interacts with structural genes to ensure that the correct globin is produced at the proper time and place during development (Clegg et al., 1997). These YAC transgenic mice are free of the restrictions inherent in single-gene cloned DNA, e.g., the genomic organization is not disrupted around the structural gene; thus, higher levels of transcription and developmental regulation of gene expression can be studied. Another method, developed by Ktihn et al., (1995), allows for the controlled expression of a transferred gene by linking an interferon-responsive promoter to control the expression of Cre recombinase. Under these conditions, treating mice with interferon activated the Mx-cre transgene, allowing expression of Cre recombinase, which deleted the floxed target gene, DNA polymerase [3 (see discussion above for C r e - l o x P system). Here the target gene was eliminated in all interferon-responsive lineages. A modification of this system was employed by Utomo et al. (2000), which integrates the advantages of tetracyclinecontrolled gene expression and Cre r e c o m b i n a s e - l o x P sitemediated gene inactivation. In this system the reverse tetracycline responsive transactivator (rtTA), which is transcriptionally active only when bound by tetracycline (or doxycycline), is placed in cis configuration to the rtTA-inducible promotor, which directs the expression of Cre recombinase. Exposure of the mouse to doxycycline provides the temporal off-on switch for tissue-specific regulation of gene expression through the C r e - l o x P system. Another system that combines cell type-specific and temporal controls consists of a fusion between Cre and a mutated steroid ligand-binding domain of the receptor controlled by tissuespecific promoters, The chimeric protein becomes activated upon binding with synthetic ligands such as tamoxifen or RU486 (Schwenk et al., 1998).

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c. R e p r o d u c i n g identical individuals. For years scientists have created inbred strains of rodents by repeated brother X sister matings. After 20 such matings the offspring are considered an inbred strain. When a transgenic mouse is developed and the investigator wants the transgene expressed on another background, depending on whether the mutation is dominant, recessive, or recessive and lethal, the transgenic mouse can be crossed into a selected strain using one of the following techniques: backcross matings, cross-intercross matings using homozygotes, cross-intercross matings using heterozygotes, or cross-intercross matings using ovarian transplantation (NRC, 1989). Techniques for cloning individual mammals have been published in studies that followed the original work conducted by John Gurden in frogs nearly 30 years ago (Wilmut, 1998). Cells of the individual to be cloned are grown in culture such as fibroblasts, and their nuclei are removed and fused into enucleated eggs. The resulting embryos are transferred into surrogate mothers, and offspring are identical to the original animal used as a source of cultured cells. This technique has been successful for cloning sheep, goats, cattle, fish, and mice (Wilmut et al., 1997; Kato et al., 1998; Wakayama et al., 1998; Steisinger et al., 1981; Bagulsi et al., 1999) and offers a much more efficient way to propagate large numbers of identical individuals, including individuals expressing the product of a transgene in milk, which may be isolated and used as a therapeutic compound in humans, e.g., human Factor IX (Wilmut, 1998). In 1999, mice were cloned from ES cells rather than from the low-passage primary cell cultures used in the past. This technology offers researchers the opportunity to create new knockout mice in a single generation rather than the three generations currently required (Vogel, 1999). However, in each of these procedures, the resulting clones show mitochondrial heterogeneity; thus, they are genetic chimeras (Evans et al., 1999). Chan et al. (2000) reported on successful clonal propagation of rhesus monkeys by embryo splitting. An 8-cell embryo was split to produce a set of identical quadruplet embryos, each consisting of two blastomeres. Two blastomeres are then inserted into an empty zona pellucida, creating one set of quadruplets. A pair of quadruplet embryos was transferred into a surrogate female rhesus monkey that delivered a healthy cloned infant. d. Cryopreservation. With the rapid accumulation of mouse strains developed by transgenesis and chemical or radiation-induced mutagenesis, cryopreservation is an inexpensive and efficient alternative to maintaining living animals. Since the first successful embryo-freezing protocol was established in 1972 by Whittingham et al., cryopreservation programs for maintaining inbred strains by embryo freezing have become commonplace. In addition to 8-cell embryos, all stages of preimplantation embryos plus sperm and oocytes may be suc-

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cessfully frozen. While maintaining stocks with frozen sperm and oocytes is more economical, embryo freezing is warranted when the full inbred background containing the transgene is desired (Crister and Mobraaten, 2000; Agca, 2000; Rall et al., 2000). Details on cryopreservation protocols along with an estimate of costs have been recently reviewed (Mobraaten, 1999). 2.

Functional Genomics: Applications to Biomedical Research

The human genome, the full set of genes that defines humans, is composed of 80,000 or so genes and 3 billion base pairs. With support from the NIH, Department of Energy (DOE), the Wellcome Trust, and others, the project to sequence the entire human genome, known as the Human Genome Project (HGP), was launched on October 1, 1990, with an expected completion date of 2005. Advances in the technology available to this project have greatly accelerated the task, and the complete, first-draft copy was finished in 2000. The entire sequence is publicly available on GenBank (accessible at ), which in 1999 was already receiving over 200,000 queries a day. Over 39,000 species are represented in the database, and over 60,000 sequence-comparison searches are conducted each day (Collins, 1999). The purpose behind the endeavor was to have a new understanding of the genetic contributions to human disease and the development of new strategies for minimizing or preventing disease phenotypes. To accomplish these goals, the HGP focused on four major initiatives: (1) develop a genetic map that establishes 6000 markers spaced within 106 bp of each other to aid in family linkage studies, (2) construct a physical map with ordered sets of DNA, properly aligned to cover each chromosome, (3) position expressed sequence tags (ESTs) on the physical map to assist in locating unknown genes, and (4) and develop the dense map of DNA variants within the population, primarily identifying insertions and deletions of nucleotides, differences in the copy number of repeat sequences, and single nucleotide polymorphisms (SNPs). One SNP is thought to occur in every 300 bp. The SNPs falling within protein-coding regions of a locus are particularly informative because they are more likely to predict changes in gene function. By 1994, the first genetic map was completed. The physical map containing 41,000 aligned sequenced-tagged sites (STS) was completed in 1998. A map containing over 38,000 ESTs was available by 1999. In 2000, the first draft of the human genome was completed, and knowledge gained through the various maps listed above led to the rapid discovery of several new genes responsible for human disease, including those that regulate families of interleukins (Loots et al., 2000) and those responsible for hearing loss (Willems, 2000). Earlier, the sequencing of mutant genes in the mouse led to the elucidation of mutant human genes causing Prader-Willi syndrome and polycystic kidney disease (Gardner et al., 1992; Moyer et al., 1994).

30. ANIMAL MODELS IN BIOMEDICAL R E S E A R C H

a. Comparative mapping. With the physical maps of the human genome now in hand, the time it takes to locate and identify a gene responsible for a disease inherited within a family has been reduced from years to weeks. In many instances, knowledge of these genetic variants has led to diagnostic tests that can be used to predict a person's increased risk of disease. Examples include the BRCA1 and BRCA2 genes and breast cancer, the hepatocyte nuclear factor 4c~ (HNF-~) in maturityonset diabetes of the young (MODY) type 1, the glucokinase (GCK) gene in MODY type 2, human Mut L homolog 1 (hMLH1) in hereditary nonpolyposis colon cancer, and ~-synclein in Parkinson's disease (Wooster et al., 1995; Yamagata et al., 1996; Froguel etal., 1993; Papadopoular et al., 1994). However, as Collins eloquently states: "Knowledge about the genetic control of cellular function will underpin future strategies to prevent or treat disease phenotypes" (1999). To better understand the function of expressed gene products (functional genomics), comparative gene mapping is critical. As the HGP began, several organisms with smaller genomes were identified for gene mapping. The entire map of Saccharomyces cerevisiae, a yeast with 6000 genes, was completed in 1996 (Goffeau et al., 1996) as was the map of Escherichia coli (Blattner et al., 1997). The map of the 19,000 genes of Caenorhabditis elegans was completed in 1998 (Chalfie, 1998; C. elegans Sequencing Consortium, 1998) and the entire map of Drosophila melanogaster was completed in 2000 (Adams et al., 2000). In fact, at the time of this writing the genomes of more than 28 microorganisms have been fully sequenced (Fraser et al, 2000). Adaptation of the dot-blot hybridization technique to DNA chips (stamp-sized silicon disks containing over 100,000 difference probes), so-called microarrays, has proven promising in approaching genome-scale studies of genetic variation. It is useful in the detection of gene expression (mRNA) in tissues and cells (e.g., the expression of cytochrome P450 genes), for the large-scale analysis of gene copy number and protein expression, and for making genomic comparisons across species (Hacia et al., 1998). Although the human genome map allows for positional candidate cloning to rapidly isolate altered genes associated with human disease (Collins, 1995), this technique may not elucidate the function of these genes. To understand the function of proteins encoded in the DNA sequence, a variety of other techniques can be useful, including (1) searching for homologs in other species where protein structure or function is known, (2) predicting protein function from structure, and (3) developing knockout animals to study function. In this context it is interesting to note that the fruit fly has orthologs to 177 of the 289 human disease genes (Rubin et al., 2000). b. Using multiple species comparisons: Research on aging. Although animal models have contributed greatly to our understanding of aging, we are still a long way from understanding the multitude of factors that contribute to long life. In 1989, Ma-

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soro pioneered studies that demonstrated increased longevity in rats on calorie-restricted diets. Investigators have also demonstrated a wide range of physiologic and endocrinologic changes apparent in rodents fed restricted diets compared to rodents fed ad libitum (Masoro, 2000). Many of these changes (e.g., lean body mass, reduced blood insulin levels, reduced blood glucose levels, reduced cholesterol and triglycerides) are themselves thought to be biological markers of fitness (Masoro, 1988, 1989). Why calorie restriction has such a dramatic effect on body function is unknown, and these observations have led others to question the relevance of calorie restriction in rodents to human longevity. However, three separate research groups have provided data on normal and calorie-restricted nonhuman primates, which had similar changes in biomarkers and reduced incidence of mortality associated with diseases such as diabetes, heart disease, and cancer (Couzin, 1998). While results on longevity in calorie-restricted nonhuman primates will require many more years of observation, these preliminary results suggest that in terms of the impact of calorie restriction on measures of fitness, primates reproduce the observations made in rodents. We now recognize that the life span of an individual is determined by the interactions between genes and the environment. We are just learning how those interactions lead to the cellular and molecular involution associated with aging. Although it is clear that individuals are subject to differences in genetic risks for age-related disorders such as Alzheimer's disease (AD), diabetes, heart disease, stroke, and cancer, it is much less clear what influence genes play on maximizing life span (Finch and Tanzi, 1997). Three categories have been proposed to classify candidate loci for human longevity: (1) genes with homologs that influence longevity in other species, (2) genes that mediate cellular maintenance and repair, and (3) genes that are associated with susceptibility to major age-related diseases (Sch~ichter et al., 1993). Using the nematode Caenorhabditis elegans, six induced mutations were found that extended life span by 40 to 100%. Each was found to increase resistance to stressors such as temperature, free radicals, and ultraviolet light (Friedman and Johnson, 1988; Larsen et al., 1995; Kenyon et al., 1993; Morris et al., 1996; Kimura et al., 1997). Age-1 appears to be associated with a phosphatidylinositol 3-kinase (Morris et al., 1996), whereas daf-2 is identified with an insulin receptor-like gene (Kimura et al., 1997). These two mutations are associated with the greatest increase in life span, both involve insulin-like signaling, and their effects are consistent with the extension of life span in food-restricted rodents (Sohal and Weindruch, 1996). The clock gene (clk) also plays a role in C. elegans, where mutations are associated with slower development, lengthened cell cycles, and modified behavior (Lakowski and Hekimi, 1996; Ewbank et al., 1997). Clk-1 encodes an 82-amino acid

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tandem repeat that is highly conserved in eukaryotes. The yeast homolog indirectly regulates the transcription of genes involved with energy metabolism. The wild-type clock gene in Drosophila and mice encodes transcription factors that are essential in the maintenance of circadian rhythms (Darlington et al., 1998; Gekakis et al., 1998). Double mutant C. elegans with daf-2/clk-1 have a fivefold increase in lifespan (Ewbank et al., 1997). The molecular pathways dependent on the expression of the transcriptional activator, clock, in mice and their relationship to longevity are now the focus of investigation (Okamura et al., 1999; Hardin and Glossop, 1999). In bakers' yeast (Saccharomyces cerevisiae) the SGS1 gene encodes a DNA helicase. Mutation of this gene causes premature aging in the yeast and redistribution of silent information regulator (sir)-3 protein from the telomers to the nucleolus (Kennedy et al., 1997; Sinclair et al., 1997). Loss-of-function mutations also occur in DNA (RecQ) helicase genes in humans, leading to impaired DNA replication and repair, decrease in telomere length, and the autosomal recessive adult-onset progeroid disorder known as Werner syndrome (Yu et al., 1997). Telomere length is inversely proportional to somatic cell proliferative capacity for all mammalian cells. Unlike in germline cells, where telomerase is constantly active, in somatic cells telomerase is not expressed (Kim et al., 1994). As a result, in somatic cells, repeats of the sequence TTAGGG/CCCTAA at the chromosome ends become shorter following each replication. One hypothesis holds that cell replicative senescence (the inability to further divide) occurs at some telomere length threshold. One in vitro experiment demonstrated that two different human cell types could be induced to greatly exceed their replicative life spans by stable transfection of the human telomerase gene (Bodnar et al., 1998). One in vivo experiment utilized telomerase deficient, m T R -/-, mice to evaluate the role of telomerase gene expression on hepatocyte proliferation and the hepatic response to chronic liver injury (cirrhosis). Following adenovirus-mediated transfer of the M T R gene (by tail vein inoculation) into m T R - / - mice, 85-100% of hepatocytes expressed M T R (but no expression was seen in spleen, consistent with hepatotropism of adenovirus). When transfected mice were compared to nontransfected controls n each undergoing partial hepatectomymthe transfected mice had increased numbers of regenerative hepatic nodules and greater numbers of mitotic figures compared to those in nontransfected M T R - / - mice. When both transfected and nontransfected mice were treated with carbon tetrachloride, the transfected mice had much less steatosis and fibrosis (cirrhosis) compared to nontransfected mice. In fact, transfected M T R - / - mice behaved like normal mice. These studies demonstrated that accelerated telomere loss was a factor leading to end-stage liver failure and cirrhosis and that telomerase gene therapy protects the liver against chronic liver disease induced surgically or by hepatotoxic drugs (Rudolph et al., 2000). Another silent information regulator, SIR2, encodes the S I R 2 P

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protein a NAD-dependent histone deacetylase in yeast. S I R 2 P is responsible for compressing chromatin and through this regulates access of many nuclear proteins to DNA and represses homologous recombination at the highly repetitive rDNA locus. Because accumulation of extrachromosomal rDNA circles is a major cause of aging in yeast, the function of S I R 2 P weighs heavily on yeast longevity. Lin et al. (2000) developed a method of imposing calorie restriction on yeast. They found the restriction was associated with a 2 0 - 4 0 % increase in life s p a n n s i m ilar to the effects of calorie restriction in mammals. Because S I R 2 P expression is dependent on NAD, its activity is linked to the energy status of the cell. The long-lived yeast had increased S I R 2 P activity and decreased rDNA circles, thus linking longevity to gene expression and energy status in the cell. These findings have placed new emphasis on the role of chromatin silencing on aging in model systems and humans (Campisi, 2000). Alzheimer's disease is a major age-related neurodegenerative disorder responsible for over 70% of all cases of late-onset dementia, afflicting over 4 million Americans and causing over 100,000 deaths per year in the United States (Martin, 1999; Shoulson, 1998). It is characterized by the deposition of amyloid [3-peptide (A[3) in plaques and in cerebral blood vessels and the development of neurofibrillary tangles (NFTs). However, both A[3 and NFT occur in the cerebral cortex of normal (nondementia) but aging individuals. Studies in human twins show a strong genetic influence in the development of AD, with concordance for AD in monozygous twins two-to threefold greater than that occuring among dizygous pairs (Gatz et al., 1997). A familial early-onset form of AD is caused by mutations in the [3-amyloid or presenilin genes. The [3-amyloid gene encodes a large protein, amyloid precursor protein (APP), a cellular transmembrane protein that gives rise to [3-amyloid, a fragment varying between 40 and 42 amino acids in length. Studies in rats have demonstrated rapid catabolism of infused A[342 to A[340 via neutral endopeptidase in normal rat brain (Iwata et al., 2000). Seven different mutations in the gene for APP have been found in human families with familial AD, and each led to increased production of the longer 1-42 [3-amyloid, which forms fibrillar aggregates that are neurotoxic. Far more common in early-onset AD are mutations in the gene for presenilin 1 and 2. The presenilins are found in the Golgi apparatus and endosomes and are postulated to interact with the y-secretase enzyme that cleaves APP to the longer 1-42 [3amyloid (Waggie et al., 1999; Martin, 1998). It has been demonstrated that the PS1 gene is identical to the tumor suppressor inhibited pathway clone 2 (TSIP2) gene and suggests that this protein is involved in the p53-induced apoptosis pathway, and possibly in the regulation of cancer-related pathways (Roperch et al., 1998). Mice transgenic for (and overexpressing) the 695-amino acid isoform of human Alzheimer [3-amyloid precursor protein had a 14-fold increase in A[31-42, numerous A[3 plaques in the brain, and impairment of memory in spatial reference and alternation


tasks by 9 months of age (Hsiao et al., 1996). In APP 23 transgenic mice there is also region-specific and age-dependent neuronal 10ss in the central nervous system (CNS) (Staufenbiel et al., 1998). Citron et al. (1997) transfected both cells and mice with several human presenilin genes associated with early-onset AD. All transfected cell lines produced greater amounts of 1-42 [3-amyloid as a result of altering the activity of 7- (but not c~- or [3-) secretase. Double transgenic mice (human PS 1 plus human APP695) expressing mutant presenilin genes deposited more A[31-42 in the brain than those expressing normal presenilin genes. Chui et al. (1999) examined the brains of aged transgenic and control mice differing in the expression of mutant presenilin 1 genes (PS 1). They discovered that despite the absence of plaques, transgenic mice had significantly more neuronal cell death, which was associated with increased intracellular A[3 in mutant transgenic mice compared to that in wild-type transgenic or age-matched control mice. They speculate that downstream events in the amyloid cascade, i.e., plaque formation, NFT, and inflammation, are not prerequisites for the induction of neuronal degeneration. It is also intriguing that apoptosis was induced in mutant presenilin 1 gene transfectants, an observation that links the previous association between PS 1 and TSIP 2 (and their role in the apoptosis cascade) and provides in vivo evidence for the previous observation that PS 1 mutations potentiate neuronal apoptosis induced by extracellular applied A[3-catenin (Zhang et al., 1998). The molecular events that regulate programmed cell death or apoptosis were first characterized in C. elegans with at least two different gene families, the caspase family of aspartate-specific proteases and the deathpromoting and death-suppressing proteins of the Bcl-2 protooncogene family. Homologous genes for these pathways occur in mammalian cells (Martin et al., 1998). Presenilins are substrates for a caspase-3 family protease during apoptosis, and the early-onset AD mutations in the PS2 gene increase susceptibility to apoptosis and further fragmentation of the PS2 protein. A homolog of the presenilin gene in C. elegans is SEL-12, the protein that facilitates the LIN-12 Notch receptor. In fruit flies, LIN-12 Notch receptor expression is modulated by catenins through the wingless (wg) signaling pathway (Axelrod et al., 1996). This observation regarding catenins in fruit flies may help explain how a nematode presenilin facilitates LIN-12 Notch receptor, and help shed light on the role of catenins in the endoproteolysis of PS 1 or PS2 in mammals and their contribution to neurodegeneration in AD (Finch and Tanzi, 1997). Other molecular studies have provided evidence for an alternative mechanism that links mutations in PS 1 and PS2 to initiation of apoptosis. Buxbaum et al. (1998) have isolated a new protein named calsenilin, a calcium-binding protein of the recovering family. Normally PS 1 and PS2 bind calsenilin, and it has been postulated that this interaction may protect against apoptosis. Overexpression of calsenilin in human neuroglioma cells, however, leads to alternative processing of PS2 in a man-

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ner similar to that seen during caspase-induced apoptosis. Further studies are necessary to determine whether the interaction between presenilins and calsenilin occurs physiologically and if so, determine the downstream consequences of this interaction. In all likelihood, transgenic mice expressing mutant PS 1 and PS2 will play an important role in these investigations. Should these exciting results linking mutant PS1/PS2 and A[31-42 to induction of neuronal apoptosis in animals be confirmed in AD patients, it will not only dispel a widely held theory on disease pathogenesis but open up opportunities to develop new therapies based on the interactions between presenilins, [3-catenin and other molecular mediators, and A[31-42 (Shoulson, 1998). c. Obstacles: Phenotyping and nanofabrication. Increasingly, discovery is being made of a considerable degree of conservatism in the activity of products expressed from homologous genes between species (see previous discussion on aging). This is particularly true when comparing mammalian species. However, it is also true that for many genetically engineered animals, particularly knockout mice, unexpected phenotypes are discovered. The mechanisms that result in these unexpected phenotypes are numerous and complex and serve to emphasize that in the postgenome era, work to understand the function of genes has only begun. In some instances knockout mice develop a phenotype quite different from that seen in humans with the same mutation. For instance, when mice are carriers of a deletion in the retinoblastoma (Rb) gene, they are prone to tumors; however, in contrast to humans where retinoblastoma is common, these mice develop tumors of the pituitary gland (Gordon, 1997). In the case of generating murine knockouts for transforming growth factor-J3 (TGF[3) genes, over 30 different murine phenotypes have been discovered by knocking out one of three structural genes (Pelton et al., 1991). This apparent nonredundancy in the TGF[3 family is due to a combination of mechanisms, including those involved with ligand processing, receptor interactions, and signaling pathways (Doetschman, 1999). Unlike the previous example, fibroblast growth factors (FGF) 1 and 2 have activities in numerous cell types and tissues; however, when FGF2 was knocked out, mice had no apparent abnormal phenotype (Zhou et al., 1998). This was unexpected since knockouts lacking Fgf 3, 4, 5, 7 or 8 have profound and unique phenotypic changes. Although living a normal, healthy life, Fgf2 -/- mice were later found to have increased platelet numbers and decreased cell density in the cerebral cortex, as well as changes in vascular tone and wound healing. Thus, unique phenotypes were eventually found for this knockout although the changes appear more important in adult life than in early development (Doetschman, 1999). In some knockout mice the lack of one gene product necessary for vertebral skeletal muscle development, MyoD or Myf5, results in rather normal muscle. While this appeared to be an example of gene redundancy, in fact it

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was an example of developmental compensation since the product of one expressed gene compensated for the lack of expression of the other (Ordahl and Williams, 1998). In other instances the mouse may not be the most appropriate species to exhibit phenotypic changes following transgenesis. Mice overexpressing the murine Ren-2 gene did not develop hypertension, while rats did (Mullins et al., 1990); likewise, mice harboring the human HLA-B27 gene did not develop inflammatory disease, whereas rats harboring the human HLA-B27 gene did (Hammer et al., 1990). Mice with a deleted hypoxanthine phosphoribosyltransferase (HPRT) gene never developed the Lesch-Nyhan syndrome seen in humans (Gordon, 1997). Strain background has also been found to profoundly change the phenotype of mice with a single gene knocked out. Although this has been demonstrated for many genes (and perhaps occurs with most), TGF[3 knockouts illustrate this point well, with modifier genes documented that affect the function of TGF[31 in preimplantation, yolk sac development, bowel and gastric inflammation, and colon tumor suppression (Doetschman, 1999). It has also been shown that knockouts on a mixed genetic background have a wider range of phenotypes. Other factors that play on the determination of phenotype in genetically engineered mice have been discussed by Roths et al. (1999). Pleiotropism is the production of multiple, often seemingly unrelated, phenotypic effects resulting from a single gene. The semidominant W locus encodes nonalbinotic white spotting, macrocytic anemia, and oocyte depletion. In addition, phenotypic similarities may be seen between mice bearing different knocked-out genes even when those genes are nonallelic. In this case, knocking out any critical gene in a pathway leading to a developmental defect may result in the same phenotype, e.g., mutations in either the Fas-encoding gene (lpr) or the Fas ligand (gld) results in lymphocyte hyperplasia (Murphy and Roths, 1977). Phenotypic divergence among mutant alleles at the same locus is also common, and among the mutant W alleles, changes in phenotype may be due to effects on transcription and RNA splicing, RNA stability and transport, translation, transport to the cell surface, or signal transduction (Roths et al., 1999). Many gene knockouts are manifested during gestation and may result in embryonic lethality or defective phenotypes at birth; however, a number of single mutations are expressed in mice only later in life. In some instances this is associated with reaching a critical threshold, such as the number of Purkinge cells in beige mice, which have progressive loss of these cells from birth to 12 months of age, when approximately 50% of Purkinge cell numbers are lost and signs of tremor, ataxia, and lethargy begin to occur. Taken together, these unexpected changes in phenotype place new emphasis on proper characterization of genetically engineered animals. Given the numerous mechanisms that can lead to different phenotypic expression of the same gene, it appears that the number of functional products will far exceed gene numbers and require the development of high throughput

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screening devices. In addition, the phenotype of the animal may well depend on a host of environmental factors that may influence the expression of genes and gene products. The laboratory animal specialist will be instrumental in the phenotyping process both in terms of controlling environmental variables and identifying those that may alter the phenotypic profile. In addition, meticulous attention must be given to development of a comprehensive screen that will help elucidate subtle abnormalities such as those originally missed in the evaluation of Fgf2 -/-. Wood (2000) has provided an overall process for assessment of phenotypes in transgenic animals based on a twotiered level of screening. The goal of a primary level assessment would be to find abnormalities, and the goal of the secondary level assessment would be to quantify and evaluate the abnormalities found in the primary assessment. The primary assessment would concentrate on clinical and pathologic (with clinical pathology) parameters and take into consideration the animal's activity, gross anatomy, reproduction, and life span, as well as a complete histopathologic and clinical pathologic examination designed to rule out the influence of pathogens. Environmental factors such as diet must also be taken into account. The secondary level assessment would involve evaluations of embryos, special pathology, biochemistry, physiology, and behavior. In all likelihood, dedicated centers will be established to conduct these specialized tests. The success of any secondary screen will depend on standardization, rapid screening, and automation. Sundberg and Boggess (1999) have also published a systematic approach to evaluation of mutant mice. Nanofabrication has led to the production of microchips used to establish microarrays in which tissue levels of specific mRNA can be detected (through binding of complementary DNA) to DNA probes in the microarray. This system provides high throughput for mRNA expression but depends on the selection of DNA probes utilized. Similarly, nanofabrication has led to microchips capable of cultivating and probing viable cells under in vitro conditions (Ghanem and Shuler, 2000). This same chip technology should be developed for measuring the expression of many proteins from tissues using the enzymelinked immunosorbent assay (ELISA) technology and adapting this tool for clinical chemistry and quantitating levels of peptides, hormones, and enzymes from blood. It is even possible to envision an automated microanalysis of hematologic parameters. These small-volume, high throughput screens, when coupled with microsurgical techniques for tissue harvesting, could satisfy the increasing demand for certain in vivo phenotypic analyses. Physiologic evaluations are also essential, and here nanofabrication of instruments especially for monitoring blood flow, pressure, tissue perfusion, and uterine contraction has made a major impact on cardiovascular monitoring (Doevendans et al., 1998). These technologies are augmented by a host of imaging techniques adapted to the mouse (Chien, 1996). However, work in this area must accelerate in order to more fully characterize the thousands of new mice engineered each year.

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One of the greatest enigmas facing the biomedical research community is deciding what assays should be employed to phenotype the many divergent organ systems of mice. The National Institutes of Health (NIH) has convened expert panels to give direction in this endeavor, and publications are emerging from various research groups illustrating the techniques available for specific organ systems. A number of papers have addressed the issue of behavioral phenotyping transgenic mice, with special emphasis on several prominent areas of research (Crawley, 2000; McClearn and Vandenbergh, 2000; Nelson and Chiavegatto, 2000, Brown et al., 2000). Among the panels to be developed are those that deal with pain and distress, for these will guide all investigations by establishing humane end points (Carstens and Moberg, 2000; Dennis, 2000).

C.

S o u r c e s of A n i m a l M o d e l s

The number and types of resources available today to aid investigators in the selection of an animal model are vast, and any attempt to list them all would be foolhardy. However, there was a time when selecting the appropriate animal model relied solely on one's knowledge of its anatomy and physiology, and lists of anatomic, physiologic, and metabolic differences between species would guide in selection (OTA, 1986). Inbreeding mice to produce strains of genetic and physiologic consistency began with Ernest Castle, who first brought them into the laboratory in 1902 when he established the Bussey Institute. Clarence Cook Little developed the first inbred strain in 1909 (Davisson et al., 1998). The tradition of using (and developing) inbred strains of mice continued under George Snell (at the Jackson Laboratories), who discovered the major histocompatibility complex (MHC) in mice and was awarded the Nobel Prize in Medicine in 1980 (Quimby, 1994). As the laboratory produced more and more inbred strains and made them available to investigators (now over 259 strains being held), spontaneous mutants were quickly recognized and saved. To date, over 1000 spontaneous mutant mice have been recognized, and over 20 new mutations are documented each year in the Jackson Laboratory colonies (Davisson et al., 1998; Quimby, 1994b). Yet before 1960, it was difficult for investigators to find a reliable supply of laboratory animals. Analysis of abstracts published in the Federation Proceedings for the year 1960 show that cats and dogs were used in 37% of all physiologic studies, rats were used in 22%, and mice in less than 3% (Schmidt-Nielsen, 1961). Many of the dogs and cats used were likely to be pound animals. In fact, until the last decade most spontaneous models of inherited disease in animals were discovered by accident, e.g., veterinarians described new diseases in companion animals and livestock (Cornelius, 1969; Stormont, 1968; Lewis and Carraway, 1992), or spontaneous mutations in rodents and rabbits were identified by their attendants in large breeding colonies (Kondo and Watanabe, 1975; Sharp and Davisson, 1994). During the early 1960s, many of the infectious diseases of ro-

dents were identified; attempts to develop specific pathogenfree (SPF) animals by cesarean section and barrier isolation resuited in a reliable commercial supply of rats and mice by the 1970s (Quimby, 1994b). Similarly, research centers throughout the United States began reconstruction of animal facilities that employed the barrier concept, and standardization of caging and sanitation practices guaranteed the valuable models remained SPF, much of this due to documents written by the Institute for Animal Resources (ILAR) at the National Academy of Sciences (NAS) and funds made available from the National Center for Research Resources (NCRR) at the National Institutes of Health (NIH) (Quimby, 1994b). As research began to flourish in the 1970s, the need for animal models increased accordingly. Attempts to identify and preserve valuable models were undertaken by ILAR, but many important large-animal models disappeared as a result of the high cost of maintenance. In 1980, Gordon et al. published the first paper demonstrating the feasibility of pronuclear microinjection into a fertilized mouse egg; the term "transgenic" arose from these studies (Gordon and Ruddle, 1981). Brinster et al. (1981) showed that foreign genes so transferred could be efficiently expressed, although expression levels were difficult to control. Mouse embryonic stem cells were first produced in 1981 (Martin, 1981; Evans and Kaufman, 1981), and via a process called homologous recombination, foreign genes could be stably integrated into the murine genome or specific mouse genes could be rendered nonfunctional by mutation (knockouts) (Smithies et al., 1985). The long-awaited dream of medical scientists had arrived, a system to produce homologs of inherited diseases of humans in a small-animal model. The numbers of new animal models created this way is approximately 1 per day in early 2000. 1.

Organizations

Several organizations in the United States have taken the lead in providing information on (or access to) animal models. The ILAR (now renamed the Institute for Laboratory Animal Research) maintains two searchable databases: an Animal Models and Genetic Stocks (AMGS) Information Program and the International Laboratory Code Registryma listing of codes that identify an investigator, a laboratory, or an institution that needs rodents or rabbits. In addition, it publishes the quarterly I L A R Journal (devoted to research animal topics) and publishes a variety of books and other documents dealing with standards for animal care, education, and occupational health and conducts workshops on current animal-related topics. Noteworthy among these books is one dedicated to mammalian models for aging research (NRC, 1981) and another on models of thrombosis and hemorrhagic disease (NRC, 1976). Volume 39 (Nos. 2 and 3) of the I L A R Journal is dedicated to comparative gene mapping and contains the gene maps for over 16 species (phone: 202-3342590; ). The National Institutes of Health (NIH) has been the major

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funding agency for animal models of human diseases. It provides updated information on national and international efforts to produce animal models at <www.nih.gov/science/models>. The Office for Protection from Research Risks at the NIH also provides a comprehensive list of animal resource links, as well as special reports and regulations at . The National Agriculture Library at the U.S. Department of Agriculture operates the Animal Welfare Information Center (AWIC). This center manages the AGRICOLA database and provides literature searches on a wide variety of subjects related to the use of animals and alternatives in research. The quick bibliography series (qb90-09) lists 189 abstracts of books and articles between January 1979 and August 1989 on the subject of animal models of disease. Between January 1988 and January 1995 (qb95-14), 330 citations were found. Using the same retrieval system and database searching for animal model citations between January 1995 and January 2000, a total of 2486 citations were found. While this serves to illustrate the growth in use and publication of animal models, it represents only a fraction of the total publications describing animal models since both the terms "animal" and "model" had to be written in the title or abstract. Access to these quick bibliography series can be made through . Custom searches can also be arranged (phone: 301-504-6212; email: [email protected]). The American College of Laboratory Animal Medicine (ACLAM) was established in 1961 (previously known as the American Board of Laboratory Animal Medicine) with the expressed goal of encouraging education, training, and research in laboratory animal medicine and of certifying specialists in the field. An extensive series of texts has been published by ACLAM, covering the biology and use of many mammalian species, including rabbits (Manning et al., 1994), hamsters (VanHoosier and McPherson, 1987), guinea pigs (Wagner and Manning, 1976), rats (Baker et al., 1980), mice (Foster et al., 1982), and nonhuman primates (Bennett et al., 1995), as well as two volumes dedicated to spontaneous models of human disease (Andrews et al., 1979). The Armed Forces Institute of Pathology (AFIP) has contributed many articles on animal models in the journals Laboratory Animal Science and the American Journal of Pathology, and it publishes a handbook of models of human disease (Jones et al., 1972). Most companies providing laboratory rodent models for research also have websites containing detailed information on the models they provide; some are listed below under websites. 2.

Original Articles and Texts

Several journals contain major segments devoted to animal models, including ILAR Journal (email: [email protected]; see above); Contemporary Topics in Laboratory Science and Comparative Medicine (formerly Laboratory Animal Science), each

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published by the American Association for Laboratory Animal Science <www.aalas.org>; Lab Animal, published by Nature America, Inc. ; Experimental Animals, published by the Japanese Association of Laboratory Animal Science <www.jalas.or.jp/index>; and Laboratory Animals, published by Laboratory Animals, Ltd., the official publication of the Laboratory Animal Science Association of the United Kingdom <www.lal.org.uk>. Other European and U.S. journals that may deal with animal models have been identified (Quimby, 1994b; NRC, 1996). A wide variety of texts have been produced that cover the various animal models of human disease; space allows for only a few of them to be mentioned here. Readers should refer to other sections of this chapter for a more comprehensive listing. In addition to the bibliographies on animal models previously cited (see National Agriculture Library, AWIC, above), Hegreberg and Leathers (1981a,b) have cataloged both the naturally occurring and induced models available in the 1980s. Festing (1993a,b) has developed an index covering the sources of over 7000 stocks of laboratory animals worldwide and separately has recorded the origins, characteristics, and sources of inbred strains of mice. Earlier listings of inbred and genetically defined strains produced by the Federation of Societies for Experimental Biology cover rats and mice (Altman and Katz, 1979a) and hamsters, guinea pigs, rabbits, and chickens (Altman and Katz, 1979b), and they are still very useful. Lyon has published "Genetic Variants and Strains of the Laboratory Mouse" (Lyon, 1996). Gay (1965-1989) has edited nine volumes titled "Methods of Animal Experimentation" and Nathanielsz (1980-1987) has edited six volumes tided "Animal Models in Fetal Medicine"; both series elaborate on the proper use of induced models in research. The use of various animal species as both spontaneous and induced models appears in each species chapter in "The Experimental Animal in Biomedical Research" (Rollin and Kesel, 1995), and a separate text is devoted to induced models (Swindle and Adams, 1988). Individual species or groups of animals used as experimental models have been described for nonhuman primates (Dukelow, 1983; King et al., 1988; Joag, 2000), squirrel monkeys (Abee, 2000), swine (Stanton and Mersmann, 1986; Swindle et al., 1992; Mount and Ingram, 1971), domestic farm animals (Doyle et al., 1968; Lewis and Carraway, 1992); spontaneous and engineered mutant mice (Roths et al., 1999; Nomura, 1997; Gordon, 1997; Colbert and Klintworth, 1997), nude mice (Fogh and Giovanella, 1978, 1982), rats (Gill et al., 1989), fish (Powers, 1989; Ishikawa, 2000; Dooley and Zon, 2000; Paw and Zon, 2000; Zhu and Sun, 2000), birds (Konishi et al., 1989; Rose, 2000; Quimby et al., 1995; Medina and Reiner, 2000), and cetaceans (Wtirsig, 1989). The reader should also refer to each species chapter in this text for a general review of its uses in research. In addition, numerous reviews are available on the use of animal models for particular types of research, including fetal research (Hansen and Sladek, 1989), cytokine research (Durum


and Muegge, 1998), autoimmune diseases (Cohen and Miller, 1994; Taylor, 1994; Antel and Owens, 1999; Kukreja and MacLaren, 1999), immunodeficiency diseases (NRC, 1989; Gershwin and Merchant, 1981; Sordat, 1984; Percy and Barta, 1993), gene therapy (Herweijer et al., 1997; Lee et al., 1999), hypertension (Bader et al., 1997), Lesch-Nyhan and other metabolic diseases (Jinnah and Breese, 1997; Haskins et al., 1992; Colbert and Klintworth, 1997; Ozaki et al., 1998), skin diseases (Norvell et al., 1997; Sundberg, 1994), amyotrophic lateral sclerosis (Dal Canto, 1997; Green and Tolwani, 1999; Sillevis and deJong, 1989), multiple sclerosis (Dal Canto, 1997; Gold et al., 2000; Wong et al., 1999a), peripheral neuropathies (Notterpek and Tolwani, 1999; Thomas, 1992), Parkinson's disease (Tolwani et al., 1999; Zigmond and Stricker, 1989), Alzheimer's disease (Price et al., 1998; van Leuven, 2000; Sommer et al., 2000; Guenette and Tanzi, 1999), Huntington's disease (Price et al., 1998; Brouillet et al., 1999), cerebral ischemia (Martin et al., 1998; Megyesi et al., 2000), corneal diseases (Smith et al., 1995; Cowell et al., 1999), retinal dystrophies (Flannery, 1999; Zack et al., 1999; Stone et al., 1999), myopia (Norton, 1999), myoclonus (Nguyen et al., 2000), cognition (Newsome and Stein-Aviles, 1999), behavior (Takahashi et al., 1994; Hunter et al., 2000), alcohol and drug abuse (Crabbe et al., 1994; Stewart, 2000), pain (Ren and Dubner, 1999; Ness, 1999; Brennan, 1999), nutrition (Beynen and West, 1988; Yamori, 1999; Montinaro et al., 1999; Wallace, 2000), atherosclerosis (Clarkson et al., 1988; Attie and Prescott, 1988; Clarkson and Klumpp, 1990; Narayanaswamy et al., 2000), hyperlipidemia (Sullivan et al., 1993; Breslow, 1993; Bauer, 1996), transplantation (Ladiges et al., 1990; Wenger et al., 2000; Locatelli et al., 2000), infectious diseases (Renegar, 1992; Gardner, 1997; Lee, 1999; Salzman, 1986; Wassom and Peper, 2000; Joag, 2000; Tsolis et al., 1999), neoplasia (Calnek, 1992; Kritchevsky, 1988; Kobaek-Larsen et al., 2000), pulmonary disease (Cantor, 1989; Chang, 2000; Forsythe and Ennis, 1999; Kon and Kay, 1999), hepatic disease (Newsome et al., 2000), ulcers (Lee, 2000), type 1 diabetes mellitus (Wong and Janeway, 1999; Wong et al., 1999a), type 2 diabetes mellitus (Ostenson et al., 1993; Kovacs et al., 1997; Bali et al., 1995; Shafrir et al., 1999; Reitman et al., 1999), and obesity (McCracken, 1988; Nicholls et al., 1996; Samad and Loskutoff, 1999). This is not meant to be an exhaustive list of either species used or the subject of their use, but rather a listing of some recent reviews that illustrate the diversity of subjects and printed material covering animal models. 3.

Websites

In addition to the websites maintained by ILAR, NIH, and AWIC (see Section I,C,1), numerous other websites have been established to disperse information on animal models available for research and genetic mapping resources, as well as more comprehensive publications• Vendors of laboratory animals maintain websites that de-

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scribe the characteristics of the animals they supply. The Jackson Laboratory <www/jax.org> database maintains a Lane List of Named Mutations and Polymorphic Loci, Neurology News, Ophthalmology News, and Dermatology News under the Animal Resources site. Within its Genetic Resources site is a searchable database on transgenic and targeted mutant mice, Mouse Mutant Resource, Neural Tube Defect Resource, Cytogenic Models Resource, Eye Models Resource, and Hearing Models Resource (Sharp and Davisson, 1994; Takahashi et al., 1994). Taconic Farms, Inc. <www.taconic.com> maintains a searchable database, Taconic Transgenic Models, providing information on resources available, as well as their R e s e a r c h Animal Review newsletter. Charles River Laboratories (email:[email protected]) also maintains a website <www.criver.com>. Internet resources for transgenic and targeted mutation research include TBASE, the transgenic/targeted mutation database maintained by Jackson Laboratories . TBASE also provides links to the following websites: Database of Gene Knockouts , and BioMedNet Mouse Knockout database . The Mouse Genome database integrates existing databases of mouse genetic information and includes mapping data, molecular probesm clones, strains, and allelic polymorphismsmas well as phenotypic data. The Encyclopedia of the Mouse Genome includes genetic linkage maps, cytogenic maps, and a text searchable tool to review information integrated from numerous centers. The Portable Dictionary of the Mouse Genome includes three separate estimates of gene position, accession numbers to GenBank sequences, data on homologs in human and other mammalian species, a complete data set on recombinant inbred strain-distribution patterns, and much more. Genetic and physical maps of the mouse genome are maintained by the Whitehead Institute at Massachusetts Institute of Technology . Other U.S. websites providing genetic information on rodents include the cybermouse project , the Gene Expression database , NCBI's Human/Mouse Homology relationship database , RATMAP , and the Mouse and Rat Research Home Page . European databases include the European Collaborative Interspecific Mouse Backcross , the Mouse Atlas Project and the Dysmorphic Human-Mouse homology database . The Japanese Animal Genome database can be accessed at .

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Nonrodent information is available at the following sources: Pigbase , Sheepbase Project , ChickGB ase, and BovGBase .

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A.

THE NATURE OF RESEARCH

Hypothesis Testing and Serendipity

1. The Progressive (and Sometimes Long) Route to Discovery The 1000-year period historically referred to as medieval or the Middle Ages was characterized by personal ambitions and bias and a preoccupation with religion and politics, which was rarely conducive to scientific discovery. Inquiry into the nature of things, fostered by Aristotle and Galen, ceased until the 1400s when once again interest in seeking facts to explain nature resumed. Realizing the situations that led to confound his predecessors, Francis Bacon (1620) proposed a process of scientific discovery based on a collection of facts and observations, followed by a systematic evaluation of these facts demonstrating them to be true. Distinction between the act and demonstration of discovery was precisely what was missing during the Middle Ages, when assertions were frequently made that the truth could be elicited from a small series of observations. Bacon's requirement for elimination of all those inessential conditions (which are not always associated with the phenomenon under study) was, in the end, unachievable, and the process of choosing facts was found to depend on individual judgment. However, Bacon did properly perceive the defects inherent in the scholastic method practiced before him, and he set the tenets for what would become the method of hypothesis testing. Arguably, the foundation for sorting fact from fiction in scientific investigations is based on hypothesis testing (a particularly weak aspect of Bacon's philosophy). Although it is never possible to directly prove a hypothesis by experimentation, but rather to disprove one (or more) alternative (null) hypotheses; history has documented the steady (although sometimes slow) progress toward understanding the world. That is not to say that certain observations made during the testing of one hypothesis have not led investigators in an altogether different direction. In fact, this happens with a certain consistency and at times results in knowledge as compelling as that originally sought by investigators. One may argue, and rightfully so, that hypothesis testing is an inefficient mechanism for discovery; however, this paradigm of generating a hypothesis based on known facts and designing experiments to disprove the hypothesis has been proven over time to generally produce meaningful and reproducible results.

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On the other hand, in looking at the outcome of one milestone in biology and medicine, such as coronary bypass surgery, and examining the history of the many facts necessary to allow experimenters to directly perform a bypass operation, it is astonishing how many individual feats and over how long a period of time it took to reach the final test. The earliest studies that contributed to the first successful bypass surgery in the 1970s go back to 1628 when Harvey described the circulation of frogs and reptiles; then in 1667, Hooke hypothesized (and later demonstrated) that pulmonary blood, flowing through lungs distended with air, could maintain the life of animals. These early observations had no impact on medicine until centuries later, primarily because other technologies necessary for successful application of extracorporeal oxygenation in humans, including antisepsis, anticoagulants, blood groups, anesthesia, etc., had not yet been discovered. Dogs played a critical role during this process of discovery and between 1700 and 1970 contributed knowledge on the differential pressures in the heart; measurements of cardiac output, cardiopulmonary function, and pulmonary capillary pressure; and development of heart chamber catheterization techniques, heart-lung pumps, angiography, indirect revascularization, direct autographs, saphenous vein grafts, balloon catheters, and floating catheters (Comroe and Dripps, 1974). While examining the history behind the 10 most important clinical advances in cardiopulmonary medicine and surgery, Comroe and Dripps (1976) selected 529 key articles (articles that had an important effect on the direction of research) in order to determine how these critical discoveries came about. They found that 41% of these articles reported work that had no relation to the disease it later helped to prevent, treat, or alleviate. This phenomenon probably contributes to the observation that few basic science discoveries, including those conducted using animals, are cited in seminal papers describing a clinical breakthrough. A recent series in the New England Journal of Medicine, called Clinical Implications of Basic Research, promises to correct this oversight (Wu, 2000). The idea that major clinical breakthroughs required a long history of basic science discoveries, often involving animals and often being conducted by individuals who were unaware of the ultimate application of this knowledge, continues to be true today. Rudolfo Llinas after reflecting on 47 years of research aimed at elucidating the nature of neurotransmission, much of it accomplished using the giant axons of squid, states: "In the end, our complete understanding of this process (synaptic transmission) will manifest itself not as a simple insight, but rather as an ungainly reconstruction of parallel events more numerous than elegant" (1999). Both Bacon and Mill, who followed him, believed it was the responsibility of scientists to find the "necessary and sufficient conditions" that describe phenomena. That exhaustive lists of circumstances had to be examined in the search of what was necessary and sufficient never concerned these philosophers


(Bacon, 1620; Mill, 1843). Both saw virtue in experimentation, the collection of data under controlled circumstances. The scientific method practiced today evolved from the principles of Bacon and Mill and was refined by the middle of the nineteenth century. The method provides principles and procedures to be used in the pursuit of knowledge and incorporates the recognition of a problem with the accumulation of data through observation and experiment (empiricism) and the formulation and testing of hypotheses (Poincare, 1905). The method excludes the imposition of individual values, unsubstantiated generalizations, and deferments to higher authority as mechanisms for seeking the truth. It also subscribes to basing hypotheses only on the facts at hand and then rigorously testing hypotheses under various conditions. Hypotheses that appear to be true today may be disproved in the future as new conditions are imposed upon them and new technologies employed in the collection of data. Although great discoveries in biology and medicine have depended on the application of these principles, progress is still often slow. As hypotheses are proven incorrect, alternative hypotheses are sought and tested. Unexpected experimental results require careful consideration; and often the reasoned explanation of this data contributes information critical for the formulation of an alternative hypothesis. In the mid-1970s, a series of breeding experiments was conducted to test the hypothesis that systemic lupus erythematosus (SLE) resulted from a mutation passed between individuals through simple Mendelian inheritance. Dogs that spontaneously developed SLE were bred and their progeny tested (Lewis and Schwartz, 1971). Surprisingly, no offspring in three generations of inbreeding developed SLE, but over half the offspring developed other autoimmune diseases, including lymphocytic thyroiditis, Sj6grens syndrome, rheumatoid arthritis, and juvenile (type 1) diabetes (Quimby et al., 1979). After careful reexamination of the data, it was hypothesized that multiple, independently segregating genes were involved in the predisposition to autoimmunity and furthermore, that certain of these genes (class 1) would affect a key component in the immune system common to several autoimmune disorders, with other genes (class 2) acting to modify the expression of class 1 genes, producing a variety of different phenotypes (autoimmune disease syndromes) (Quimby and Schwartz, 1980). Data collected over the next 15 years, using techniques unavailable in the 1970s, have generally upheld this hypothesis and elucidated genetic mechanisms unimaginable at the time (Datta, 2000). 2.

Taking Advantage of Unexpected Findings

Serendipity also contributes to important discoveries. In 1889, a laboratory assistant noticed a large number of flies swarming about the urine of a depancreatized dog and brought it to the attention of VonMering and Minkowski. Minkowski discovered, on analysis, that the urine contained high concen-

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trations of sugar. This chance observation helped VonMering and Minkowski discover that the pancreas had multiple functions, one being to regulate blood glucose (Comroe, 1977). In the late 1800s, Christiaan Eijkman was sent to the Dutch Indies to study the cause of beriberi, a severe polyneuritis affecting residents of Java. While conducting studies, Eijkman noticed that chickens housed near the laboratory developed a similar disease. He tried and failed to transfer the illness from sick to healthy birds; however, shortly thereafter the disorder in chickens spontaneously cleared. Eijkman questioned a laboratory keeper about food provided the chickens and discovered that for economy, the attendant had previously switched from the regular chicken feed to boiled polished rice, which he obtained from the hospital kitchen. Several months later the practice of providing boiled rice to the chickens was discontinued, which correlated with disease recovery in the birds. This chance observation led Eijkman to conduct feed trials demonstrating that a factor missing in polished rice caused beriberi and that the disease could be cured by eating unpolished rice. These studies led to the discovery of the vitamin thiamin, and were the first to show that disease could be caused by the absence of something rather than the presence of something, e.g., bacteria or toxins (Eijkman, 1965). These examples reinforce the necessity for making careful observations, investigating unexpected findings, and designing careful follow-up experiments. Eijkman was awarded the Nobel Prize in Medicine in 1929, and Banting and Macleod received the Nobel Prize in 1923 for their discovery of insulin, made possible by the previous observations of VonMering and Minkowski (Leader and Stark, 1987).

B. 1.

Breakthroughs in Technology

Paradigm Shifts

In "The Structure of Scientific Revolutions," Kuhn (1970) makes a case for scientific communities sharing certain paradigms. Scientific communities consist of practitioners of a scientific specialty that share similar educations, literatures, communications, and techniques and as a result, frequently have similar viewpoints, goals, and a relative unanimity of judgment. Kuhn believes that science is not an objective progression toward the truth but rather a series of peaceful interludes, heavily influenced by the paradigms (call them theories) shared by the members of a scientific community and interrupted, on occasion, by intellectually violent revolutions that are associated with great gains in new knowledge. Revolutions are a change involving a certain sort of reconstruction of group (community) commitments. They usually are preceded by crisis (from within or outside the community) experienced by the community that undergoes revolution. Kuhn explains that scientific communities share a disciplinary

1202 matrix composed of symbolic generalizations (expressions, displayed without question or dissent by group members), beliefs in particular models, shared values, and exemplars (those concrete problem solutions that all students of community learn during their training). This disciplinary matrix is what provides the glue that keeps members of the community thinking (problem solving) alike. However, it is also what prevents members from taking high-stake chances and proposing new rules that counter prevailing opinion. Precisely when two members of a community disagree on a theory or principle because they realize that the paradigm no longer provides a sufficient basis for proof is the debate likely to continue in the form it inevitably takes during scientific revolutions. What happens during revolutions is that the similarity sets established by exemplars and other components of the disciplinary matrix can no longer neatly classify objects into similar groups. An example is the grouping of sun, moon, Mars, and Earth before and after Copernicus, where a convert to the new astronomy must now say (on seeing the moon), "I once took the moon to be a planet, but I was mistaken." As a result of the revolution, scientists with a new paradigm see differently than they did in the past and apply different rules, tools, and techniques to solve problems in the future (Kuhn, 1970). We are now at the threshold of such a revolution in biology. The crisis is beginning, and it takes the form of computerassisted biological modeling. Two diverse technologies are approaching maturity, computerized information processing and genetics (from recombinant DNA to functional genomics). They have converged as a result of the Human Genome Project where analysis of data provided by high throughput nucleotide sequencing (where the sequencing itself necessitated integrating the engineering fields of nanofabrication and computer sciences) requires computer-directed selection of intron-exon segments, protein modeling, associative screens matching models to existing protein families--so-called data mining (and thus linking structure to function)-- and rapid screens for nucleotide homology with other nucleotide (or amino acid) sequences in the same or another species. On the biological side of this research enterprise are techniques ranging from rapid throughput amino acid sequencing, X-ray crystallography (and other molecular imaging techniques contributing to structure), and genetic manipulation of biological models. On the computational side are analysis of gene expression using cDNA microassays, computer-assisted modeling (3-D imaging) from crystallographic images, and prediction of new molecular mimics (molecules that may simulate or inhibit the putative activities of another molecule). The end result is the new discipline of bioinformatics, which merges biology with mathematics, computer science, and engineering (Spengler, 2000). The merging of these technologies will be as profound in this century as were the technologies created by Galileo and van Leeuwenhoek in the seventeenth century. Clever combinations of technologies have led to novel methods for predicting protein function. One method called

FRED W. QUIMBY phylogenetic profiling (Pellegrini et al., 1999)" looks for the correlation of protein inheritance across species, while the second method, the Rosetta stone method, looks for correlation of protein domains across species. By use of these methods, proteins have been identified with particular complexes and pathways that suggest common functions for homologs in other species (see Section II,C). Likewise, comparing genomes of different species provides valuable information concerning noncoding regions in the DNA sequence. These correlations have revealed > 70% identity for noncoding regions in the mouse compared to human genomes (Spengler, 2000). The algorithm called Moving Average Point Analysis allows for global alignment of sequences, their comparison, and the display of their identity and has determined conserved sequences in mouse, dog, and human genomes. Likewise, programs have been written to evaluate complex physiologic phenomena, e.g., genes (and proteins) regulating DNA replication or simulation of chemical kinetic systems. But the revolution has just begun; the future shall fully integrate these various disciplines to achieve goals far more lofty than the association between DNA, protein structure, and function; bioinformatics will address complex mechanisms at the cellular, tissue, and population levels, as well as the interaction between genes, their products, and the environment. This level of investigation, unimaginable now, may lead to understanding the biological basis for learning and memory and a better understanding of how genes and the environment interact in processes such as obesity (see Section II,C). What is so revolutionary about integrating mathematics and computer science with biology? While not completely obvious now, the entire paradigm of hypothesis-driven research will change. Computers will make the comparisons, sort out the facts, and from them assist in generating hypotheses. For example, the new computer-assisted technology may input new nucleotide sequences from mouse chromosome 5 and after selecting for the coding region and Rosetta stone screening, demonstrate homology between the unknown sequence and a similar gene encoding the transmembrane receptor, Toll, in Drosophila. While Toll is known to affect dorsal-ventral polarity in Drosophila embryos, it also protects adults from attack by fungi. The cytoplasmic domain sequence of Toll has homology with the interleukin (IL)-1RI cytoplasmic domain in mammals. Human homologs are called Toll-like receptors (TLR), and mutations in TLR4, causing constitutive gene activation, led to expression of IL-1, IL-6, IL-8, and B7-1, proteins involved with inflammation and innate immunity. Both Toll (in Drosophila) and TLR4 share a signal transduction pathway involving activation of NF-×B (Wright, 1999; Medzhitov et al., 1997). One would thus postulate that disruption of the new mouse gene, through homologous recombination of ES cells, would lead to founders incapable of appropriate activation of NF-×B pathway and thus abrogation of the production of inflammatory cytokines essential in innate immunity. In fact, nature has already performed this experiment by creating a nonconservative point mutation in Tlr4 of C3H/HeJ

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30. ANIMAL MODELS IN BIOMEDICAL RESEARCH mice and a null mutation of T1P4 in C57BL10/ScCr mice (Poltorak et al., 1998; Qureshi et al., 1999). Both strains share an inability to properly respond to endotoxin, a lipopolysaccharide (LPS) in the cell walls of gram-negative bacteria, and in both strains genetic mapping has localized the defective gene (previously known as lps) to chromosome 5. Tlr4 is posulated to be a coreactor along with the LPS binding receptor, necessary for activation of signal transduction pathways leading to inflammatory cytokine production via NF-×B (Ulevitch, 1999). One may anticipate that much of the serendipity that characterized discovery in the past will be eliminated in this new age of bioinformatics, but that remains to be seen. The biologist ultimately must see if computer predictions are accurate and, rather than eliminate alternative hypotheses, test directly the consequences of knocking out or knocking in genes. For this work, the mouse will become the standard model of the future, having the advantage of similar physiology, similar genomic structure, high fecundity, and ease of maintenance. Before leaving the subject of scientific revolutions, it is prudent (and consistent with Kuhn's hypothesis) to ask if the members of the community of geneticists are witnessing a crisis. The answer is clearly yes. The crisis involves two different but related phenomena, information overload and speaking a com.mon language. The amount of information (call them facts) presented to geneticists today is overwhelming and is manifested by the proliferation of scientific journals, websites, and scientific meetings. In fact, the volume of data in various sequence data banks is so great that existing computers are often insufficient to analyze large data sets in reasonable time frames. There is also no easy way to transfer vast amounts of data between different institutions. As a result, individual investigators will have to become more focused and selective in the techniques they use and questions they ask and depend on collaboration with others to unravel complex or more generalized questions. Another generation of computers operating at higher speeds may make data analysis more efficient, and the Internet may ease the transfer of information between institutions. For the time being, anxiety is running high as scientists deal with the frustrations mentioned above (Malakoff, 1999). Equally critical is the interdependence between biologists, computer scientists, and engineers, each of whom uses a different language for expression. The full integration of several scientific communities into a single community has not been achieved, and frustrations are mounting as members of different communities attempt to work together. This problem was recognized by the Biomedical Information Science and Technology Initiative, which recommends a new paradigm in education where future biomedical scientists seek cross-disciplinary training (Malakoff, 1999). Several institutions in the United States have begun such programs where engineers, computer scientists, and biologists train together and gain the rudiments of each discipline.

2.

Making Use of Past Achievements

The time could not be more perfect for the next scientific revolution. If indeed the mouse (and possibly the rat) serves as the mammalian model that ultimately links gene and protein structure with function, it comes at a time when the discipline of laboratory animal science is reaching maturity. Since 1966, the year the Animal Welfare Act was passed into law, the world of biomedical research, in particular the use of rodent models, has dramatically changed. At that time spontaneous diseases of laboratory animals were the primary focus of articles published by Laboratory Animals and Laboratory Animal Science, and infectious diseases of laboratory mice and rats were prevalent, with 75% of institutions reporting respiratory disease in rats alone (Seamer and Chesterman, 1967). Several critical technologies, including the flexible-film isolator for cesariean rederivation of rodents (Trexler and Reynolds, 1957) and the defined bacterial "cocktail" (Schaedler et al., 1965), which was used to overcome intestinal complications frequent in germfree rodents and in which all eight strains have been defined by 165 rRNA sequence analysis (Dewhirst et al., 1999), were adopted early by a newly spawning industry to meet the demands of the scientific community (Foster, 1980). Professional societies were being formed in the United States and Europe to develop standards for laboratory animal care (Quimby, 1994). The Institute of Laboratory Animal Research (ILAR) of the National Academy of Sciences (NAS) held meetings devoted to laboratory animal housing and published a "Guide for Laboratory Animal Facilities and Care" (Hill, 1963), which served as the predecessor of the "Guide for the Care and Use of Laboratory Animals," a standard used by institutions throughout the United States (NRC, 1996). In the 1960s, the National Center for Research Resources (NCRR) of the National Institutes of Health (NIH) awarded grants to develop animal diagnostic/investigative laboratory programs to universities throughout the United States. These programs provided diagnostic support for the rapidly growing research animal enterprise and fostered research on disease pathogenesis and diagnostics. In the early 1970s, the NCRR developed a funding mechanism for development and improvement of institutional animal resources (Quimby, 1994b). Another program of the NCRR funded institutional training grants to educate and train competent laboratory animal veterinarians. The American College of Laboratory Animal Medicine (ACLAM) was established in 1961 to certify competency and encourage continued education (Quimby, 1995). During this same period, cage manufacturers met with scientists and laboratory animal specialists to design and construct optimum primary containment for laboratory animals, especially rodents. Although polycarbonate shoe-box cages became an early standard in the industry, new challenges to maintain rodents in germ-free environments (immunocompromised rodents) and contain agents used in infectious disease studies led to microisolator caging and the independently ventilated cage unit. The latter is becoming a

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standard in transgenic mouse facilities. Together with standardization of feeds and bedding, these changes have led to efficient, safe, and productive housing of the many SPF rodent models used today. Mouse genetics today is based on a long history of developing inbred strains, characterizing spontaneous mutations, characterizing phenotypic differences among strains, and mapping genetic loci. Equally important contributions have arisen as a result of recombinant DNA research and induced mutagenesis in the mouse, making it the ideal candidate for nonhuman mammalian genomics (see Section I,B for details). 3.

Looking at the Future

The impact of bioinformatics and its role in complementing functional genomics are likely to finally add to biology the same quantitative component that fostered 200 years of advances in theoretical physics and chemistry. As a better understanding is gained for each of the biochemical pathways controlling such fundamental processes as cell cycling, signal transduction, and transcription (including the proteins that modulate the activity of these biochemical pathways and the kinetics involved in these reactions), mathematical models will arise that may eventually predict certain outcomes. Eventually such models may anticipate host responses to such diverse entities as environmental chemicals, climatic change, and nutrition, but that is in the distant future. Certain fundamental changes are occurring already as new, more powerful tools are made available to scientists. The notion expounded by Robert Koch, which required the isolation and characterization of infectious agents from suspected cases and then re-creation of the illness by inoculation of the pure agent and its subsequent reisolation (the so-called Koch's postulates), is rapidly being replaced by the much simpler procedure of polymerase chain reaction (PCR) to identify microbes from tissues of infected individuals (Bradford, 1998). Genetic fishing using degenerate probes to identify putative agents of human (and animal) disease is more rapid and overcomes problems associated with low tissue concentration of the microbe and inability to culture certain organisms; however, occasionally mistakes are made. Although this technique will likely decrease the number of animals needed to validate a causative agent using Koch's postulates, recent studies have shown that in vitro model systems for investigating microbial evaluation (and in particular, mutations in bacteria conferring resistance and susceptibility to antibiotics) are not faithful replicas of the in vivo environment (Bull and Levin, 2000). As the number of infectious agents with complete genome sequences increase, new opportunities for investigation of microbial pathogenesis will arise (Strauss and Falkow, 1997). At this writing, complete genomes are available for Neisseria meningitidis (Tettelin et al., 2000), Escherichia coli (Collins et al., 1998), Helicobacter pylori (Alm et al., 2000), and C a m p y l o b a c t e r jejuni (Parkhill et al., 2000), and for 9 other pathogenic bacteria, with partial se-

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quences for another 28 bacterial pathogens (Weinstock, 2000). Technologies, including nucleic acid probe arrays and overlapping lambda clones of bacterial genomes, allow for rapid screening of thousands of bacterial genes temporally expressed during infection in in vivo models. Other rapid and specific technologies that can measure gene expression in the host under conditions of infections include in vivo expression technology (IVET), differential fluorescence induction (DFI), and the signature-tagged transposon method (STM), each designed to detect and follow specific virulence factors at discrete stages of interaction between mammalian host and the invading microorganism (Strauss and Falkow, 1997). Combining information on the genomes of the host and the pathogen will allow determination of genes for host resistance and susceptibility and provide the tools necessary to rapidly screen populations for individuals at risk. One example is the increased susceptibility to mycobacterial and salmonella infections in people who have IL-12R deficiency and reduced interferon (IFN)-y secretion but are otherwise healthy (deJong et al., 1998). Here the human findings were predicted based on published observations using IL-12R knockout mice (Magram et al., 1996; Wu et al., 1997; Flynn et al., 1993; Cooper et al., 1997). Information gathered through various genomics initiatives is already being used to improve gene therapy for inherited diseases and cancer (Geddes et al., 1997; Greenberg and Riddell, 1999; Ram et al., 1997; Roth et al., 1996), to modify organs and tissues for transplantation (Bracy et al., 1998), and to engineer vaccines against malaria and a host of other diseases (Hoffman et al., 1998; O'Donnell et al., 2000). In each case above, animal models were used to demonstrate efficacy. In addition, functional genomics is rapidly providing thousands of new molecular targets for future drug development, and animals remain as important models for efficacy and safety (Wetterau et al., 1998; Drews, 2000). After reflecting on future discoveries in science and the commotion that would follow the discovery of living things in other parts of our galaxy, Sir John Maddox continues: "But there will be more subtle surprises, which, of necessity, cannot be anticipated. They are the means by which the record of the past 500 years of science has been repeatedly enlivened. They are also the means by which the half-century ahead will enthrall the practitioners and change the lives of the rest of us" (1999).

CQ Taking Advantage of Interspecies Similarities: Research on Obesity

An example of a complex physiologic trait in which genetic, behavioral, and environmental components each play a role is that of obesity. Obesity, which is associated with decreased longevity in humans, is a complex disorder affecting 32% of adults and 40% of children in the United States (Campfield et al., 1998); it deals

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with the regulation of energy balance and fat mass, i.e., when intake exceeds expenditure, a state of positive energy balance is achieved, resulting in increased body weight. Some estimate that 40-70% of the variations in obesity-related phenotypes in humans are heritable (Allison et al. 1996; Comuzzie and Allison, 1998). Currently, three genome scanning efforts are underway that have obesity phenotypes as their central focus. Initial results suggest the existence of several genes that have an effect on obesity. Examples include a linkage region in human chromosome 2 that encompasses the gene for the prohormone proopiomelanocortin and a region on chromosome 8 that encompasses the gene for the [33-adrenergic receptor (Comuzzie and Allison, 1998). Efforts to map obesity genes in humans have been fueled by the discovery of genes affecting body weight and composition in animals, as well as by the large body of information available on feeding behavior and energy homeostasis collected from animal models (Woods et al., 1998). In the 1970s, it was discovered that cholecystokinin (CCK) regulated satiety in rats (Gibbs et al., 1973). However, while CCK had a potent effect on food intake, it had a limited effect on fat mass, implying that other factors affecting energy homeostasis were also essential for the long-term control of adiposity. Two central nervous system pathways control energy balance in response to body fat and include the anabolic neuropeptide Y axis (which stimulates food intake and promotes weight gain) and the catabolic hypothalamic melanocortin system (which reduces intake and promotes weight loss). Spontaneously mutant and knockout mice have contributed greatly to the current understanding of these two CNS pathways. Obese (ob/ob) and diabetic (db/db) mice were found to have single-gene defects affecting the expression of leptin (ob) and the function of the leptin receptor (db) (Zhang et al., 1994; Chua et al., 1996). Using these models, it was found that leptin is a hormone secreted by adipocytes that binds receptors in the hypothalamus (and cross the blood-brain barrier via receptors on brain capillary endothelial cells). Leptin was found to share many properties with insulin, the latter influencing secretion of the former. Both insulin and leptin reduce feed intake, body weight, and fat stores. Except in leptin-deficient obese mice, obese mammals have elevated plasma leptin levels and appear to be resistant to leptin-induced anorexia. Studies in the obese Zucker (fa/fa) rat, which has a mutation in the leptin receptor gene, suggest that the CNS effects of insulin require a functional leptin system (Woods et al., 1998). Human homologs for the defects seen in obese and diabetic mice have been characterized (Montague et al., 1997; Clement et al., 1998) and like the murine models, little effect is seen in obese humans following administration of exogenous leptin, except in those who are leptin-deficient (Himms-Hagan, 1999). Many other actions of leptin in mice are not seen in humans. Neuropeptide Y (NPY) is a neurotransmitter present throughout the brain; however, in the hypothalamus, NPY mediates effects on energy homeostasis. Central administration of NPY promotes positive energy balance and increased fat stores. Neu-

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ropeptide Y also reduces sympathetic nervous system outflow to brown adipose tissue and thus lowers energy expenditure while increasing enzymes necessary for lipogenesis of white fat; NPY is overexpressed in ob/ob and db/db mice, and its response is attenuated by leptin in ob/ob mice. Thus, leptin provides negative feedback to the NPY system (Woods et al., 1998). The NPY Y1 receptor knockouts have changes in fastinduced feeding behavior, cardiovascular responses to NPY, and energy expenditure, suggesting this receptor (as opposed to NPY Y5) is principally involved in the control of food intake (Pedrazzini et al., 1998). Melanocortins, peptides cleaved from proopiomelanocortin precursor, play a role in the central anabolic pathways where they participate in energy homeostasis and elicit anorexia in animal models (Sehioth, 1997). Genetic deficiency of the melanocortin 4 receptor in mice results in hyperphagia and obesity (Cone et al., 1996). However, studies in the ob/ob mouse have shown that melanocortin neurons are a target of leptin action. Corticotropin-releasing hormone (CRH) is a catabolic hypothalamic neuropeptide that also contributes to energy homeostasis. Although endogenous CRH is involved with stress and illness, central administration of CRH reduces food intake and body weight. Leptin administration increases and glucocorticoids inhibit CRH gene expression in animals. (Seeley et al., 1996). Furthermore, although overproduction of CRH has been implicated in anorexia, underproduction promotes the actions of glucocorticoids, which cause weight gain and obesity. As with other neuropeptides involved in central control of energy homeostasis, leptin and insulin are intimately involved in CRH metabolism and activity. Observations made in mice with defective expression of insulin receptors in the central nervous system show that these animals have increased body fat content and are predisposed to obesity (B~ning et al., 2000). Finally, administration of leptin or insulin to animals potentiates the reduction in meal size seen following CCK administration. Thus, the size of fat stores can influence feeding behavior, resulting in animals being more sensitive to meal-generated signals following bouts of excessive eating or after gaining excess weight. Although it is unclear how satiety signals integrate with central anabolic and catabolic hypothalamic pathways, it is clear that the influence of adipose tissue signals on food intake is not responsive to individual meals, but rather exerts its effects over several days. These findings from animal research have helped elucidate critical components in the control of energy homeostasis and feeding behavior. The complexity of this system, with its redundancies and numerous points of negative feedback, has contributed to a program that efficiently responds to weight loss and replenishes depleted fuel stores. While the mechanisms for defending feed stores have probably conferred a selected advantage to individuals (and populations of animals) possessing them, finding a simple antiobesity molecular target for drug interaction is not likely to be easy and will certainly involve

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changes in environmental components, e.g., modified diets and physical exercise. The goal of all antiobesity drugs is to induce and maintain a state of negative energy balance (Campfield et al., 1998). Generally they fall into one of four classes of compounds: inhibitors of energy intake (appetite suppressants), inhibitors of fat absorption, enhances of energy expenditure, and stimulators of fat mobilization. Currently, only a small group of compounds remains approved and available for treating obesity, and each acts centrally as an appetite suppressant to reduce food intake by modulating monoamine neurotransmitters (serotonin and norepinephrine, or norepinephrine alone) in the brain. These drugs work by inhibiting the reuptake of serotonin or norepinephrine. Reports of heart valve disease in obese patients taking inhibitors of serotonin reuptake have resulted in three (of five) of these drugs being withdrawn. As a result, there is keen interest in development of drugs targeting other central (or peripheral) pathways leading to alteration in energy balance. Based on animal experimentation, new molecular targets for antiobesity drugs include among others, ob receptor agonists, NPY receptor antagonists, melanocortin 4 receptor agonists, agouti-related peptide agonists, proopiomelanocortin antagonists, melanocyte-concentrating hormone receptor antagonists, CRH receptor antagonists, urocortin antagonists, and CCK-A receptor agonists - - all of which are designed to suppress appetite. Drugs designed to enhance energy expenditure include stimulators of uncoupling protein 2 (UCP2) and 3, proton transporters that are expressed in peripheral tissue and cause increased thermogenesis; stimulators of protein kinase A (PKA), and agonists of the [33-adrenergic receptor. Finally, drugs that stimulate fat mobilization include those that agonize the obreceptor, stimulate PKA, agonize the [~3-adrenergic receptor, or agonize the growth hormone receptor (Campfield et al., 1998). Further elucidation of the integrated network of neurotransmitters, hormones, and receptors controlling energy balance may help define which target above is most likely to produce negative energy balance without compromising other systems. Alternatively, future studies may obviate new molecular targets that can safely be modulated therapeutically to achieve weight loss in overweight individuals.

III.

HISTORY OF ANIMAL USE IN

BIOMEDICAL RESEARCH A.

Early History

Humans have a history of close interaction with animals that extends back over 20,000 years (with the domestication of poultry in China) and includes the domestication of buffalo, cattle, sheep, and dogs between 6000 and 10,000 years ago. The earli-

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est written records date to 2000 BC when Babylonians and Assyrians documented surgery and medications for humans and animals. True scientifc inquiry began in the intellectually liberal climate of ancient Greece where the teachings of Aristotle, Plato, and Hippocrates symbolized a move to understand natural phenomena without resorting to mysticism or demonology. In this environment, philosophy was conceived and wisdom was admired. Early animal experimentation was conducted in 304 BC by the anatomist Erasistratos, who demonstrated the relationship between food intake and weight gain in brids. In the second century AD, the physician Galen used a variety of animals to show that arteries contained blood and not air, as believed by his contemporaries. During this period, physicians carried out careful anatomic dissections, and on the basis on the comparative anatomy of animals and humans, accumulated a remarkable list of achievements, including a description of embryonic development; the establishment of the importance of the umbilical cord for fetal survival; and the recognition of the relationship between the optic nerves, which arise from the eyes, and the brain. The Greeks, and later the Romans, developed schools of higher learning (including medical schools), created museums, and documented their findings in libraries. Physicians from this period recognized that fever aided the healing process, recognized the inherited nature of certain disorders and classified them, and practiced intubation to prevent suffocation and ligation and excision for the treatment of hemorrhoids. This brief period of scientific inquiry in Europe gave way to the Middle Ages, a 1200-year period characterized by war, religious persecution, and unsavory politics. During the Middle Ages until the Rennaisance, the writings of ancient Greece and Rome remained the final word on science and medicine. Medical education was revived in tenth-century Salerno, Italy, but because of a prohibition on human dissection that lasted into the thirteenth century, animals were substituted for humans as models in the instruction of anatomy. Because no investigations took place, virtually no new discoveries in medicine were made. Imagine how handicapped these medieval physicians must have been. They still did not know that the filling of lungs with air was necessary for life, that the body was composed of many cells organized into tissues, that blood circulated and the heart served as its pump, and that blood traverses from arteries to veins in tissues via capillaries; these facts were revealed by Hook in 1667, Swammerdam in 1667, Van Leeuwenhoek in 1680, Harvey in 1628, Malpighi in 1687, and Pecquet in 1651, respectively, each using animals to demonstrate these basic principles. In part, this return to the process of scientific discovery was built on the foundations established by Francis Bacon, a foundation based on collecting facts, developing hypotheses, and attempting to disprove them via experimentation (see Section II,A,1). The pace of biomedical research increased during the 1700s as Priestley discovered that the life-promoting constituent of air

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was oxygen. Scientists such as Von Hailer, Spallanzani, Trembly, and Stevens, each using animals, discovered the relationship between nerve impulses and muscle contraction, recorded cell division, and associated the process of digestion with the secretions of the stomach. Hales made the first recording of blood pressure in a horse in 1733, Crawford measured the metabolic heat of an animal using water calorimetry in 1788, and Beddoes successfully performed pneumotherapy in animals in 1795 (although it was not until 1917 that Haldane would introduce modern oxygen therapy for humans). By 1815, Laennec had perfected the stethoscope, using animals. Despite these dramatic gains in medical knowledge, physicians were still not aware of the germ theory of disease (and of course could not avoid, prevent, or treat infections) (Quimby, 1994a).

B.

F r o m P a s t e u r to P r u s i n e r

In the 1860s, the French scientist Louis Pasteur discovered that microscopic particles, which he called vibrions (i.e., bacteria), were a cause of a fatal disease in silkworms. When he eliminated the vibrions, silkworms grew free of disease--the first demonstration of the germ theory of disease. In 1877, Pasteur turned his attention to two animal diseases, anthrax in sheep and cholera in chickens. In each disease he isolated the causative agent, reduced its virulence by exposure to high temperature, and showed that on injection the attenuated organism imparted protection against the disease. Pasteur referred to this process as vaccination (from Latin vacca, "cow") in homage to the English surgeon Edward Jenner, who discovered that injection of matter from cowpox lesions into humans protected them against smallpox. Pasteur went on to develop the first vaccine against rabies, in which the virus was attenuated by passage through rabbits. This vaccine was shown to impart protection in dogs and later in humans. Pasteur's work with microscopic organisms as agents of disease quickly led to two other important discoveries. John Lister, having read of Pasteur's discovery, hypothesized that these microorganisms were responsible for wound infections. He impregnated cloth with an antiseptic of carbolic acid and showed that when used as a wound dressing, the antiseptic prevented infection and gangrene. This led to the generalized use of antiseptics before surgery and sterilization of surgical instruments. In 1876, Robert Koch would demonstrate a technique for growing bacteria outside of an animal (in vitro) in pure culture. This would reduce the number of animals required to conduct research on infectious agents, and it allowed Koch to establish postulates for definitively associating a specific agent with a specific disease (see Section II,B,3). Using these postulates, Koch discovered the cause of tuberculosis, Mycobacterium tuberculosis, and he developed tuberculin used to identify infected animals and people. Between 1840 and 1850, Long and

Morton demonstrated the usefulness of ether as a general anesthetic first in animals and later in humans. 1.

Contributions to Inheritance

The second half of the nineteenth century began a new era in biology and medicine. In addition to such medical developments as vaccination, testing for tuberculosis, anesthesia, and blood transfusion, each of which depended on animal experimentation, two other events changed the direction of biological science forever. In 1859, the English naturalist Charles Darwin published "On the Origin of Species," in which he hypothesized that all life evolves by selection of traits that give one species an advantage over others. Around the same time, the Austrian monk Gregor Mendel used peas to demonstrate that specific traits are inherited in a predictable fashion. Nearly half a century later, the English biologist William Bateson reached the same conclusion by selectively breeding chickens and reported his result, as Mendel's work was being generally recognized. Mendel proposed two laws of heredity: first, that two different hereditary characters, after being combined in one generation, will again segregate in the next; and second, that hereditary characteristics assort in new daughter cells independently (Sourkes, 1966). Unfortunately, Bateson's investigations with chickens did not always give the numerical results of two independent pairs of characters. This led Sutton and Bovery, at the turn of the century, to conclude that the threadlike intracellular structures seen duplicating and separating into daughter cells carried the hereditary characters. Later Thomas Hunt Morgan, using cytogenetics and selected breeding in fruit flies, clearly demonstrated the phenomenon of genetic linkage (Morgan, 1928). Others went on the verify these observations in plants and animals. During the first half of the twentieth century, revelations concerning the discovery of nucleic acids by Kossel, using salmon sperm and human leukocytes (Sourkes, 1966); the structure of nucleotides by R A. Levine; and the structure of DNA by Watson, Crick, Wilkins, and Franklin depended on advances in chemistry and X-ray crystallography (Watson and Crick, 1953). In fact, it was the application of X-ray diffraction techniques that finally allowed scientists to deduce the double helical structure of DNA. When Watson and Crick saw Franklin's photographs, it galvanized them into action; by building models of the nucleotides and hypothesizing the points for hydrogen bonding between purines and pyrimidines, they quickly assembled the three-dimensional structure of DNA. Their insight into how the diffraction pattern correlated with helical symmetry allowed for a practical solution to a very complex and, until then, elusive problem. They reinforced the meaning of the term "great science," as expressed by Lisa Jardine, "Great science depends on remaining grounded in the real" (1999). There were 50 years between the isolation of "nuclein" in leukocytes by Kossel and the discovery of the double helical

1208 structure of DNA, which recognized that the pattern of purine and pyrimidine coupling contained the code for heritability. Likewise, there were 50 years between the hypothesis by Garrod in 1902 that family members with alkaptonuria had inherited a deficiency in a particular enzyme that metabolizes homogentisic acid and Beadle and Tatum's proof, using Neurospora, that indeed X-ray-induced genetic mutations affected the production of specific enzymes (Lederberg and Tatum, 1954). To a certain extent, these latter studies depended on the demonstration that bacteria (and other lower organisms) in fact contained genetic information that controlled protein synthesis in a manner similar to that in eukaryotes (Lwoff, 1953). This breakthrough provided the fuel for the revolution in molecular genetics, which included the biological synthesis of deoxyribonucleic acid (Kornberg, 1959) and the genetic regulation of protein synthesis (Jacob and Monod, 1961)meach depending on work in bacteria. Later, the details on the control of gene expression were heavily dependent on Escherichia coli and its well-studied lac operon, yeast, and the fruit fly, Drosophila melanogaster. No wonder these three organisms were among the first to have their entire genome sequenced. With the mechanisms in hand for synthesizing nucleotide sequences and creating constructs with precise regulatory sequences for insertion into the mammalian genome, another revolution is about to occur; however, this time the mouse will guide investigators interested in discovering the function of proteins encoded by new genes and unraveling the mechanisms behind inborn errors in humans (see Section II,B, 1). For their achievements in genetics and molecular biology, the following scientists have won the Nobel Prize: Thomas Morgan; Albrecht Kossel; George Beadle, Edward Tatum, and Joshua Lederberg; James Watson, Francis Crick, and Maurice Wilkins; Andre Lwoff, Francois Jacob, and Jacques Monod; and Severo Ochoa and Arthur Kornberg. After a century of research demonstrating that all "living" things replicate based on information encoded in DNA or RNA, imagine how heretical Stanley Prusiner's hypothesis appeared to the scientific community when he proposed an infectious disease agent composed entirely of protein (prions) that caused disease in humans and animals as a result of abnormal protein folding. Prusiner used many types of animals in pursuit of the prion, but laboratory hamsters and mice played a particularly important role, as did the disease-causing agent of scrapie in sheep (Liu et al., 1999; Supattapone et al., 1999). As in the scientific debate concerning the humoral versus cellular theory of immunity (see Section III,B,2,b), the scientific community continues to debate the prion hypothesis, and again as in the earlier debate, the Swedish Academy cast its vote by awarding Prusiner a Nobel Prize in 1997. 2.

Progress in the Field of Immunology

a. Origins. The concept of adaptive immunity, developing protection after exposure to an infectious agent or poison, dates back to at least 430 Bc when Thucydides writes of the plague of

FRED W. QUIMBY Athens, "Yet it was with those who had recovered from the disease that the sick and dying found most compassion. These knew what it was from experience and had now no fear themselves; for the same man was never attacked twice D never at least fatally" (1934). Despite this early recognition, the association of disease with infectious agents was missing. During the 1200s, the Black Death in Europe and the East was attributed to a conjunction of Mars, Saturn, and Jupiter; and later in the fifteenth century the appearance of syphilis in Europe was attributed to another conjunction of the same planets (Silverstein, 1989). Even during the Renaissance, when cities were adopting the principles of quarantine in the face of an epidemic, influenza was still ascribed to the influence of the stars, and mal-aria meant "bad air." It was not until the end of the nineteenth century that studies using animals allowed investigators such as Pasteur, Koch, Ehrlich, von Behring, and Metchnikoff to demonstrate the phenomenon of acquired immunity, the association between infectious agent and disease, the principles of vaccination, and the treatment of diphtheria (and tetanus) with antitoxins. It was not until the twentieth century that many other scientists unraveled the molecular basis for acquired immunity and extended its practical application to the fields of transplantation and vaccinology, and developed treatments for autoimmune, immunodeficiency, and hypersensitivity diseases, as well as treatments to prevent graft rejection and therapies for shock and cancer. b. Pioneers of humoral immunity. Emil von Behring (1854-1917) was a student of Robert Koch and went on to demonstrate that animals (guinea pigs, rabbits) vaccinated with diphtheria or tetanus organisms developed immunity to infection and to the detrimental effects of their toxins. In two manuscripts published in December 1890, he describes how he produced antitoxins to diphtheria and tetanus and how cell-free serum from immune animals protected nonimmune animals after passive transfer. Within 1 year the first human was successfully treated for diphtheria, and soon after, serum treatment came into general use. The death rate from diphtheria fell from 35 to 5%, and among those with laryngeal involvement, from 90 to 15%. Von Behring was awarded the Nobel Prize in 1901 (Sourkes, 1966). Another student of Koch was Paul Ehrlich, who by studying antisera made in animals (particularly guinea pigs) against plant and bacterial toxins developed a standardized test in 1897 to quantitate toxin and antitoxin. In doing so Ehrlich postulated the unique stereochemical relationship between active sites on antibody and antigen and introduced the concepts of antibody affinity and of functional domains on antibody molecules. Finally, he postulated that antibody formation was the cellular response to the binding of antigen to its surface receptors. He was awarded the Nobel Prize in 1908 along with Elie Metchnikoff (who discovered the antibacterial properties of phagocytes in a variety of animals) (Ehrlich, 1900). The mentor of both von Behring and Ehrlich, Robert Koch

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made many cohtributions, particularly in the new field of bacteriology; however, he also developed tuberculin and devised the standard tuberculin skin test, which was one of the first demonstrations of cellular immunity (Silverstein, 1989). He was awarded a Nobel Prize in 1905. In the mix with a group of scientists primarily devoted to the humoral theory of immunity, Elie Metchnikoff proposed a cellular theory of immunity. This was based on his observations of starfish phagocytic cells and their activity in the presence of bacteria. Furthermore, Metchnikoff theorized that immune activation produced a substance that heightened the activity of phagocytes. The work of Pasteur, Koch, Ehrlich, and von Behring all implicated humoral factors as protective in immunity, and the debate between humoral and cellular theories began. It did not help matters that during this debate, which lasted nearly 2 decades, the discoveries of bacterial agglutination (by Max von Gruber and Hurbert Durham), anaphylaxis (by Paul Portier and Charles Richet), the arthus phenomenon (by Maurice Arthus), and serum sickness (by Clemens von Pirquet and Bela Schick) all supported the humoral theory (and all involved animal research). Subsequently, the Nobel Prize would also be given to Charles Richet for his discovery of anaphylaxis, using dogs, and Jules Bordet for his discovery of complement, using guinea pigs (Bordet, 1909). Many years passed between Richet's discovery of experimental anaphylaxis and Bovet's discovery of histamine as a major mediator of that phenomenon. Bovet developed an ex v i v o assay based on exposing strips of sensitized animal uterine tissue to antigen. Later he developed antihistamines for the treatment of allergies such as asthma and atopy, based on the concept that synthetic molecules that resemble a metabolite of an active agent, e.g., histamine, may block or antagonize the effects of the active compound. Later, while studying the mode of action for curare in rabbits and dogs, he developed curare-like relaxants, tranquilizing drugs, and anesthetics. Daniel Bovet was awarded the Nobel Prize in 1957 (Sourkes, 1966). In retrospect, the arguments against a cellular theory seem antiquated, with the discovery of complement receptors on phagocytic cells, the role of complement as an opsonin for phagocytosis by Wright and Douglas, and even Koch's demonstration of the cellular infiltration at the site of a tuberculin reaction. Unfortunately, Metchnikoff was a lone voice in the cellularist doctrine, and in the end most early investigators of the twentieth century turned to investigations involving antibody. For nearly 50 years the important area of cellular immunity and the role of lymphocytes in immunity were placed on the back burner. This is an excellent example of Kuhn's contention that scientific communities tend to reinforce the familiar and reject hypotheses that are more controversial and require taking risks (see Section II,B,1; Kuhn, 1970). During the next 30 years investigators would study how immunologic (antibody) specificity was expressed and its biological implications (precipitation, agglutination, hemolysis, allergy), how immunologic specificity is structurally determined,

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and how the information for immunologic specificity is encoded. Major contributions were made by many, but perhaps none had a more profound effect on immunology (and especially medicine) than Karl Landsteiner. Born and educated in Vienna, he trained in the laboratories of Emil Fischer (in Wurzburg) and E. Bamberger (in Munich) before becoming an assistant to Max von Gruber (in Vienna). He traveled to Holland following World War I to be a pathologist at the Hague (19191922), and in 1922, he became a member of the Rockefeller Institute where he worked until his death in 1943. Landsteiner studied the phenomenon of red cell agglutination in humans and nonhuman primates. He discovered the ABO blood group and the isoagglutinins associated with them. He later discovered the MN and Rh factor blood groups. The consequences of this work allowed for proper typing of blood for transfusions (which has saved millions of lives from hemorrhage due to trauma, surgery, ulcer, ectopic pregnancy, bleeding after childbirth, and bleeding due to hemorrhagic disease, as well as by treatment for anemia and carbon monoxide poisoning). Although the MNP group is important primarily in forensic medicine, knowledge of the Rh factor and the antibody therapy developed from it has dramatically reduced the incompatability disease known as erythroblastosis fetalis (hemolytic disease of the newborn) (Landsteiner, 1945). Other major contributions made by Landsteiner include the demonstration of the first antitissue antibodies (antisperm), description of the first autoimmune disease--paroxysmal cold hemoglobinuria (with Donath in 1904)--the phenomenon of hapten inhibition, and the ability to passively transfer delayed hypersensitivity (with Merrill Chase in 1942). Landsteiner was one of the first to show that poliomyelitis and syphilis could be induced in nonhuman primates. Landsteiner was awarded the Nobel Prize in 1930. Other advances in immunology that centered primarily on antibodies and their function include the work of Tiselius and Kabat, classifying antibodies as high molecular weight gamma (y-) globulins; and the work of Porter and Edelman, using myeloma proteins of humans and mice, as well as guinea pig immunoglobulin, to demonstrate the basic structure of antibodies through selective chain cleavage with enzymes. This allowed for primary amino acid sequencing of antibodies, with the resulting acknowledgment of constant and variable regions, repeating domains, and sites for secondary biological activities, e.g., complement fixation (Silverstein, 1989). Once the structure of antibodies was known, many investigators began a search for the molecular (genetic) basis for proteins with constant and hypervariable regions. Calculations of the amount of DNA required to generate antibodies with all the diversity seen in animals and humans at times exceeded the size of the genome; therefore in 1965, Dreyer and Bennett proposed the existence of multiple variable region genes that could combine with the constant region gene to produce a unique isotype. Tonegawa and colleagues first discovered the presence of the constant region locus, plus multiple variable and joining region genes, which assemble such that a single variable region gene

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and a single joining region gene combine with the constant region gene to produce a unique light chain. With the assistance of Leroy Hood, Tonegawa later found a fourth gene cluster, called diversity, which contributed a single gene to form the longer heavy chain of immunoglobulin. This important work not only demonstrated how the enormous diversity of antibodies could be encoded in a compact segment of DNA, but also opened the doors to those searching for the elusive T-cell receptor. For their contributions, Porter and Edelman were awarded the Nobel Prize in 1972, and Tonegawa received the award in 1987. Two other groups made contributions involving antibodies that would revolutionize many areas of the biological sciences. For their contribution of the radioimmunoassay, a technique requiring specific antibodies made in animals (usually rabbits and goats), Rosalyn Yalow, Roger Guillemin, and Andrew Schally won the Nobel Prize in 1977. In 1984, the Nobel Prize was awarded to Cesar Milstein and Georges Kohler for their contribution of monoclonal antibodies and the hybridoma technique. These two assays, and the enzyme-linked immunosorbent assay (ELISA) that followed, revolutionized the detection of specific antigens in tissues and biological fluids and serve today as the basis for disease diagnosis. Monoclonal antibodies have a multitude of purposes, from disease diagnosis to the purification of proteins and therapy for cancer (Dickman, 1998). Of course, many other investigations concerning the role of antibodies in health and disease occurred during this period, including the discovery of immunoglobulin deficiency disease in 1952 by Ogden Bruton and the bursa of Fabricias as the site of antibody-producing "B" cells in the chick (the mammalian counterpart was fortunately found to be the bone marrow, thus preserving the B-cell designation). The discovery of mucosal immunoglobulin A and later discoveries that demonstrated the role of secretory piece and transepithelial transport of the molecule (much of this dependent on the use of various animals) were made, as well as the discovery of IgE by Ishizaka and Ishizaka in 1966; calculations on the size of the antibody combining site by Kabat; and the existence of idiotypes by Kunkel, Mannik, Williams, Oudin, Michel, Gell, and Kelus in 1964. However, 50 long years went by before another major investigation of the cellular theory of immunity was to occur, c. Pioneers o f cellular immunity. World War II stimulated research to improve the survivability of grafted tissues, particularly skin for wound victims. Peter Medawar was interested in tissue grafting and was the first to document that second grafts from the same donor to recipient were rejected more quickly than the first graft, whereas a third-party graft was not. This finding supported the view of immunologic specificity and the secondary response. He and colleagues Brent and Billingham established the field of transplantation biology. When Ray Owen documented that dizygotic cattle twins, which were red cell chimeras, were unable to reject each other's organs, the Australian physician MacFarlane Burnet proposed that im-

FRED W. QUIMBY

munologic responses arise late in animal development and that during early development, cells of the immune system would catalog available antigens as self (and thus not respond to them). Later in development, the introduction of new antigens would be considered nonself or foreign and elicit an immune response (Burnet, 1959). This hypothesis was studied by Medawar and colleagues subsequently by exposing neonates to antigens of another strain and then transplanting skin between these inbred strains as adults. He confirmed that early antigen exposure prevented the immune system from recognizing the antigen later as foreign; he called this acquired immunological tolerance (Billingham et al., 1953). These observations were among the first to be made in the new field of transplantation. They paved the way for characterizing the immune responses typical of rejection (which are primarily cellular in allograft rejection), the antigens that elicited these responses, and mechanisms for suppressing the response (such as acquired tolerance and immunosuppressive drugs). MacFarlane Burnet and Peter Medawar shared the Nobel Prize for Medicine in 1960. In the 1950s and 1960s, George Snell and Peter Gorer discovered the genetic locus in mice important in allograft (between individuals of the same species) rejection. The work of many who followed refined the information concerning this very complex locus, the major histocompatibility locus (MHC). However, this feat occurred because George Snell had the inventiveness to inbreed mice, thus generating strains (identical twins) and congenic lines that differed between one another by only a single gene. From this point onward, inbred strains and lines of mice would be the animal model of choice for those studying immunogenetics. They allowed for many of the studies in oncology, transplantation, and molecular biology. Among those who used inbred strains to study the MHC was Baruj Benacerraf of Harvard University. He studied the loci in mice that determined the immune response to synthetic polypeptides and named them immune response (IR) genes. It was later shown that the IR region fell into the MHC class 1 regionm a locus that controlled the intercommunication between various immunocytes. Jean Dausset, a French immunologist, discovered that the human leukocyte antigen (HLA) system in humans controlled graft rejection. These HLA antigens were later found to be encoded in the MHC. Benacerraf, Snell, and Dausset shared the Nobel Prize in 1980. Later, inbred strains of rats, rabbits, and guinea pigs would be available to aid future studies, and histocompatibility typing was extended to many species of animals. Also during the 1960s, Jacques Miller described the role of the thymus in immunity, the origin of the T cell, which would later be found to mediate graft rejection, modulate antibody synthesis, and directly participate in the eradication of infectious agents and cancer (Miller, 1961). By 1966, Claman, Chaperon, and Triplett, as well as Mitchell and Miller, had described T-cell subsets that discriminated between the types of T cells that assisted B cells in antibody production (helper cells) and those with direct cytotoxic activity. In 1970, Gershon and Kondo described suppressor T cells, which modified the activ-


ity of other lymphocytes. Now the availability of monoclonal antibodies was allowing scientists to detect surface markers on lymphocytes that could be used to distinguish functional subpopulations of lymphocytes, i.e., CD4 for helper cells and CD8 for cytotoxic lymphocytes. Availability of inbred strains also allowed investigators to evaluate immune mechanisms by passive transfer of subpopulations of cells between individuals. Early in these investigations, Peter Doherty and Rolf Zinkernagel discovered that certain immune responses were MHC-restricted, that is, the lymphocytes of one MHC type could not recognize foreign antigens presented by cells of another MHC type. This led to the discovery that antigen-presenting cells express foreign peptides together with MHC proteins on their surface. Cells presenting MHC II molecules stimulated CD4 + cells, and those presenting antigen together with MHC I molecules stimulated CD8 + cells. These discoveries explained how different cells of the immune system were selected to attack a particular foreign invader and would aid in the understanding of intrathymic education of newly developing T cells to become tolerant to self antigens (Zinkernagel and Doherty, 1997). For their achievements, Doherty and Zinkernagel were awarded the Nobel Prize in 1996. First described by Flanagen in 1966, the nude mouse occurred as a spontaneous mutation in which there is developmental failure of the thymic anlage, resulting in a mouse completely devoid of functional T cells. The scid mouse was described in 1983 by Bosma et al., and due to a mutation in r a g l gene encoding recombinase activity, this mouse failed to rearrange immunoglobulin or T-cell receptor genes and thus developed a combined (T- and B-cell) immunodeficiency. Use of these mice during the past 2 decades has greatly assisted in unraveling the cellular basis of immunity (Fogh and Giovanella, 1978, 1982; NRC, 1989; Carballido et al., 2000). As molecular techniques and the knockout technology became available, scientists probed deeply into the genetic mechanisms controlling immune function (Preckel et al., 1999; Pappu et al., 1999; Schaeffer et al., 1999; Philpott et al., 1992; DeTogni et al., 1994; Pages et al., 1999; Tamada et al., 2000). Once again, the mouse was the favored species for this work. The fine details on how animals (and humans) develop and maintain self-tolerance are unraveling and with them, new ideas for future therapy for autoimmune disease (see below). Likewise, the importance of a new class of immune modulators, the interleukins, has been recognized primarily through animal research. The ability to modulate levels of various interleukins has major implications for the treatment of inflammatory illnesses and shock; in the augmentation of immune responses against infectious agents, including parasites; and in the battle against cancer (Weinblatt et al., 1999; Hoffman et al., 1997; Tokman and Quimby, 1995). d. Transplantation biology. The reader must now be brought back to the 1960s and the pioneer work of George Snell, to pick up where Peter Medawar, of graft-rejection fame,

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left off. Several factors came together during this period to make the allografting of organs successful. First, the preservation and tissue typing of the donor organs were critical. Next, surgical manipulation of organs to allow prompt reestablishment of the blood supply was necessary. Finally, a mechanism of suppressing the cellular immune response was essential. Woodruff, a transplant surgeon in Edinburgh enthralled by the experiment of Medawar and colleagues, found a pair of dizygotic twins who shared each other's red cell types. He hypothesized that they had shared placental circulation and found, after cross-skin grafting them successfully, that humans, like cattle and mice, could develop acquired immunologic tolerance (Woodruff, 1959). During the first half of the twentieth century, Carres, Quinby, Dempster, and Simonsen each attempted renal autografts and allografts in nonimmunosuppressed dogs. Although autografts remained functional for longer periods than allografts, all transplants ceased to function due to lack of innervation, lymphatics, or both. It was the many investigations conducted by Joseph Murray during the early 1950s that resulted in a surgical technique that would leave autografted kidneys in dogs completely functional after 2 years (Murray et al., 1956). However, allografts were still quickly rejected. Lawrence (1959) was the first to liken the allograft rejection response to delayed hypersensitivity reactions. Attempts to suppress immune rejection with steroids, anticoagulants, or both, failed. Following trials in mice and rabbits (Main and Prehn, 1955), a protocol involving total body irradiation followed by bone marrow transplantation and kidney transplantation achieved variable success in human kidney graft recipients. In 1959, Schwartz and Dameshek reported on the ability of 6-mercaptopurine (6-MP) to prevent rabbits from producing an antihuman serum albumin antibody response. Calne (1960) in London used 6-MP to suppress the rejection of allografted canine kidneys with success, although the drug itself was toxic. Calne then urged G. H. Hitchings and G. B. Elion of Burroughs Wellcome Laboratories to become collaborators. New Wellcome drugs greatly improved allograft survival, with dogs surviving normally for years. Hitchings and Elion had developed the imidazole derivative of 6-MP, known as Imuran (azathioprine). This became the mainstay of transplant surgeons for the next 20 years (Murray, 1992). Following investigations in dogs, orthotopic liver allografts were performed in humans by Moore and Starzl. In 1960, Lower and Shumway developed a surgical technique for transplant of the heart in dogs. This was followed in 1970 by successful heart transplantation in man. Later achievements included transplantation of the bone marrow, pancreas, and portions of the intestinal tract. The advent of reagents to histocompatibility type donors and recipients reduced the immunologic barrier between them and led to more appropriate selection of grafts, with a resultant increase in functional longevity of the graft. Also, improved drugs, such as cyclosporin A, rapamycin, and FK506, targeted

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specific cellular events necessary for initiating the rejection process, causing fewer side effects and leaving the host must less immunologically compromised (Carpenter, 2000). Other drugs, such as mycophenolate mofetil and anti-IL-2R, are now in trials (Beniaminovitz et al., 2000). In addition, monoclonal antiCD3 antibodies have been used to prevent acute rejection since the early 1980s (Ortho Multicenter Transplant Study Group, 1985). However, the dream of transplant surgeons is to induce acquired immunologic tolerance in the recipient to the graft, what Medawar called "the Holy Grail." Starzl demonstrated early that kidney rejection in dogs treated with azathioprine could be reversed 88% of the time by injection with steroids (Marchioro et al., 1964). Similar rates were published for humans undergoing the same therapy. It was also shown that delayed hypersensitivity skin tests (against tuberculin, histoplasmin, coccidiodin) that were positive in the donor but not the recipient crossed over to the recipient about 77% of the time following a kidney transplant (Starzl, 1993). Microchimerism involving the survival of donor leukocytes in the body of the recipient following transplantation was proven using polymerase chain reaction (PCR) and persisted up to 29 years following transplantation (Starzl, 1993). Even more surprising was the observation that some liver graft recipients discontinued their immunosuppressive medication 1-6 years following transplantation and were normal 5-13 years later. These patients had achieved lasting immunologic tolerance, and all were shown to be lymphocyte chimeras. The movement of donor leukocytes out of the transplanted graft into immune compartments of the host had been demonstrated in animal models (Qian et al., 1994). It had also been shown that longstanding peripheral tolerance could be achieved in mice made chimeras by neonatal infusion of donor leukocytes (Silvers et al., 1975). These observations fostered the notion that for completely successful organ engraftment, four interrelated phenomena must occur in close temporal sequence: clonal deletion of the recipient immune (antigraft) response, clonal deletion of the donor's leukocyte response, maintenance of clonal exhaustion, and reduction in the immunogenicity of the transplanted organ over time (Starzl and Zinkernagel, 1998). However, it is not clear how these events are controlled. For some time it has been known that acute allograft rejection is mediated by CD4 + and CD8 + cells. The former are activated by binding foreign antigen in association with MHC class II molecules n i n this case, on the donated graft. For proper activation, costimulatory signals mediated via CD28 on T cells and B7 on antigen-presenting cells plus CD40 present on antigenpresenting cells binding CD40L on T cells must be engaged. Once activated, CD4 lymphocytes produce many cytokines that act in an autocrine fashion to stimulate more CD4 cells and a paracrine fashion to activate CD8 cells. Many of these cytokines, such as TNF, may be liberated by activated CD4 cells within the graft, causing rejection even without the cytotoxic effects of CD8 cells. These immune mechanisms were largely dis-

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covered through research on mice and rats (Sayegh and Turka, 1998). Investigators have demonstrated that blocking the costimulatory pathways for T cells in allografted animals (rodents and nonhuman primates) greatly prolongs graft survival and ablates the acute rejection phenomenon (Kirk et al., 1999). Although prevention of T-cell activation and cytokine release can explain acute graft survival, it does not really explain the long-term survival of grafts observed by Starzl in microchimeric animal and human transplant recipients. One explanation comes from the studies of Li and Wells, where peripheral allograft tolerance was established in mice by a combination of costimulatory blockade and the use of rapamycin. In this study, mice received an MHC-incompatible cardiac transplant plus monoclonal antibodies against CD40L (CD154) plus CTLAIg (which blocks CD28). In this case, heart grafts survived rejection, but skin grafts were rejected. When rapamycin is added to the regimen, permanent engraftment is seen for both heart and skin; however, if cyclosporin A (CsA) is substituted for rapamycin, neither graft survives rejection. The inclusion of CsA thus antagonized the tolerizing effects of both costimulatory blockade and rapamycin. Using cell labeling studies, these investigators found that costimulatory blockade alone inhibited proliferation of alloreactive T cells in vivo while allowing cell cycledependent T-cell apoptosis of proliferating T cells. Addition of rapamycin resulted in massive apoptosis of alloreactive T cells, but addition of CsA abolished T-cell proliferation and apoptosis. Subsequent studies demonstrated that the combination of blockade plus rapamycin did not induce tolerance in IL-2 or in bcl-XL transgenics. In both instances, these transgenic mice failed to be induced into T-cell apoptosis. Activation-induced cell death (by apoptosis) occurs when primed cells are repetitively activated by antigen and requires previous exposure to IL-2. Unlike CsA, rapamycin does not block IL-2 production, and it does not block antigen priming for apoptosis. The authors conclude that stable peripheral tolerance can be induced as long as alloreactive T cells are suppressed from initial cytokine production and eliminated by apoptosis (Li et al., 1999; Wells et al., 1999). How these observations fit into the microchimera model of Starzl is unknown, but one hypothesis is that the apoptotic alloreactive T cells trigger an immunoregulatory effect that serves to maintain the state of tolerance (Ferguson and Green, 1999). This is supported by the finding that apoptosis and immune tolerance are linked through the activation of immunoregulatory mechanisms mediated by inhibitory cytokines (ILl0, TGF-[3). In this model, tolerance is now stable because of a double hit--deletion and regulation. The initial blockade of acute rejection with immunosuppressive drugs coupled with the tremendous continuing antigenic stimulation caused by alloantigen-bearing donor cells in the lymphoid tissues of the host may lead to apoptosis of alloreactive cells and production of antigen-specific suppressor cells on a continuing basis. For their achievements in transplantation biology, Joseph


Murray and E. Donnall Thomas were awarded the Nobel Price in 1990. For their accomplishments in the field of pharmacology (including the development of drugs to treat high blood pressure and gastric ulcer, and immunosuppressant and antiviral drugs) Sir James Black, Gertrude Elion, and George Hitchings were awarded the Nobel Prize in 1988. e. Vaccinology. Intimately associated with advances in immunology were advances in vaccinology. Following in the footsteps of Jenner, Pasteur, and Bouquet, Calmette, and Guerin (BCG vaccine), Max Theiler was able to develop a mouse protection assay for yellow fever virus and attenuated viral strains in mice and chickens. Efficacy for these attenuated strains was demonstrated in monkeys and ultimately led to the first vaccine in the 1930s. Theiler was awarded a Nobel Prize in 1951 for his efforts (Strode, 1951). During the second half of the twentieth century, vaccines were developed and utilized to protect against diphtheria and pertussis (in the 1940s), poliomyelitis (in the 1950s), rubella or German measles (in the 1960s), pneumococcal, meningococcal diseases, measles, and mumps (in the 1970s), hepatitis A and B and Haemophilus influenzae (in the 1980s), and varicella or chicken pox and Lyme disease (in the 1990s). This history of vaccine development depended heavily on the use of animals, especially nonhuman primates (Hilleman, 1998). Despite this record and the hundreds of millions of human lives saved through vaccination, a safe, effective vaccine against HIV-1 and HIV-2 has been elusive. Vaccinologists are borrowing from all the concepts in modern immunology to devise recombinant vaccines that, via point mutation, immunize without leading to fully assembled virions, creating new vectors that will generate MHC class I-restricted cytotoxic T-lymphocyte responses, in addition to antibody responses and constructs that encode structural sequences for antibodyenhancing cytokines (Liu, 1998). Other approaches being used in vaccine development include polysaccharide protein conjugates for H. influenzae, Streptococcus, and Neisseria meningitidis; new adjuvants and delivery systems that would enhance mucosal immunity; edible-plant vaccines to inexpensively deliver vaccination without refrigeration to tropical countries; and therapeutic vaccines that make use of dendritic cells as vaccines for HIV (Liu, 1998). Once again, vertebrate animals are essential in evaluation of efficacy for each innovation (Seder and Gurunathan, 1999; Langermann, 1998; Guranathan et al., 1998; Hislop et al., 1998; Fehr et al., 1998; Marinaro et al., 1999). Two other experimental applications of vaccines have also arisen, based on new knowledge of the molecular basis of autoimmune disease and cancer. Based on prior work in mice, recombinant fusion DNA vaccines are now being evaluated in humans afflicted with lymphoma and myeloma (King et al., 1998; Falo and Storkus, 1998; Pardoll, 1998). In some instances, dendritic cells spiked with tumor-specific antigens are used as a vaccine against melanoma (Timmerman and Levy, 1998; Nestle et al., 1998). In other instances, monoclonal antibodies

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themselves are used to attack the tumor or enhance autologous T cells to attack the tumor (Houghton and Scheinberg, 2000; French et al., 1999; Dickman, 1998). New discoveries in the molecular pathogenesis of type 1 diabetes mellitus and multiple sclerosis, based on extensive analysis in animal models and humans, implicate clones of selfreactive T cells as mediators of these diseases (Bach et al., 1998; Sela, 1999; Wong et al., 1999b); as a result, the feasibility of eliciting T-cell responses against the self-reactive T-cell receptors is being evaluated (Weisman et al., 1996). Another approach to prevent autoimmune diseases in humans is based on two observations supported by animal studies; (1) genotyping family members at risk may identify candidates for disease before symptoms, and (2) certain autoantigens, when delivered by the oral route, induce tolerance (Lund et al., 1990; Miller et al., 1992; Bowman et al., 1994). Clinical trials using these approaches have people at risk of developing multiple sclerosis orally vaccinated with immunodominant peptides of myelin basic protein and those at risk of developing type 1 (juvenile) diabetes orally vaccinated against insulin or glutamic acid decarboxylase (Noseworthy, 1999). f Asthma. Much has been learned about allergic reactions since the discoveries by Richet and Bovet. The molecular pathogenesis of early and late responses, characterized by biphasic reactions mediated by IgE; release of various mediators, and the influx of different populations of cells, has been studied primarily in rats and mice (including knockouts) (Matsuoka et al., 2000). During the past 2 decades, new therapies, including deep bronchial inhalers to modulate the bronchoconstrictive response of inflammatory mediators, have aided in the reduction of signs and symptoms for those suffering from asthma. Clinical trials are underway in chronically asthmatic people, using a humanized mouse monoclonal anti-IgE antibody, and the first successes have been reported (Milgrom et al., 1999). Work that led to the development of this therapy involved studies in rats and cynomolgus monkeys (Fox et al., 1996). Of course, it would be impossible to recite, in any meaningful way, all the incredible accomplishments in biology and medicine that occurred during the twentieth century, using animal models. A former surgeon general of the United States has stated that every major achievement in medicine during this century has depended in some fashion on animal research. Over two thirds of all Nobel Prizes in Physiology and Medicine have been awarded to scientists who used vertebrate animals to accomplish their goals (Leader and Stark, 1987). In a survey of living Nobel laureates, 97% responded that animal experiments have been vital to the discovery and development of many advances in physiology and medicine, and 92% felt strongly that animal experiments are still crucial to the investigation and development of many medical treatments (Seriously Ill for Medical Research, 1998). The twentieth century saw an explosion of activity in every

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area of the biological sciences. Virtually all modern medical treatments and devices, including most that are now taken for granted, were developed through animal research. Advances in the science of cell biology, ecology, developmental biology, respiratory physiology, cardiovascular physiology, endocrinology, biochemistry, bacteriology, virology, parasitology, psychology, ethology, neurobiology, and nutrition and metabolism have enriched and supported the medical disciplines of cardiology, dermatology, surgery, orthopedics, pediatrics, anesthesiology, pharmacology, microbiology, psychiatry, neurology, dentistry, hematology, medical genetics, and women's health. Reviews documenting the progress in biomedical sciences are available (Singer and Underwood, 1962; Sourkes, 1966; Keen, 1914; Moore, 1995; Camac, 1959; Schmidt, 1959; Reiser, 1981; Bliss, 1982; McGehee, 1981; Comroe, 1983; Fox and Fox, 1988; Weisse, 1991; Robinson, 1976).

C.

Animals as Recipients of Animal Research

Because humans and nonhuman animals share common physiologic responses, many of the advances sought for humans through the use of animal models also benefit animals themselves. Humans and other vertebrates are susceptible to a large number of infectious diseases, and many disease agents infect both animals and humans. These are called zoonotic agents and are frequently passed between humans and other animals. Certain of these zoonotic diseases are prevented by vaccination in humans and animals, including rabies, anthrax, tetanus, and Lyme disease. Monkeys continue to be necessary in the evaluation of polio vaccine, although they may soon be replaced with transgenic mice bearing the human poliovirus receptor; in addition, human polio vaccine has been used to protect wild chimpanzees in East Africa (Quimby, 1994). Since the adoption of the Nuremberg Code in the 1950s, humans have been protected by first testing the safety of new drugs and medical devices in nonhuman animals (Spicker et al., 1988). As a result, an enormous amount of data is known on the safety and pharmacokinetics of these drugs in various animal species. It is therefore not surprising that drugs designed for human use, such as antibiotics, tranquilizers, steroids, sulfonamides, anesthetics, analgesics, chemotherapeutics, anticoagulants, antiparasitics, antiepileptics, and antihistamines, are all commonly used in veterinary practice (Quimby, 1998). Many surgical techniques intended for humans were first perfected in animals and subsequently used to treat animal disorders. Included among these techniques are repair of spinal cord, hip replacement, fracture repair, repair of congenital heart defects, treatment for burns, and organ transplantation. Nonhuman animals, particulary dogs, develop a variety of autoimmune and hematologic conditions that are identical to the human counterparts, and as a result, these animals benefit from blood transfusions, immunosuppressant therapy, purified blood

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components, and hormones, much like humans with the same disorders (NRC, 1994). These same animals benefit from both diagnostic procedures and, in some instances, surgical intervention originally designed for humans. In fact, dogs with disorders such as severe combined immunodeficiency, hemophilia A, and hemophilia B are currently the subjects of experimental gene therapy to evaluate its safety and efficacy before widespread deployment in humans. Pet animals are the beneficiaries of advances in biomedical imaging, and veterinary practitioners utilize X-ray machines, computed tomography, ultrasonography, and fiber-optic endoscopy for animal disease diagnosis. Knowledge of artificial insemination, semen evaluation and storage, egg incubation, and behavioral adaptation were employed in the successful captive breeding and reintroduction of the eastern peregrine falcon, which has now been removed from the endangered species list. Similarly, biomedical techniques such as in vitro fertilization are being used in zoological gardens to help restore populations of animals threatened with extinction. Occasionally, a compound designed for use in animals is discovered to aid in the treatment of human disease. Levamisole, developed as a cattle wormer, is now used together with the chemotherapeutic agent 5-fluorouracil for the treatment of colon cancer. Ivermectin, developed as a preventive for heartworm disease in dogs, is now being used to treat millions of people in the tropics infected with onchocerciasis (river blindness). This sharing of diagnostic methods, preventives, drugs, surgical techniques, and medical devices between humans and other animals adds credence to the philosophy of Sir William Osier, a physician and Dean of Johns Hopkins University School of Medicine and an outspoken advocate and practitioner of comparative medicine, that there is only "one medicine" (see Chapter 31 for further details) and calls into question the rationale of those who seek to eliminate the use of animals in biomedical research. This seems especially true if one considers that each year only half as many animals, of all species, undergo an experimental medical procedure as the number of pets in the United States that endure surgery for cosmetic reasons (Conn and Parker, 1998).

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Chapter 31 Research in Laboratory Animal and Comparative Medicine Henry J. Baker and J. Russell Lindsey

I.

II.

III.

IV.

V.

VI.


Introduction .................................................

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Historical Perspective

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1228

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Comparative Medicine .....................................

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R e s e a r c h as a F o u n d a t i o n of the Specialty

The Research Process

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The Research Cycle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

B.

Research Design

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A n i m a l Subjects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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R e p o r t i n g R e s e a r c h Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Research Training ............................................

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F r o m the D.V.M. to L a b o r a t o r y A n i m a l Veterinarian

B.

C h a r a c t e r i s t i c s of L a b o r a t o r y A n i m a l M e d i c i n e T r a i n i n g

.............

C.

G r a d u a t e E d u c a t i o n and L a b o r a t o r y A n i m a l M e d i c i n e . . . . . . . . . . . .

1233

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S u p p o r t for T r a i n i n g

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Research Resources ...........................................

1234

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Animal Resources

1234

B. C.

Research Laboratories ..................................... Diagnostic Laboratories . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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D.

L i b r a r y and Electronic Literature S e a r c h i n g . . . . . . . . . . . . . . . . . . . .

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........................................

S u p p o r t for R e s e a r c h . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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A.

Grantsmanship ...........................................

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B.

Public Sources of S u p p o r t for R e s e a r c h

.......................

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C. D.

Private Sources of S u p p o r t for R e s e a r c h . . . . . . . . . . . . . . . . . . . . . . . Intellectual P r o p e r t y . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Summary ...................................................

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References ..................................................

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HENRY J. BAKER AND J. RUSSELL LINDSEY

I.

A.

INTRODUCTION

Historical Perspective

Laboratory animal medicine is the only specialty of the American Veterinary Medical Association in which diplomates are expected to practice their specialty almost entirely in biomedical research institutions and who are constantly involved in the research process. Consequently, it is reasonable to expect that laboratory animal specialists, particularly diplomates of the American College of Laboratory Animal Medicine (ACLAM), should be trained thoroughly in research and capable of functioning as research scientists. The founders of ACLAM recognized the importance of research skills for laboratory animal specialists and incorporated this key element into all aspects of standards that define training and experience necessary for certification in the specialty. Today, ACLAM's requirements for training, application for membership, certification examination, educational programs, and recertification emphasize scholarship and achievement in research relevant to the specialty (see ACLAM website ). In addition to an appreciation for research, which is needed to function successfully in an intensive research environment, the very foundation of the specialty and its future viability depends on the acquisition of new knowledge that will improve the use of animals in science. For example, 30 years ago it was common for suppliers of laboratory mice to be unable to fill orders during the summer because rotavirus infection, known as epizootic diarrhea of infant mice (EDIM), killed entire populations of neonatal laboratory mice. Solution of this vexing problem required the pioneering work of laboratory animal scientists, who discovered elegantly simple methods that prevented these zoonotics that were so disruptive to biomedical research (Kraft et al., 1964). Today, the exclusion of such pathogens is expected by laboratory animal specialists and investigators alike, and zoonotics of this type would not be tolerated by the scientific community. Abundant examples can be cited of research and development contributions of a highly productive cadre of research scientists within and outside of ACLAM that have been responsible for the high level of sophistication in animal experimentation enjoyed today. Although laboratory animal scientists can take much of the credit for research and development responsible for the current state of high-quality laboratory animal resources, an equally sustained effort will be required in the future if progress is to be made in meeting the laboratory animal challenges that accompany each advance in biomedical research. It is the responsibility of laboratory animal specialists to assure that the commitment, resources, and talent are available and applied to this important mission of the specialty, if laboratory animal medicine and science are to endure and flourish.

B.

Comparative Medicine

The conceptual basis for comparative medicine can be found in the visionary statements of some of the greatest innovators in biology and medicine at the turn of the twentieth century. Rudolf Virchow (1821-1902) captured the essence of comparative medicine in the much-quoted statement: "Between animal and human medicine there is no dividing line, nor should there be. The objective is different, but the experience obtained constitutes the basis of all medicine." Sir William Osier (18491919), Chairman of Medicine and Dean of the Johns Hopkins School of Medicine, was not only a notable proponent, but also a practitioner of the comparative approach, which he called "one medicine." In the 1870s and 1880s, he published numerous articles on comparative pathology of nematodes in dogs, hog cholera, echinococcosis, bovine tuberculosis, and parasites of pork (Harvey et al., 1989). Even though Claude Bernard (1813-1878) is reviled by modern opponents of animal research, he is considered to be the founder of experimental physiology and expressed his strong conviction about comparative medicine in 1865: "I not only conclude that experiments made on animals from the physiological, pathological, and therapeutic points of view have results that are applicable to theoretical medicine, but I think that without such comparative study of animals, practical medicine can never acquire scientific character." The concept of comparative anatomy is easily understood as the study of similar and dissimilar morphology of two or more species, but the dominion of comparative medicine may not be as clear or intuitive. In essence, comparative medicine includes characterization of similarities and differences in disease processes affecting humans and other animals. In the same way that the scope of comparative medicine is not generally understood, practitioners of the specialty are also not well defined. Obviously, successful application of the comparative approach to science requires knowledge of disease processes in both animals and humans. For veterinarians, this means at least an introduction to human diseases and in-depth study of the literature pertaining to specific diseases. Conversely, those trained in medicine or dentistry must gain a fundamental understanding of animal diseases and animal modeling. To some, comparative medicine represents the academic and research specialty companion to laboratory animal medicine. Although a division of emphasis can be made in this way, it serves no useful purpose and tends to segregate rather than integrate these companion, mutually complementary activities. The need for research is as critical for improving the suitability and health of animals used in research as it is for discovering new animal models of human diseases. Therefore, in this chapter, use of these terms will be overlapping and not meant to distinguish between research or training activities in animal care or modeling. Although laboratory animal scientists have embraced the concept of comparative medicine and advanced it more than most other medical

31. RESEARCH IN LABORATORYANIMALAND COMPARATIVEMEDICINE specialties, it is certainly not an exclusive domain, and continued recognition and acceptance by the biomedical community for its place as a veterinary specialty will demand sustained, noteworthy contributions to the field. Unfortunately, the recent increased emphasis on regulation of animal research has so seriously distracted laboratory animal specialists from participation in productive research that a dangerous trend is in progress, which, if allowed to progress unchecked, could disenfranchise laboratory animal veterinarians from leadership in comparative medicine. In spite of the lack of clarity about the boundaries and participants of comparative medicine, the value of this approach to medical science cannot be disputed. Clarence Cook Little's effort early in the twentieth century to genetically "purify" the mouse as a model for cancer research exemplifies the power of the comparative approach (Little, 1913; also see Mobraaten and Sharp, 1999). The eventual success in achieving genetic homozygosity in laboratory mice and the largely unanticipated occurrence of a multitude of spontaneous diseases, including cancer, in these strains may have contributed more to advancing the health of humans and animals than any other achievement in comparative medicine in the twentieth century. Inbred mice have been key to understanding cancer, susceptibility to infections, inherited diseases of all types, and the basic nature of the genome in health and disease. There are numerous examples of contributions made by laboratory animal scientists that have significantly altered biomedical research using animals (see Chapter 30 and McPherson and Mattingly, 1999). With the advent of powerful research technologies, particularly those of molecular biology, opportunities abound to continue contributions to comparative medicine. Laboratory animal specialists have unlimited opportunities, and in fact an obligation, to contribute to this incredibly exciting and important part of the specialty.

C.

Research as a Foundation of the Specialty

Subspecialization is not recognized or endorsed by ACLAM. However, there appears to be an informal trend toward division of laboratory animal scientists who emphasize clinical and service activities and those who concentrate their effort on research. This division of interests and activities has been accentuated by an increasing emphasis on regulatory and compliance issues raised by the U.S. Department of Agriculture's enforcement of the Animal Welfare Act, the National Institutes of Health (NIH) requirements to comply with the U.S. Public Health Service policies on animal welfare, accreditation of animal care programs by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC), and institutional policies and procedures such as

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the Institutional Animal Care and Use Committee review and approval of all research projects using animals. One justification that has been advanced to support this inclination toward division of effort is that success in today's highly competitive research arena requires more than part-time participation, which is usually required by specialists who have a substantial commitment to clinical and service activities. The hazard of this arrangement is development of a two-tiered system that encourages separation of interests, discourages interaction, and can be a basis for adversarial attitudes. In the long run, the specialty and its members would suffer from such divisions for a variety of reasons. First, the further the distance between colony management and disease problems, which clinicians know best, and research to solve those problems, the greater the likelihood that research will become less relevant to advancing laboratory animal science. Second, the proportion of specialists who participate in research is directly related to the emphasis placed on scientific advancement of the specialty. If a high proportion of specialists isolate themselves from research, the specialty is in danger of losing a pillar of its founding precepts. Eventually, complete loss of research productivity by any specialty presages conversion of a profession to a trade. When the profession is practiced in academic and research institutions, this contrast is accentuated, and the loss of acceptance as a peer group could be rapid. Regrettably, movement in this direction is currently in progress, and there does not appear to be a concerted effort to slow or reverse the trend. Although the ACLAM and other organizations representing laboratory animal medicine recognize the importance of clinical medicine, the specialty must aggressively preserve, encourage, and enhance participation of specialists in productive research, if the scientific character of the specialty is to be maintained. Institutions that are unable to employ more than one specialist severely restrict opportunities for participation in research. In contrast, at institutions where several specialists share responsibilities, opportunities for research are much improved. Therefore, larger institutions must share greater responsibility for assuring research productivity of the specialty.

II.

THE RESEARCH PROCESS

A.

The Research Cycle

The research process can be considered to be a cycle of activities starting with a statement of a problem or question and ending with an answer to that question. The steps intervening between the beginning and ending activities include (1) searching the scientific literature for existing information that addresses

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B. R e s e a r c h Design the problem, (2) critically assessing relevant literature to distinguish between valid conclusions based on sound data and errors Skillful research design is fundamental to all successful exin experimental execution or interpretation, (3) formulating a hypothesis or statement that addresses the problem and that will periments. A comprehensive discussion of the elements of good be tested, (4) designing one or more experiments to test the hy- experimental design is beyond the scope of this chapter, and the pothesis, (5) conducting the experiments according to a well- reader is referred to excellent publications on this subject defined experimental protocol, (6) evaluating results, (7) reach- (Navia, 1977). Good design starts with a clearly stated research ing conclusions by interpretation of the results, and (8) sharing problem amenable to testing by a series of manipulations that results and conclusions through presentations and publications. will result in clear differences between experimental and conNote that of these eight major steps, only one (step 5) involves trol groups. From the outset, methods to control confounding physically working in the laboratory. This disproportionate ra- variables independent of the subject parameter must be emtio of "thinking" and "doing" is contrary to the common per- ployed if small differences between experimental and control ception of the research process, but the investment in thinking groups are to be distinguished. Of all the subjects in experiand planning cannot be overestimated in importance (Navia, mental design of research using animal subjects, control of biological variables should be the dominion and expertise of the 1977). Discovery and characterization of naturally occurring animal laboratory animal specialist. This topic is worthy of extensive models of human diseases and other types of clinical investiga- discussion, and the reader is referred to Chapter 29 of this voltion may follow a different course of research activities than ume, "Factors That May Influence Animal Research," for com"hypothesis-driven" research, but although the emphasis may be prehensive descriptions of environmental factors that can different, the basic precepts remain the same. The fundamental influence research results and can be controlled by skillful pardifference is that testing a hypothesis by experimental manipu- ticipation by laboratory animal veterinarians. Selection of the lation of subjects allows for greater control of all variables other most appropriate animal subject or model is also a key element than the single parameter being studied. Clinical investigations in research design and the natural domain of the laboratory anmay not permit such elegant control of confounding variables. imal specialist. To a large extent, this requires an encyclopedic Nevertheless, clinical research can yield results as informative knowledge of the biology of several species, genetics of experand valuable as experimental designs. In the past, discovery of imental animals, detection of occult infections, and naturally disease models happened by serendipitous opportunities pre- occurring or induced animal models of human diseases. Chapsented to individuals with a mind-set prepared to recognize and ter 30 of this volume addresses some of the issues of animal pursue the opportunity. In contrast, genetic engineering using modeling. Other publications include "Spontaneous Animal transgenic technology has made it possible to selectively insert Models of Human Disease" in the ACLAM/Academic Press seforeign genes or disable endogenous genes ("knockout") in ries (Andrews et al., 1979; Desnick et al., 1982) and the Armed mice. In fact, this powerful new technology makes it possible to Forces Institute Registry of Comparative Pathology "Animal design and test hypotheses using cleverly modified transgenic Models of Human Disease" (Capen et al., 1985). These refermice. In addition to new professional opportunities created by ences provide a starting point that should be supplemented by transgenic technology, there is an increasing need to character- exhaustive search of the literature for details pertaining to a ize transgenic mice using histopathology, biochemistry, and specific research objective. molecular biology. These professional opportunities should attract specialists in comparative medicine and pathology. AlC. A n i m a l Subjects though transgenic animal models provide enormously important research opportunities, it is important to recognize that Unless the research topic involves the development of in vitro there are many examples of mice modified in this way that remethods to replace animal use in research, then animal subjects spond very differently than human and animal patients with corbecome a central theme for research pursued by laboratory aniresponding inherited diseases. Transgenic mice may show no clinical signs of disease or have lethal consequence of the gene mal medicine practitioners and trainees. The process of condeletion (Phaneuf et al., 1996). To be certain, these differences ducting animal research involves basic skills and issues that all in the response of mice can be instructive by identifying alter- research scientists deal with continuously but that are critically nate metabolic pathways that enable mice to circumvent patho- important for laboratory animal veterinarians to master. The logical consequence of a mutation. However, the experiments of special skills and insights required for successful research innature (naturally occurring mutations and other diseases in do- volving animals include (1) understanding the biology of spemestic animals) remain a valuable source for modeling the hu- cies in the context of their use in specific types of research, man condition and remain worthy of pursuit when they are en- (2) selecting appropriate experimental animal subjects, (3) developing skills required for manipulating animals in expericountered (see Chapters 28 and 30).

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31. RESEARCH IN LABORATORY ANIMAL AND COMPARATIVE MEDICINE

mental situations, (4) assuring that the animals to be used are healthy and not subjected to conditions that might confound the research objectives, (5) practicing humane treatment of animals, (6) and satisfying regulatory requirements. The literature that addresses many of these issues is widely dispersed but must be searched for specifics about selecting and using animals in biomedical research. The ACLAM text series published by Academic Press is an excellent starting point to find such information (see ACLAM website for a listing of titles). In addition to locating and understanding pertinent literature, firsthand experience gained through conducting research is an essential element of a well-rounded training program. Laboratory animal scientists are on the front lines of institutional commitment to compassionate and humane use of animals in research. Good science and good ethics demand that investigators select the least-invasive and most humane methods of animal research. Eaboratory animal veterinarians must be leaders in this vital research issue through collaboration or consultation. This can be accomplished only by careful consideration of research objectives and innovation to achieve these objectives with the least risk of pain or discomfort to animal subjects. Proceedings of an ACLAM symposium on the topic of "Advancing Science and Animal Welfare in the 21st Century" addresses advances made in the development of more humane methods of animal experimentation (Kraus, 2001).

D.

Reporting Research Results

Arguably, sharing research results through presentations and publications is the most important step in the research process. If results are not shared with others in the field, then the effort expended is completely wasted, other than personal gratification gained by the investigator performing the research! Value of the reporting process includes (1) formally organizing data and formulating conclusions; (2) providing an opportunity for critical review of results by others, such as meeting attendees, manuscript reviewers, editors, and readers; (3) assisting other investigators in developing better research designs or preventing unnecessary duplication of research; and (4) enabling granting agencies to recognize competence in a field for which research support may be sought. Careful consideration should be given when selecting meetings where results are reported and journals where manuscripts may be published to maximize the distribution of information to the segment of the scientific community who are most likely to be interested in and influenced by a study. In the laboratory animal and comparative medicine community, scientific meetings sponsored by ACLAM, American Association for Laboratory Animal Science (AALAS), and American Veterinary Medical Association (AVMA) are particularly relevant. Journals serving the specialty include Comparative Medicine (formerly Laboratory Animal Science), the

American Journal of Veterinary Research, the American Journal of Pathology, the American Journal of Medical Genetics (Animal Models series), Comparative Immunology, Microbiology and Infectious Diseases, Veterinary Immunology and Immunopathology, Journal of Comparative Pathology, Research in Veterinary Science, etc. Information that may have high impact on specific disease specialities should be submitted for presentation at meetings and publication in journals serving that particular discipline.

III.

A.

RESEARCH TRAINING

From the D.V.M. to Laboratory Animal Veterinarian

The traditional veterinary professional curriculum provides an outstanding foundation for development of laboratory animal specialists, but does not include the specialized knowledge and skills required for successful participation in clinical laboratory animal medicine or research. Veterinary students can augment their introductory course in laboratory animal medicine by serving externships, preceptorships, and summer fellowships at institutions that have departments of comparative medicine or laboratory animal medicine. Experience gained from these short-term training opportunities helps to solidify emerging interests in the specialty and provide insights into the culture of the specialty that cannot be gained from the lecture room. Such short-term experiences also provide faculty of laboratory animal medicine training institutions with the opportunity to gain firsthand knowledge about students who may proceed to seek extensive postdoctoral training in the specialty. Other types of research opportunities can be found at academic departments of medical and veterinary schools, research centers, national primate centers, and industrial settings. Detailed information about institutions offering short-term training experiences that are supported by NIH can be found at under listings for "ShortTerm Training Awards for Students of Health Professional Schools," and under "Institutional Training Awards," "Regional Primate Research Centers," and "Laboratory Animal Science Centers." Individual colleges of veterinary medicine and major pharmaceutical firms should be contacted for information about summer research fellowships, preceptorships, and similar preD.V.M. training opportunities that they may offer.

BO Characteristics of Laboratory

Animal Medicine Training Graduate veterinarians aspiring to become laboratory animal medicine specialists should seek a well-designed postdoctoral

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training program in an institution that provides a curriculum that meets the ACLAM standards. Even though on-job experience is an acceptable alternative to formal training to qualify for taking the ACLAM specialty board examination, it is less efficient and is usually not as comprehensive as formal training programs. A list of institutions offering formal training in laboratory animal medicine that are able to meet standards recognized by ACLAM can be found in the ACLAM website address cited. These should be viewed as minimal standards, since most training programs offer opportunities in addition to criteria prescribed for preparation to be certified by ACLAM. Potential applicants for training should inquire about the characteristics of each program being considered. Points of interest should include (1) experience of the institution and faculty in training laboratory animal specialists; (2) emphasis of the program on research, clinical, and administrative skills; (3) clinical expertise of the faculty; (4) opportunity to earn a graduate degree; (5) number of trainees currently in the program; (6) names and current positions of former trainees; (7) availability and amount of training stipends; (8) division of effort between clinical, service, research, and didactic activities; (9) institutional resources, such as diagnostic laboratories, as well as numbers and types of species being used in research; and (10) research strengths of training faculty, as Well as those of the entire institution. Didactic courses offered by a candidate-training institution should be compared with the core of knowledge identified by ACLAM as required for certification. Resources at a training institution that are critical for a good training experience include laboratories equipped with modern research instrumentation, diverse species used as experimental subjects, diagnostic laboratories staffed by qualified experts in laboratory animal clinical and anatomical pathology, a working library with substantial holdings pertaining to animal research, experimental surgery support laboratories, and adequate veterinary-assistant technical support. After receiving formal information about training programs of interest, applicants should schedule a telephone conversation with the appropriate contact person listed on the ACLAM website and discuss items of special interest or those needing clarification. A personal visit to the department or division offering training, which includes discussions with faculty and trainees, is highly advisable. A formal application process and interview are normally required by most training programs. Recommendations from individuals who know an applicant well and who have experience in the specialty can be very influential in the competition for training positions. Because of the unique role that laboratory animal specialists play in biomedical research, all training programs must include instruction and experience in both clinical and research topics, regardless of the intention of the trainee to emphasize one activity or the other after training. In fact, the credentials requirements of ACLAM demand evidence of a broad exposure to both activities. It is not necessary that both training activities occur

HENRY J. BAKERAND J. RUSSELL LINDSEY concurrently. Actually, some programs prefer to start postdoctoral training by emphasizing clinical experience for the first year. Since the trainee contributes substantially to the service activities of the institutional animal resources program, institutional support for the trainee's stipend during this time is well justified. All postdoctoral fellows supported by the U.S. Public Health Service (PHS) are required to devote at least 75-80% of effort to research training for the term of this award. Therefore, clinical training must be emphasized before or after, but not during PHS-sponsored training. The number and diversity of animals used in research can be key elements in assuring a broad training experience. In most institutions, rodents constitute the largest populations of research animals, but exposure to other species such as primates, lagomorphs, birds, amphibia, etc., is equally important. Even though graduate veterinarians are well versed in the health care of dogs and cats, the special considerations in housing and care of these species can be unique and unfamiliar. For example, the chapter by Griffin and Baker on use of cats as experimental animals (Chapter 12) emphasizes the unique aspects of research and breeding-colony management, many of which are not taught in the veterinary curriculum. The importance of interacting with other trainees with common aspirations, difficulties, and solutions to everyday problems cannot be overemphasized. Therefore, programs that have a cadre of veterinarians in training will provide a rich training environment. During training, the more senior trainees become increasingly influential and helpful to those trainees with less experience. Informal and formal meetings of trainees from a single institution or neighboring institutions can be a key characteristic of an excellent environment for training in laboratory animal medicine. Group interaction becomes especially important in preparation for specialty board examinations. Mentoring by an experienced laboratory animal veterinarian during the training period is an exceedingly important part of the training process. The mentor is responsible for providing a broad overview of the specialty, giving personal insights about current events and issues, protecting the trainee from political or intrapersonal incidents, assuring that there is balance in time commitments between varying duties competing for attention, monitoring the trainee's progression in acquiring skills and knowledge, and being an advocate for the trainee in the transition from training to first full-time professional appointment. The laboratory animal specialist may or may not be the trainee's research supervisor, but if possible, the research activity should be integrated closely with the other activities of the training program. If the research experience occurs in a laboratory distant from the laboratory animal medicine faculty, a clearly defined schedule and plan must be in place to assure that the requisite integration is achieved. It is not satisfactory for the research experience to be far afield of laboratory animal or comparative medicine in terms of goals pursued, insights gained, and skills acquired.

31. RESEARCH IN LABORATORYANIMALAND COMPARATIVEMEDICINE C.

Graduate Education and Laboratory Animal Medicine

Earning a graduate degree should be an option, but not necessarily a requirement of specialty training. Graduate education adds many didactic course requirements, examinations for admission to candidacy, and preparation of a dissertation, which are very time-consuming, rigorous, and not necessarily relevant to the goal of becoming a laboratory animal specialist. If an institution offers a graduate degree, and if the trainee is eager to pursue graduate education, then the additional demands of the graduate program must be discussed with the training faculty and mentor to be certain that all of the varied and sometime conflicting demands of the total training program are accommodated and resolved from the outset. The trainee must understand that an additional 3 to 5 years of time commitment are usually required to achieve the combination of specialty and Ph.D.-level graduate training, and 1 to 3 additional years for M.S.-level training. Benefits of a graduate degree include (1) organized, compulsory, in-depth training in research; (2) recognition for having accomplished a rigorous research training experience; and (3) an earned academic degree that is universally accepted as evidence for expertise in research and that some academic institutions regard as necessary for advancement in rank. D. I.

Support for Training

Pre- and Postdoctoral Training

As discussed earlier, salary or stipend support for trainees must comply with specific guidelines concerning the objectives of particular training grants or awards. For example, National Research Service Awards (NRSAs) funded by the National Institutes of Health (NIH) are intended to support only research training, but allow up to 20-25% effort in other academic activities. Therefore, if a substantial portion of effort is directed to development of clinical skills or performance of clinical service, then salary support must not be derived from NIH training grants (institutional or individual training grants). Because institutions benefit directly from trainee participation in clinical care of research animals, it is appropriate for salary support to come from institutional sources. In fact, a common practice is to appoint trainees to full-time clinical duties for the first year of experience and to convert after that time to a predominantly research-based training program. This allows the trainee to gain important skills and experience in clinical care, become familiar with the training department and institution as a whole, and learn about research laboratories that could be considered for research experience. A system that allows trainees to focus their attention and effort on either clinical or research activities can be helpful in this early stage of professional development. The

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NIH offers programs of support for research training, which encompass all stages of research career development. A complete listing of these programs and details, such as training program goals, eligibility, application procedures, etc., can be obtained from the website . The Comparative Medicine Division of the National Center for Research Resources (NCRR) of the NIH provides support for training veterinarians who wish to pursue post-D.V.M, training in research pertaining to laboratory animal science and comparative medicine. Specific programs that are commonly used for training in this specialty are National Research Service Award Short-Term Institutional Research Training Grants (T35), National Research Service Award to Individual Postdoctoral Fellowships (F32), National Research Service Award Institutional Research Training Grants (T32), Mentored Clinical Scientist Development Award (K08), and the Mentored Research Scientist Development Awards (K01). As described in the introductory paragraph of this section, a brief (1-3 months) exposure to the specialty can be extremely useful for students who are interested in gaining firsthand experience and insights. These awards are made to the training institution, and students interested in participating in this type of experience should inquire directly to institutions offering this training (see listing at ). Postdoctoral (postD.V.M.) training can be supported through institutional training grants, or individuals may apply directly for support. The primary distinction between these awards is that appointment can be made by institutions immediately, while the individual awards require at least a few years of research experience and development of a research training plan by the applicant and mentor prior to application. A listing of the institutions that are currently funded to provide this training, instructions for preparation of an application for individual training grants, and eligibility requirements for these awards can be found online at the NIH Comparative Medicine website . 2.

Advanced Research Career Development Awards

In the context of scientists who earn the Ph.D., a "postdoctoral" experience is intended to add to their research training, which was the primary goal of their graduate education. This postdoctoral experience is designed to allow progression from a mentored student to an independent scientist. The D.V.M. who enters a postdoctoral training program usually has had little or no research experience on which to build. Consequently, research training starts at the postdoctoral stage rather than refining research skills previously acquired. In addition, the postdoctoral experience for laboratory animal specialists divides attention to skill building between both clinical and research areas, further diluting time and attention available to become proficient in research. Nonetheless, the D.V.M. trainees

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bring to the postdoctoral stage of their career extensive education and experience in medical sciences. Therefore, the acquisition of strength in research is an achievable goal but may require more time than the Ph.D. "postdoc." The extensive protected time necessary for laboratory animal specialists to gain this critically important in-depth research experience requires longterm (3-5 years)salary and research support. Fortunately, such funding mechanisms exist to assist in research training beyond that supported by T32 or F32 awards. The Comparative Medicine Division of the NCRR, NIH offers Special Emphasis Research Career Awards (SERCA) in pathology and comparative medicine, also known as the Mentored Research Scientist Development Award (K01) and Mentored Clinical Scientist Development Award (K08) (see and ). Candidates for these awards must be graduate veterinarians who have completed fundamental training in the specialty, particularly clinical training. Institutions nominate candidates who demonstrate exceptional potential for achievement in research and provide the required rich training environment that will assure the success of the research trainee. These awards are given with the expectation that the first 3 years will be focused on mentored research training and the final 2 years will be devoted increasingly to independent research projects. In addition to salary support, these awards provide research funds. Competition for these awards is keen and requires a candidate with exceptional potential in research, an institutional environment that can support and facilitate this type of training, and a project proposal that is meritorious. Although these awards are relatively recent and few in number, their impact on increasing the numbers and qualifications of research scientists in laboratory animal science is already apparent. More extensive use of this excellent training mechanism should be a high priority for the specialty.

IV.

RESEARCH RESOURCES

A.

Animal Resources

Meritorious research cannot be performed without support from a variety of resources. Of these, animal resources are central to productive research in laboratory animal and comparative medicine. Characteristics of desirable animal resources include (1) a sound animal resource-management program that addresses the health and welfare of animals and the needs of investigators; (2) facilities that are adequate in size, design, and maintenance to accommodate the required species and number of animals needed by the institution's research faculty; (3) an animal science program that provides high-quality domiciliary

care of animals; (4) ability to accommodate special needs, such as barrier exclusion of pathogens and isolation of animals used in hazardous experiments at biosafety level 2 and higher; (5) surgery and support laboratories; (6) gnotobiotic laboratory; (7) transgenic laboratory; and (8) breeding colonies for production of special strains or stocks, including perpetuation of inherited diseases. One measure of the overall quality of animal resources is accreditation by the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). This does not necessarily mean that resources that have not sought or achieved accreditation are deficient, and accreditation may not address the specific needs of investigators. Therefore, judgment must be used in assessing the adequacy of animal resources for support of research. Since laboratory animal specialists can wear two hats, as investigator and animal resource manager, the animal resources should be especially attuned and responsive to the needs of laboratory animal scientists as well as investigators in other disciplines.

B.

Research Laboratories

Research laboratories are second only to animal resources in importance and must be available and equipped to serve the needs of laboratory animal scientists. These laboratories may overlap in function and capability with the diagnostic resource discussed below. Basic laboratory functions and equipment should include (1) analytical biochemistry, (2) molecular biology, (3) mammalian cell culture, (4) microbial culture, (5) physiological function tests, (6) radioisotope handling and detection, (7) computer resources with software for date acquisition and statistical analysis, (8) gross and microscopic pathology, and (9) photographic capability. Special procedures that should be available through institutional shared resources include (1) flow cytometry; (2) electron microscopy; (3) quantitative densitometry; (4) DNA synthesis, sequencing, and detection; (5) gross and microscopic digital imaging and quantitation; (6) protein synthesis and sequencing; (7) body-imaging modalities, such as X-ray, bone densitometry, and ultrasonography; (8) statistical support; (9) confocal or other types of quantitative microscopy; (10) biohazard isolation laboratory; and (11) computer graphics and digital projection. In some cases, specialized research resources may be accessed through interinstitutional agreements. Examples of such specialized resources include regional primate research centers, specialized rodent resources, invertebrate resources, and special animal colonies. The scientific literature provides guidance to investigators who specialize in maintaining and using unique colonies of animals or who perform procedures such as cryopreservation of embryos or sperm (also see the Institute for Laboratory Animal Research, Animal Models and Genetic Stocks Information Program ).

31. RESEARCHIN LABORATORYANIMALAND COMPARATIVEMEDICINE

C.

Diagnostic Laboratories

Laboratories that provide clinical and pathological support necessary to maintain healthy laboratory animal colonies are a fundamental resource found in most large-animal care and use programs. Such laboratories also provide valuable research support because in the process of evaluating health problems, excellent research opportunities are likely to be revealed. In addition, these laboratories provide many of the analytical procedures that are commonly used in laboratory animal and comparative research projects. Professionals who operate these laboratories are often laboratory animal and/or comparative pathology specialists who are highly qualified to collaborate in research with others and serve as principal investigators of independent projects. In fact, in most institutions with these diagnostic resources, there is close integration with academic departments of laboratory animal medicine or comparative medicine, as well as with the animal services programs. These laboratories can also play a pivotal role in training veterinary students and postdoctoral students in laboratory animal diseases.

D.

Library and Electronic Literature Searching

Recent advances in computer-assisted searching of the scientific literature have virtually solved the difficult problem of finding and obtaining publications pertaining to highly circumscribed or specialized research topics. This is especially important for the laboratory animal specialist because topics of interest frequently are published in a variety of biomedical science journals and monographs that do not focus on laboratory animal science or comparative medicine. As discussed earlier, understanding work reported by others in the scientific literature is a critically important prerequisite to initiating a new investigation. Therefore, electronic computer-searching capability is an absolutely essential resource for research. Bibliographicsearching software is designed to allow investigators to perform successful searches themselves; however, assistance of a skilled librarian can be extremely helpful in both searching for citations and acquiring the corresponding articles. Vendors such as Ovid, EBSCO, and Cambridge Scientific Abstracts provide libraries as well as individuals with powerful online literature search programs or platforms. Each vendor includes various subject databases accessible through its platforms. For example, some of the databases pertinent to biomedical and veterinary sciences provided by Ovid are Journals@Ovid Full Text, Medline, AGRICOLA, Biological Abstracts, International Pharmaceutical Abstracts, and CAB. Vendors also provide access to full-text versions of articles of a limited number of journals. CARL is a vendor providing literature search capabilities using one database that crosses all subject areas. In addition, it provides a rapid and economical

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document delivery service. Literature search limited to specific journals or publishers can be accessed through online services such as Wiley Interscience Journal Finder , Springer Link Information Services , IDEAL (International Digital Electronic Library) , and Science Direct . These services usually allow free searching of their journals for citations and their corresponding abstracts. To obtain a full version of the article, users must subscribe to the service or pay a fee per article. Institutional libraries usually subscribe to one or more of these services and provide free access to their pa-. trons. Some services are now beginning to allow free access to full-text articles of older issues of their journal collection. Other sources of free full-text articles are Highwire Press and BioMedNet , although this list undoubtedly will increase in time. Citations accessed electronically can be saved and stored electronically in personal bibliographic databases, such as Reference Manager, Endnote, and Procite. Details about these products, marketed by the Institute for Scientific Information (ISI), can be found at . These personal bibliographic databases allow for organizing citations electronically to a journal's specifications when preparing manuscripts for publication. Enormous time and effort are saved by this advancement because it eliminates the laborious reorganization of references during manuscript revisions and avoids opportunities for typographical errors. Research scientists must make full use of these new library resources to be competitive in research today.

V.

SUPPORT FOR RESEARCH

A.

Grantsmanship

The term "grantsmanship" refers to the sum total of all strategies and procedures used to successfully compete for funding to support research and training. Success or failure in securing funding depends largely on (1) knowing where to apply, so as to focus an application toward an agency interested in the specific objectives of the application; and (2) writing a persuasive description of the research problem, a logical approach to its solution, and a high probability that the outcome of the proposed project will materially advance understanding of the topic's field. The skills required for writing a successful application have some similarities to those for writing a scientific paper, but there are distinct differences. First, the scientific paper describes work completed and includes supporting data. To be competitive, the research grant application must have preliminary data to demonstrate feasibility, but the reviewers are


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required to make judgments about the applicant's ability to perform the proposed experiments and evaluate the results. The second major difference is that the grant application includes a request for financial support that must be justified for specific expenditures. Grantsmanship must be learned by the budding laboratory animal scientist since future research pursuits depend on this skill as much as on the research skills practiced in the laboratory. The process of learning skills of grantmanship is much like that of other aspects of research, that is, through association with a mentor who has mastered the skills and demonstrated this mastery by successful competition for grant support. Initially, a basic understanding of the process can be found in introductory guides to grant writing. Guides of this type can be found in "Proposal Writer's Guide" by Don Thackery , "Guide for Writing a Funding Proposal" by S. Joseph Levine <wysiwyg://15/http:// www.canr.msu/edu/ace/dissthes/proposal.htm>, and "Guide to Grant and Proposal Writing" by Ann Martinez . In addition to understanding the objectives and requirements of granting agencies, it is also essential to understand the policies and procedures of the institution that will host the grant and provide administrative and business support. These details can usually be found through an institution's Office of Sponsored Programs. Two particularly complete and informative websites of such programs can be found at Pennsylvania State University and the University of Michigan . Although the information on these sites pertains to details for those particular institutions, much of the information is of general interest and very informative to grant applicants from other institutions.

B.

Public Sources of Support for Research

As detailed elsewhere in this chapter, the NIH, particularly the Comparative Medicine Division of the NCRR, is a major source of public support for research in laboratory animal medicine, comparative medicine, and comparative pathology. Investigators who study animal models of human diseases, or who wish to develop specialized resources for animal research or train students in the specialty should understand all of the various granting mechanisms available through this program. The online descriptions are a good starting point for current information that can be supplemented with direct consultation with staff, who are identified on the website . Comparative Medicine staff members are eager to assist potential applicants interested in one of their granting mechanisms, and they provide important advice about the intent of grant solicitations, format of the application process, and other admin-

istrative details, but they are not able to provide opinions about the merits of a proposal. The large variety and number of granting mechanisms provided by the various institutes of the NIH can be daunting to the new investigator. Nevertheless, familiarity with these mechanisms is essential for successful preparation and direction of applications to the appropriate agency within the NIH. Details about each of these mechanisms are available directly from the NIH through its website or from the NIH Office of Extramural Grants , which provides the latest information on funding opportunities, grant policies, receipt deadlines, etc. The NIH also provides information about past and current grants awarded on various topics (CRISP: Computer Retrieval of Information on Scientific Projects), which can be helpful in locating other investigators working on research of mutual interest.

C.

Private Sources of Support for Research

Research funding can be obtained from foundations and societies that focus on specific research goals. Excellent sources of information about funding from foundations can be found at websites for the Foundation Center and the Grant Advisor . Personal contact with an individual knowledgeable about the goals, policies, and procedures of a granting agency is always an important strategy but is absolutely mandatory for applications to private foundations. For laboratory animal medicine, grants are awarded by the American College of Laboratory Animal Medicine. Other organizations include the American Cancer Association, the Muscular Dystrophy Association, the American Heart Association, etc. Organizations that are less focused on a single disease or organ system include the Geraldine Dodge Foundation, the American Kennel Club, and the Mark Morris Foundation. Commercial firms that depend on research conducted under contract with academic institutions will search for academic-commercial partnerships, which are usually designed to accomplish a specific goal. Commercial contracts frequently are highly focused on product development, but some of the larger firms have foundations that fund projects that may not be product-oriented. Institutional grant-information offices and administration offices should be asked to provide information and periodic announcements of granting opportunities on topics of specific interest. Some of these offices subscribe to grant-alert services, such as Community of Science and Science Wise Alert . Each user provides the service with a profile of research interests, and the service automatically filters out the specific grant opportunities that pertain to those interests and emails the investigator directly with details of selected grant solicitations.

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31. RESEARCH IN LABORATORY ANIMAL AND COMPARATIVE MEDICINE

D.

Intellectual Property

During the past decade, academic institutions have become acutely aware of the enormous value of research findings produced by their faculty that have potential for commercialization. The current estimate is that the top 160 research universities in the United States spend more that $24 billion on research annually, and this research leads to 3500 patents per year. On the other hand, commercial firms are constantly searching for new findings that can be exploited commercially. Unfortunately, academic scientists have been slow to understand the need for protection of intellectual property, which they and their university own. Human and animal health care are prime topics for discoveries with high potential for valuable intellectual property. Most institutions have an office of industrial relations that serves the faculty by soliciting information about discoveries of potential value, assisting in evaluating the commercial value of research findings, gaining patent protection, and finding commercial firms who are interested in funding research that may be needed to bring an idea to market or who may be interested in licensing more advanced discoveries for commercialization. Laboratory animal research scientists are no less likely to make discoveries of commercial value and must become aware of all issues related to protecting intellectual property.

VI.

SUMMARY

Research is a foundation of laboratory animal and comparative medicine that has contributed enormously to the state of sophistication in animal experimentation currently enjoyed by biomedical scientists. Progress in biomedical science continues to advance at an ever increasing rate, making it imperative that laboratory animal and comparative medical specialists continue to contribute new knowledge required to solve complex problems introduced by new research concepts and technology. Although these major advances in biomedical science create problems, they also provide exciting opportunities for laboratory animal medicine specialists to demonstrate their expertise in animal experimentation and animal modeling. Regulatory and administrative distractions must not allow laboratory animal medicine to lose its momentum and reputation as a major contributor to the science of animal research. The next generation of specialists must accept the responsibility to become pre-

pared intellectually and gain the research skills needed to lead the specialty successfully into the twenty-first century. Fortunately, there are sufficient specialists who are successful research scientists and are willing to mentor trainees. Institutions with organized training programs offer excellent opportunities for those entering the field to become skilled in the art of research. Financial support during training is available to competitive candidates. Therefore, all of the essential ingredients required to sustain progress in developing research talent are available. All that remains is a commitment by those established in the specialty and those entering the specialty to maintain the scientific character of laboratory animal medicine.

REFERENCES

Andrews, E. J., Ward B. C., and Altman, N. H., eds. (1979). "Spontaneous Animal Models of Human Disease." Academic Press, New York. Bernard, C. (1865). "An Introduction to the Study of Experimental Medicine" (trans. H. C. Green. Reprinted by Dover, New York, 1957). Capen, C. C., Jones, T. C., and Migaki, G., eds. (1985). "Animal Models of Human Disease." Registry of Comparative Pathology, Armed Forces Institute of Pathology, Washington, D.C. Desnick, R. J., Patterson, D. E, and Scarpelli, D. G. (1982). "Animal Models of Inherited Metabolic Diseases." Alan R. Liss, New York. Harvey, A. M., Brieger, G. H., Abrams, S. A., and McKusick, V. A. (1989). "A Model of Its Kind: A Centennial History of Medicine at Johns Hopkins." Johns Hopkins Univ. Press, Baltimore. Kraft, L. M., Pardy, R. R, Pardy, D. A., and Zwickel, H. (1964). Practical control of diarrheal disease in a commercial mouse colony. Lab. Anim. Care 14, 16. Kraus, L. (2001). "Laboratory Animal Medicine: Advancing Science and Animal Welfare in the 21 st Century." American College of Laboratory Animal Medicine, Chester, New Hampshire. Little, C. C. (1913). Experimental studies of the inheritance of color in mice. Carnegie Institute Publ. 179, 17-102. McPherson, C. W., and Mattingly, S. E, eds. (1999). "50 Years of Laboratory Animal Science." American Association for Laboratory Animal Science, Memphis. Mobraaten, L. E., and Sharp, J. J. (1999). Evolution of genetic manipulation of laboratory animals. In "50 Years of Laboratory Animal Science" (C. W. McPherson and S. E Mattingly, eds.), pp. 129-135. American Association for Laboratory Animal Science, Memphis. Navia, J. M. (1977). The design of experiments. In "Animal Models in Dental Research," pp. 13-27. Univ. of Alabama Press, Tuscaloosa. Phaneuf, D., Wakamatsu, N., Huang, J. Q., Borowski, A., Peterson, A. C., Fortunato, S. R., et al. (1996). Dramatically different phenotypes in mouse models of human Tay-Sachs and Sandhoff diseases. Hum. Mol. Genet. 5, 1-14.


Chapter 32 Laboratory Animal Behavior Kathryn A. L. Bayne, Bonnie V. Beaver, Joy A. Mench, and David B. Morton

I. II.

III. IV. V. VI.

Introduction .................................................

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Rodents

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....................................................

A.

Mice ...................................................

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B.

Rats

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C.

Guinea Pigs

D.

Hamsters

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Rabbits .....................................................

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Laboratory Dogs and Cats . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Nonhuman Primates

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Farm Animals

..........................................

...............................................

References ..................................................

Increased awareness among the community using laboratory animals of the need for objective information regarding animal behavior resulted from federal regulations calling for promoting the "psychological well-being" of nonhuman primates. Although the literature was rich with scientific studies documenting the behavior of many species of primates in free-ranging conditions and the relatively extensive housing conditions in zoos, little was known about their behavioral profiles in the laboratory. In consequence, attention was focused on captive primate behavior and ways to enrich their environment to increase the breadth of normal behaviors exhibited and reduce or eliminate atypical behaviors. Federal regulations also called for laboratories to provide the opportunity for exercise for dogs. Again, a great deal of information was known about abnormal canine behavior, essentially due to the occurrence of undesirable/abnormal behaviors in companion dogs. Thus, new work LABORATORY ANIMAL MEDICINE, 2nd edition

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1252 1256

was done to evaluate the behavioral effects of providing canines the opportunity for exercise. The publication of the seventh edition of the National Research Council's (NRC) "Guide for the Care and Use of Laboratory Animals" (NRC, 1996) expanded the concern for laboratory animal behavior and environmental enrichment to all species used in research, testing, and education. This chapter presents a comprehensive overview of the species-typical and atypical behaviors in common laboratory animals. Where evidence has been shown in the literature, topics covered for each species include types of behavior problems, the effects of these problems on research, and environmental enrichment techniques. "It is difficult to determine to what extent behavior altered by captivity is simply another adaptive change to a different environment rather than an indication of adversity" (Shepherdson, 1998). Copyright 2002, Elsevier Science (USA). All rights reserved. ISBN 0-12-263951-0

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KATHRYN A. L. BAYNE, BONNIE V. BEAVER, JOY A. MENCH, AND DAVID B. MORTON

I.

INTRODUCTION

The study of laboratory animal behavior has increased steadil~r over the last decade. In the United States, this trend was initially focused on species for which there was a regulatory requirement to consider normalizing behavior, e.g., the U.S. Department of Agriculture requirement to promote the psychological well-being of nonhuman primates--the 1991 Animal Welfare Regulations (AWRs). With the advent of the seventh edition of the "Guide" (NRC, 1996), more emphasis has been placed on addressing the structural, social, and activity elements in all laboratory animals' environments. The European counterpart of the "Guide," Council Directive 86/609/EEC (European Directive, 1986), states that "any restriction on the extent to which an experimental animal can satisfy its physiological and ethological needs shall be limited to the absolute minimum." Indeed, the Europeans have made significant progress in addressing the behavioral needs of numerous laboratory animal species vis-a-vis environmental enrichment techniques. The material presented in this chapter summarizes the state of knowledge of animal behavior for a number of commonly used laboratory animals and provides suggestions for promoting normal behavior. Scientists should be concerned about the behavioral state of animals kept in laboratories, not only for ethical reasons but for reasons of science, as behavioral abnormalities may be accompanied by physiological or immunological variations from the norm, thereby potentially confounding research data. For example, it is now clear that the central nervous system has a significant direct effect on the immune system independent of corticosteroids (Dantzer and Kelley, 1989; Kingston and Hoffman-Goetz, 1996). Moreover, there is a wealth of literature on the effects on behavior of enriched and impoverished environments showing that they affect brain development, memory, learning ability, problem solving, and social interactions with humans and other animals. Enriched environments can also mitigate the effects of undernutrition and old age; promote recovery from brain trauma; and alter drug responses, tumor latency, LD 50%, and the development of athersclerosis (see, e.g., Chance, 1957; DePass et al., 1986; Renner and Rosenzweig, 1987; Claassen, 1994; Kempermann et al., 1997). Three important methodologies have been used to help determine what animals require, and each has its own advantages and limitations (Mason et al., 1998; Dawkins, 1990). The first is simply to observe what animals do and to prepare an ethogram of those behaviors based on the time they spend carrying them out and the time at which they perform those behaviors. One can then compare time budgets in different environments, with perhaps the natural species ethogram being the "gold standard" (see, e.g., Lawlor, 1984; Stauffacher, 1997b; Poole, 1992, 1998). This approach can also be used to determine whether and how animals interact with an environment and objects in that environment, such as furniture and toys (Mench, 1994). Also

with this type of approach, one can measure physiological variables or use psychometric tests (such as the open field test for anxiety, attack latency) and then see how the results deviate from those obtained in other environments (see Broom and Johnson, 1993; Cooper and Hendrie, 1994). The disadvantage of this approach is that the standard for what is "normal" may be subjectively based on the observer's knowledge and experience. The second approach is to offer animals a choice of environments or aspects of an environment to see which they prefer to spend their time in--so-called preference testing. This has an honorable tradition going back to the work of Craig (1918). In preference tests, animals have free access to different choices, and the amount of time spent with each choice is measured. However, the results should be treated cautiously (Duncan, 1978; Dawkins, 1990; Fraser, 1996) as animals may not indicate what is in their long-term interests and can choose only from the environments offered. Also, an animal's choice may vary with experience. The third method to assess animal needs builds on preference testing by determining how hard the animal will work to reach a certain environment and comparing this effort with other behaviors, i.e., testing the strength of their preferences. Animals will continue to work hard for essentials like food and water but make less effort for different substrates, environments, or social interactions. In this manner it may be possible to rank the relative importance of various activities in the behavioral repertoire to separate needs from wants from luxuries (see Dawkins, 1990, 1992). Limitations to this test are based on the inherent variability in the stimuli used to test the animals and in making equivalent comparisons between them. Behavior problems encountered in captive animals can be classified broadly and simply as "qualitative" or "quantitative" aberrant expressions of behavior (Erwin and Deni, 1979). This classification scheme implies either that species-typical behavior is modified in the amount expressed, such as an excessive amount of time spent in the behavior or the absence of the behavior, or that the behavior itself has been modified such that it is expressed in response to an atypical stimulus or is directed to an inappropriate target. In either case, a mismatch between the response and the stimulus is evident (Bayne, 1996). Fox (1968) has stated that a behavior that initially began as a means for the animal to adapt to the conditions that elicited the behavioral change frequently becomes maladaptive and can "become emancipated or released independent of the o r i g i n a l . . , stimuli." Crockett (1998) includes altered activity cycles in the list of behavioral measures that may reflect inadequate well-being. The effects of stereotypies on health and well-being are less clear-cut than for many other kinds of abnormal behaviors, such as self-biting (Mason, 1991; Lawrence and Rushen, 1993; Mench and Mason, 1997). For example, there is conflicting evidence as to whether the performance of stereotypic behavior results from stress or actually reduces stress levels (Ladewig

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32. LABORATORY ANIMAL BEHAVIOR

et al., 1993). Stereotypies are a heterogenous category of behaviors that can include a variety of locomotor, postural, or gestural patterns. Their development can be influenced by a number of factors, such as neurological predispositions, exposure to stressors, impoverished environmental conditions, and frustration of the motivation to perform particular behaviors. Once they are established, stereotypies can be difficult to stop. Since there is general agreement that stereotypies generally indicate that the animal's environment is or has been inadequate in some way, a better approach is to minimize or prevent the development of stereotypies by providing opportunities for the expression of species-typical behaviors (Duncan et al., 1993). For a broader perspective on environmental factors likely to impinge on animal welfare and science, see Clough (1982) and Rose (1994). The goals of enrichment are to decrease the incidence of abnormal behaviors and to increase the diversity of normal behaviors, and the evaluation of any enrichment can be carried out in any of the three ways described above (Bayne et al., 1992c; Beaver, 1989; Benn, 1995; Hart, 1994; Markowitz and Gavazzi, 1995; Newberry, 1995; Poole, 1998; Scharmann, 1991; Stauffacher, 1997a). However, it has to be remembered that simply changing an animal's behavior pattern does not necessarily mean the change is for the better. It may simply result in one stereotypic behavior being substituted for another. Therefore, a basic understanding of the species-typical behavior of the animal and an ongoing program to evaluate the effects of an enrichment program are key to improving the behavioral wellbeing of laboratory animals. This chapter will review some fundamental behaviors of the more common laboratory animals and link these to environmental enrichments that can improve animal well-being and provide a more refined animal model for research.

II.

RODENTS

Rats and mice comprise the most common mammals used in research in most countries (see Her Majesty's Stationery Office, 1997). Other rodents used in research include gerbils, guinea pigs, hamsters, voles, and various wild species. The systems of husbandry used for these animals have changed little since they were first kept in laboratories, but in the last 10 years or so, efforts have been made to make their immediate caging environment more in keeping with their behavioral needs. Rodents have been kept in captivity for at least a century and some species far longer. Despite this, they retain many of their natural characteristics and instincts that evolved in their wild ancestors, as evidenced from studies of their behaviors when they are placed in more "natural" habitats (e.g., Boice, 1977). This may generate ideas about how to modify their environ-

ments to suit them better and so reduce any adverse effects they may feel (Brain, 1992). However, the fact that there are several hundred strains of rodents (specifically, rats and mice, both inbred and outbred) raises the question of whether the environmental requirements are the same for all strains. Some general principles and guidelines for some of the commonly used species of rodents are described below.

A.

Mice

Mice show a range of behaviors (Brain et al., 1989; Jennings et al., 1998), but those that cause most concern involve stereotypies such as wire gnawing and jumping and aggression (particularly among male mice of certain strains). Expression of aggression, stereotypies, and natural behaviors may be influenced by cage design, cage furniture, and substrate. It may also be desirable to satisfy some select natural behaviors, such as nest building. 1.

Influences on Behavior

Mice kept in traditional cages may gnaw on the cage wire, jump and circle around the tops of cages, chew, and dig (e.g., Tuli, 1993; Wiirbel et al., 1996; Hobbs et al., 1997). Significant differences between strains in the kinds of behaviors expressed have been observed. For example, nude mutants jump more than the parent wild-type strain (Zur:ICR), which in turn, gnaw more; and DBA/2 mice exhibit more eating, grooming, and exploring than do CD-1 or B6CBAF1/J strains (Hobbs et al., 1997). Some behaviors may be modified through the provision of nesting materials, and empty water bottles and tunnels can be used as resting areas (Hobbs et al., 1997; Ward and DeMille, 1991). Wiirbel and colleagues (1998) also found that providing a cardboard tube significantly reduced wire gnawing in ICR mice. The tube provided shelter, resulting in increased resting periods, which probably indicated a feeling of security. In another study, mice (BALB/c and Crl:NMRI strains) kept in an enriched environment (polyvinyl chloride [PVC] tube, nesting material, and a metal grid) showed more climbing and eating than the controls kept in standard caging; but there were strain differences in that B ALB/c mice spent more time than NMRI climbing and moving about, but NMRI spent more time resting and grooming (Van de Weerd, 1996). Barbering (hair nibbling and whisker chewing) in mice is not uncommon and usually manifests as an area of hair loss over the upper back (shoulder and neck); the whisker bed is not as commonly affected (Hauschka, 1952; Long, 1972; Thornberg et al., 1973; Litterst, 1974). Barbering is considered to be a social behavioral effect with the dominant mouse being the perpetrator, and several common strains of mice are affected (e.g., SwissWebster, C57BL, C3H, CDF). Sarna et al. (2000) studied this effect in c57BL/6 mice and found that separating the mice

1242


reversed the condition, and that when mice were rehoused together (in pairs), the barbering recurred as a result of mutual grooming. The recipients were passive in accepting barbering, and even pursued conspecifics for more grooming. Brain analysis showed differences between the barbers and the barbered animals, and the results were discussed in relation to social dominance and the consequences of barbering on brain function. Another form of noninfective alopecic (little erythema or swelling) hair loss occurs when animals push their noses through the hopper bars to access food. In these cases, the alopecia is around the muzzle. Aggression in male mice can hardly be called an aberrant behavior, but it is certainly a welfare problem for the animals lower in the hierarchy, resulting in bites to the tail, rump, ears, and shoulders. It can also occur between females and between sexes (e.g., when defending a litter). There are wellcharacterized strain differences, with inbred C57BL/10 and DBA/2, outbred Swiss, and TO mice considered more agonistic, whereas CBA/Ca, C3H/He mice are typically more docile (Jones and Brain, 1987). Aggressiveness has been shown to be affected by intrauterine position of the pups and is thought to be related to local placental transfer of hormones between the sexes (Vom Saal, 1991). Mice use olfactory clues, rather than sight or sound, to establish a pecking order. Disturbing the cage environment, by cleaning for example, can therefore precipitate a bout of fighting while scent marking is carried out and the order reestablished (Hurst et al., 1993). Blom (1993) found that while NMRI mice preferred clean to soiled cages that had not been cleaned for 4 to 6 days, 2-day soiled cages were preferred to clean ones. This may have implications for routine husbandry of animals and would be interesting to reevaluate in ventilated cages. Aggression has been shown to be influenced by strain, age, and prior encounters. Participation in aggressive encounters can affect levels of pituitary hormones, including the reproductive gonadotropins (animals may become infertile), adrenocorticotropic hormones (ACTH), as well as catecholamine levels in the adrenal medulla (Brain, 1990). Corticosterone levels can be higher in defeated subordinate mice, especially when they are kept in noncomplex conditions, such as bedded polycarbonate cages (Durschlag and Stauffacher, 1996), but can also be higher in dominant animals with a large territory to defend (Bishop and Chevins, 1989). Any enrichment to reduce aggression has to be carefully designed, as simply providing objects that mice can occupy may increase the amount of territory to defend and so promote aggression (Haemisch et al., 1994). However, if adequate space is provided in addition to shelters, the degree of injury may be reduced by allowing the opportunity for submissive posturing rather than overt fighting (Durschlag and Stauffacher, 1996). Other ways shown to successfully reduce aggression include housing mice together before puberty is reached, using more docile strains, and replacing conspecifics as soon as possible if they have to be separated, since even the removal of an

animal for 24 hr can increase the level of aggression on its return (Brain, 1997). 2.

Environmental Enrichment

Enriched environments do not appear to have any long-term effects on the performance of mice in open field tests, their docility, corticosterone levels, or adrenal gland weights. However, there is evidence that mice kept singly have compromised immune systems (Schwartz et al., 1974) and develop tumors faster than when kept in groups (Riley, 1981). Considerable work has recently been undertaken to look at what sorts of environments and substrates mice choose and how hard they will work to access them. The traditional housing system for rodents has been wire-bottom cages, but these are increasingly being replaced, when appropriate, by solid-bottom cages. This raises the question of what sort of bedding material is appropriate and liked by the animals. Given a choice, mice avoid wire-bottom cages, preferring solid-bottom cages with shredded filter paper (Blom, 1993, B lom et al., 1996), which they will work hard to obtain (Roper, 1973). When a cage was constructed as half mesh and half solid, the mice used the mesh half more as a latrine and rested in the solid part, a tendency confirmed in later studies where mice in enriched cages urinated in certain locations, unlike animals kept in standard conditions (Van de Weerd, 1996). Furthermore, in rank order, mice preferred shredded filter paper to wood chips to sawdust, and there were no strain differences in these preferences between BALB/c and C57BL/6 mice. B lom et al. (1996) attributed the preference for the paper to the higher irritancy of fine sawdust particles, but mice were also able to manipulate the paper and build nests, which might have contributed to their preference. When nesting materials were specifically tested, it was found that paper was preferred to wood products (i.e., paper tissues, towels, and strips to wood wool or shavings), and combinations of nesting materials were preferred. Certainly, pregnant and lactating females should be given nesting materials that they can manipulate, but it appears that all mice like to build nests (Van de Weerd et al., 1997). Sherwin (1996a) further emphasizes the potential importance to the animal of being able to build its own nest out of appropriate materials rather than being given one ready-made, and that this may be a behavioral need. It is important to note that some bedding materials may cause problems for neonatal mice because the materials may be very absorbent, thereby potentially dehydrating the pups (Hessler and Moreland, 1984). Van de Weerd (1996) evaluated the use of nest boxes and found that mice preferred a cage with a box to one without, but still preferred a nesting material on a grid floor to a grid floor with a nest box. However, findings in relation to whether mice choose dark or light nest boxes; open, closed, or meshed ones; or metal rather than plastic are at variance and may be straindependent, as the choice may reflect the balance of olfactory

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32. LABORATORY ANIMAL BEHAVIOR

to visual or tactile cues (see Buhot, 1989). Strain differences appear to exist in the design of the nest built. For example, in BALB/c and C57BL strains, the shape of the nest differed--the BALB/c's nest was domed and the C57BL's nest was bowlshaped (Van de Weerd, 1996). Sherwin (1996a) found that mice were prepared to work as hard, within the imposed cost of having to traverse 30 cm of shallow water, to reach a running wheel, deep sawdust (6 to 7 cm), a conspecific (unfamiliar male of the same strain), or increased space or shelter (plastic cup), as for food. The author interpreted these findings to indicate that the animals place a value on all of these items. Standard environments for group-housed mice have also been supplemented with cans (although rusting and physical hazards are concerns), glass bottles, plastic pipes, soft wood pieces, and cardboard tubes. Such environments, when compared with standard caging, have been shown to reduce aggression (providing that objects are renewed to remove olfactory clues; see Ambrose and Morton, 1997), emotionality, anxiety (open field test, freezing), and adrenal gland weight, and to promote exploratory activity (hole board tests, cage emergence time) and alertness (rearing) (Chamove, 1989; Ward and DeMille, 1991; Van de Weerd et al., 1994; Van de Weerd, 1996). However, a strain difference is apparent, with BALB/c mice appearing to be more anxious than C57BL mice. Another interesting finding is that standard-caged animals sleep huddled together, whereas enriched animals sleep in groups of two or three (Van de Weerd, 1996). Preferences for type of shelter have been studied in some detail, and it was found that individually housed mice prefer to sleep in loose sawdust rather than in tubes, and that there is no overall preference for shape, opacity, or openness of tube (Sherwin and Nicol, 1995; Sherwin, 1996b).

B. 1.

Rats


Rats are the second most common rodent used in research laboratories, yet few studies on their behavioral needs and enrichment have been done, although many caretakers are intuitively looking at ways in which the animals' environments can be made more stimulating. Rats are nocturnal animals that show a range of natural active behaviors, such as exploration, inquisitiveness, digging, aggression, rearing, climbing, and jump/ pounce/roll/wrestle/hold behaviors, etc., in play, with a welldefined circadian rhythm (Blanchard et al., 1975, 1988; Flannelly and Lore, 1977; Silverman, 1978; Cowan, 1983; Weihe, 1987; Lore and Schultz, 1989; Batchelor, 1994). Choice tests have confirmed that they are inquisitive about novel environments (e.g., Hughes, 1968) and that they are gregarious animals (Latane, 1969; Latane et al., 1972). They are social animals that can live in well-defined, compatible groups (Barnett, 1967).

It is well known among caretakers and pet owners that when rats are kept in isolation, they tend to become more aggressive and may exhibit increased susceptibility to disease (Hatch et al., 1963; Baer, 1991). Hatch and colleagues (1963) observed that not only did the rats become more intractable, but their adrenal and thyroid glands increased in weight, their spleen and thymus decreased in weight, and their tolerance to chemical toxicity decreased, as shown by a reduced LDs0. Damon et al. (1986) showed that the LDs0 was also reduced if animals were not acclimated to a new environment, such as a metabolic cage, and acclimation has been shown to stabilize urinary excretion of steroids (Gomez-Sanchez and Gomez-Sanchez, 1991). Such noticeable effects are evident even after a few days and can be affected by overcrowding as well as isolation, resulting in increased corticosterone levels (Capel et al., 1980a,b; Holson et al., 1991). Brain and Benton (1979) have argued that it is not easy to determine whether these effects are truly due to isolation, as the variance could also be due to factors such as individual responses, strain, sex, and previous housing conditions. Work by Hurst and colleagues (1998) examining social sexual strategies suggests that the effects of social isolation may be different for males and females. Single housing of females had much less effect on time budgeting and corticosteroid levels than for males, e.g., tail chasing was less for females than males, but escape behaviors such as bar chewing were higher. Rose (1993) concludes that there is abundant evidence that individual housing of rats produces significant behavioral changes that impair physical and psychological fitness for research. The wealth of literature on the effects on behavior of enriched and impoverished environments has also shown substantial effects on brain development, including morphological and physiological aspects, synapse density, memory, learning ability, problem solving, brain responses to undernutrition and old age, recovery from brain trauma, and altered corticosterone levels (e.g., see Renner and Rosenzweig, 1987; Claassen, 1994; Kempermann et al., 1997; Hurst et al., 1998), and interestingly, increased benzodiazepine receptor binding (Wadham and Mortin, in press). Restraint is also an adverse condition for rats and has been shown not only to increase corticosteroids but to predispose animals to developing gastric ulcers (Gamello et al., 1986). The phenomenon is restrainer-dependent, with different types of restraint altering heart rate, blood pressure, and body temperature to varying degrees (Gardiner and Bennett, 1977; Wadham, 1996). 2.

Preferred Environments

Blom and colleagues (1995) determined on the basis of preference tests that females preferred cages with low heights (80 mm as opposed to heights of 320 mm; time spent in each was 29.9% compared to 19.2%), and males preferred the lowest height (38.6% in 80 mm) but also spent considerable time in higher cages (25.3% in 320 mm). Low light intensities were

1244


chosen by all rats regardless of whether they were albino or pigmented strains (Wistar Cpb:WU and a pigmented Wistar hybrid), although the albino animals spent more time in the reduced light. When grid floor mesh size was examined, female Wistar rats preferred smaller rather than larger mesh (i.e., 10 mm x 10 mm, compared to 10 mm x 30, 50, or 80 mm; see Blom, 1993). When kept on grid floors, rats also appear to like cage furniture such as plastic tubes and raised wooden platforms, and they tend to use any form of shelter to take cover in and to climb on (Bradshaw and Poling, 1991; Scharmann, 1991; Batchelor, 1993, 1994; Manser et al., 1998a). Old mouse boxes placed in rat cages are used as hides and climbing objects. The rats gnaw holes at the corners, which provide at least two escape routes, and will turn the box on its edge or upside down (D. B. Morton, personal observation). Rats like to gnaw on wooden sticks (Orok-Edem and Key, 1994; Chmiel and Noonan, 1996) as well as furniture, which may pose a theoretical confounding variable for toxicology studies. Rats choose to spend more time in more complex environments (Denny, 1975). Rats prefer to work for their food, and thus will press a lever rather than eat food placed in front of them (Carder and Berkowitz, 1970; Singh, 1970) and will also remove the husk from a sunflower seed rather than eat one that is already prepared (Shettleworth and Jordan, 1986). The type of flooring preferred by rats is coincident with their long-term health, as solid floors are preferred to grid floors (Van de Weerd et al., 1996), and it is generally observed that grid floors are associated with superficial foot lesions leading to ulceration, inflammation, pain, and swelling (Kohn and Barthold, 1984). Regardless of previous experience of flooring type, rats spend more time resting on solid floors compared with the wire floors (88% :12%), but wire floors are nearly equally used during the rats' active period (55%: 45%) and for defecation and urination. Furthermore, a comparison of rats kept on the two types of flooring revealed no differences in weight gain, food or water consumption, ease of handling, and many physiological parameters, including immune function and catecholamine, testosterone, and corticosteroid levels (Manser, 1992; Nagel and Stauffacher, 1994; Manser et al., 1995; Stauffacher, 1997a; Van de Weerd et al., 1996). Therefore, there would seem to be no reason not to use solid-bottom cages given the increased potential for animal well-being. Moreover, rats will work (by lifting a weighted barrier) as hard to reach solid-bottom floors as they will to reach a novel environment (Manser et al., 1996). Solid-bottom cages require bedding, and choice tests have shown that rats prefer cages with wood shavings and paper bedding to those with sawdust (Blom et al., 1996; Van de Weerd et al., 1996). Paper bedding may also be more acceptable in toxicological studies. B lom et al. (1993, 1996) showed that rats preferred materials with large fibrous particles, that were manipulable, and that may produce ultrasound by particles rubbing against each other (see also Manser et al., 1998a). The type of nest box preferred by rats has also been investi-

gated. Manser et al. (1998a,b) found that rats spend considerable time in the nest (60 to 80% of the time) and that they will work harder to reach a cage with a nest box, whether or not it contains nesting material, than they will to reach an empty cage. Manser and colleagues went on to develop an opaque plastic nesting box with a roof and three sides as a practical piece of furniture, incorporating the preferred features. This box was not chewed and was easy to clean.

C. 1.

G u i n e a Pigs


Compared with work on rats and mice, there has been little experimental work on guinea pigs in terms of their preferences and abnormal behavior patterns. Guinea pigs are social and crepuscular animals (active at dawn and dusk) but appear not to burrow. Manning and colleagues (1984) found that guinea pigs like to lie beside each other. Although not normally aggressive in the laboratory, they can be under certain conditions, e.g., when males are in the presence of females (Sachser and Lick, 1991). When disturbed, guinea pigs may squeak and rush for cover, but they also show freezing behavior in response to a stressor (Fara and Catlett, 1971; Suthedand and Festing, 1987), which may mistakenly be taken to indicate that they do not find the situation particularly aversive. Fears may be communicated through ultrasound vocalization. Guinea pigs do not appear to sleep for long periods of time but rather take short rests (White et al., 1989). Sachser (1986) looked at certain anatomical and physiological parameters when guinea pigs either were kept in colonies or were singly caged. He found that singly caged animals showed evidence of atrophy of the reproductive organs, with males having lower testosterone levels, which would lead to lower accessory gland weight. The singly caged guinea pigs also had lower corticosteroid levels and adrenal gland weights. 2.

Preferred Environments

Since they are social animals that like to be in contact with conspecifics, it is not unreasonable to think that guinea pigs would prefer to be kept in groups, but as stated previously, little experimental work has been carried out. White et al. (1989; White, 1990) suggested that guinea pigs did not utilize the whole of the cage area; however, Scharmann (1991) found that when the cage was enriched through the addition of hay and straw, then they burrowed and hid in it. Furthermore, Scharmann observed that guinea pigs spent some time chewing on the bars and hoppers of the cage. This behavior could be stopped by adding pieces of wood, which gave the guinea pigs something else to gnaw on. Whether this simply displaces one type of oral stereotypy with another is not clear. As guinea pigs are

32. LABORATORYANIMAL BEHAVIOR rather timid animals, they like cage or pen furniture that enables them to hide, such as boxes or plastic pipes (Noonan, 1994; Meyer, 1995).

D.

Hamsters

Hamsters have a reputation for fighting when housed in groups, but stable groups can be established if animals are grouped early in their lives. Little work has been done on what these animals prefer, but one study has shown, based on occupation times, that they prefer solid-bottom cages with bedding, regardless of their age or sex (Arnold and Estep, 1994). In the solid-bottom cage environment, hamsters spent more time engaged in behaviors such as sleeping, grooming, gnawing, eating, hoarding, and exploring. However, that preference could be influenced by experience. Forty percent of those reared on wire spent more of their time on wire mesh (see Arnold and Estep, 1990), although most chose the solid-bottom cages. Hamsters spend much of their time asleep (41%), older animals sleeping more than younger ones (44% compared to 37%). Hamsters reared on bedding rarely chose wire on which to sleep (

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