Molecular and Diagnostic Procedures in Mycoplasmology Molecular Characterization Vol 1

Molecular and Diagnostic Procedures in Mycoplasmology Volume II

DIAGNOSTIC PROCEDURES

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Molecular and Diagnostic Procedures in Mycoplasmology Volume II

DIAGNOSTIC PROCEDURES Edited by

JOSEPH G. TULLY Mycoplasma Section Laboratory of Molecular Microbiology National Institute of Allergy and Infectious Diseases Frederick Cancer Research and Development Center Frederick, Maryland

SHMUEL RAZIN Department of Membrane and Ultrastructure Research The hiebrew University-Hadassah Medical School Jerusalem, Israel

ACADEMIC PRESS San Diego

New York

Boston

London

Sydney

Tokyo

Toronto

Front cover photograph: Vero cells infected with M. hyorhinis, stained by a double stain method using DNAF and fluoresceinated anti-M hyorhinis antibodies, and viewed with the filter set for fluorescein. Courtesy of Dr. Gerald K. Masover and Frances A. Becker, Genentech, Inc., South San Francisco, CA.

This book is printed on acid-free paper. ©

Copyright © 1996 by ACADEMIC PRESS, INC. 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.

Academic Press, Inc. A Division of Harcourt Brace & Company 525 B Street, Suite 1900, San Diego, California 92101-4495 United Kingdom Edition published by Academic Press Limited 24-28 Oval Road, London NWl 7DX Library of Congress Cataloging-in-Publication Data Molecular and diagnostic procedures in mycoplasmology / edited by Shmuel Razin, Joseph G. Tully. p. cm. Includes indexes. Contents: v. 1. Molecular characterization - v. 2. Diagnostic procedures. ISBN 0-12-583805-0 (v. 1: alk. paper) ISBN 0-12-583806-9 (v. 2: alk. paper) 1. Mycoplasma diseases-Diagnosis. 2. Mycoplasma diseases-Molecular aspects. I. Razin, Shmuel. II. Tully, Joseph G. [DNLM: 1. Mycoplasma—physiology. 2. Mycoplasma—pathogenicity. 3, Molecular Biology-methods. 4. Mycoplasma Infections-diagnosis. QW 143 M718 1995J QR201.M97M63 1995 589.9-dc20 DNLM/DLC for Library of Congress 95-4586 CIP

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Contents

Contributors

xi

Preface

xv

Contents of Volunne I

xvii

Mollicute-Host Interrelationships: Current Concepts and Diagnostic Implications Joseph C. Tully

SECTION A Diagnostic Genetic Probes A1

Introductory Remarks

25

Shmuel Razin A2

Oligonucleotide Probes Complementary to 16S rRNA

29

Karl-Erik Johansson A3

Cloned Genomic DNA Fragments as Probes

47

David Yogev and Shmuel Razin A4

PCR: Selection of Target Sequences

53

Remi Kovacic, Odile Crau, and Alain Blanchard A5

PCR: Preparation of DNA from Clinical Specimens

61

Bertille de Barbeyrac, Christiane Bebear, and David Taylor-Robinson A6

PCR: Amplification and Identification of Products Bertille de Barbeyrac and Christiane Bebear

65

VI A7

Contents PCR: Application of Nested PCR to Detection of Mycoplasmas

75

Ryo Harasawa A8

PCR: Random Amplified Polymorphic D N A Fingerprinting

81

Steven I. Geary and Mark H. Forsyth

SECTION B Bl

Immunological Tools

Introductory Remarks

89

Joseph C. Tully B2

ELISA in Small Animal Hosts, Rodents, and Birds

93

M. B. Brown, j. M. Bradbury, and I. K. Davis B3

ELISA in Large Animals

105

I. Nicolet and I. L. Martel B4

ELISA in Human Urogenital Infections and AIDS

115

Richard Yuan-Hu Wang and Shyh-Ching Lo B5

ELISA in Respiratory Infections of Humans

123

Gail H. Cassell, Ginger Gambill, and Lynn Duffy B6

Monoclonal Antibodies as Diagnostic Tools

137

Ghester B. Thomas, Monique Gamier, and John T. Boothby B7

Microimmunofluorescence David

B8

147

Taylor-Robinson

Immunoblots and Immunobinding

151

David Thirkell and Bernard L Precious B9

Differentiation of Mycoplasma Mycoplasma

pneumoniae

Joseph G. Tully

genitalium

from

by Immunofluorescence

169

Contents

SECTION C Cl

vii

Antibiotic Sensitivity Testing

Introductory Remarks

181

Christiane Bebear C2

Problems and Opportunities in Susceptibility Testing ofMoliicutes

185

George E. Kenny C3

Determination of M i n i m a l Inhibitory Concentration

189

Christiane Bebear and Janet A. Robertson C4

Cidal Activity Testing David

Taylor-Robinson

SECTION D D1

199

Diagnosis of Specific Diseases

Introductory Remarks

207

Joseph C. Tully D2

Laboratory Diagnosis of Mycoplasma R. J. Harris, j. Williamson,

D3

Infection

211

C. Hahn, and B. P. Marmion

Diagnosis of Sexually Transmitted Diseases David

D4

pneumoniae

225

Taylor-Robinson

Diagnosis of Neonatal Infections

237

Ken B. Waites and Gail H. Cassell D5

Mycoplasmas in AIDS Patients

247

Shyh-Ching Lo D6

Mycoplasma Infections of Cattle Ed A. ter Laak and H. Louise Ruhnke

255

VIM D7

Contents Mycoplasma Infections of Goats and Sheep

265

A, I. DaMassa D8

Mycoplasma Infections of Swine

275

Richard F. Ross and Gerald W. Stemke D9

Mycoplasma Infections of Poultry

283

Stanley H, Kleven and Sharon Levisohn DIO

Diagnosis of Spiroplasma Infections in Plants and Insects

293

C. Saillard, C. Barthe, ]. M. Bove, and R. F. Whitcomb Dll

Detection of Phytoplasma Infections in Plants

299

£. Seemueller and B. C. Kirkpatrick D12

Identification of Mollicutes from Insects

313

Robert F. Whitcomb and Kevin /. Hackett

SECTION E Experimental Infections El

Introductory Remarks

325

Joseph G. Tully E2

Experimental Mycoplasmal Respiratory Infections in Rodents

327

Gail H. Cassell and A. Yancey E3

Urogenital Infections in Rodents Patricia M. Furr and David

E4

337

Taylor-Robinson

Experimental Models of Arthritis

349

Leigh Rice Washburn E5

Experimental Infections in Poultry Janet M. Bradbury and Sharon Levisohn

361

Contents E6

Experimental Infections of Swine

ix 371

Marylene Kobisch and Richard F. Ross E7

Experimental Infections in Cattle

377

Ricardo F. Rosenbusch and H. Louise Ruhnke E8

Experimental Infections of Plants by Spiroplasmas

385

X. Foissac, I. L Danet, C. Saillard, R. F. Wbitcomb, and I. M. Bove E9

Experimental Phytoplasma Infections in Plants and Insects

391

Alexander H. Purcell ElO

Mycoplasmas and in Vitro Infections of Cell Cultures w i t h HIV Shyb-Ching Lo and Alain

SECTION F

Fl

399

Blancbard

Diagnosis of Mycoplasma Infections of Cell Cultures

Introductory Remarks

407

losepb G. Tally F2

Isolation of Mycoplasmas from Cell Cultures by Axenic Cultivation Techniques

411

Ricbard A. Del Giudice and losepb G. Tully F3

Detection of Mycoplasmas by D N A Staining and Fluorescent Antibody Methodology

419

Gerald K. Masover and Frances A. Becker F4

Detection of Mycoplasma Infection by PCR

431

Connie Veilleux, Sbmuel Razin, and Laurie H. May F5

Antibiotic Treatment of Mycoplasma-lnfected Cell Cultures Ricbard A. Del Giudice and Roberta S. Gardella

439

X

Contents

F6

Prevention and Control of Mycoplasma Infection of Cell Cultures

445

Ann Smith and Ion Mowles

APPENDIX Table I

Genus Mycoplasma and Major Characteristics

455

Table II

Genera Ureaplasma and Acholeplasma

458

Table III

Genera Anaeroplasma and Asteroleplasma

458

Table IV

Genera Entomoplasma and Mesoplasma

459

Table V

Group Classification of Genus Spiroplasma

460

Index

463

Contributors

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

C. Barthe (293), Laboratoire de Biologic Cellulaire et Moleculaire, Institut National de Recherche Agronomique, F-33883 Villenave d'Ornon, France Christiane Behear (61, 65, 181, 189), Laboratoire de Bacteriologie, Universite de Bordeaux II, F-33076 Bordeaux, France Frances A. Becker (419), Department of Quality Control, Genentech, Inc., South San Francisco, California 94080 Alain Blanchard (53, 399), Department du SID A et des Retrovirus, Institut Pasteur, Unite d'Oncologie Virale, 75724 Paris, France John T. Boothby (137), Department of Biological Sciences, San Jose State University, San Jose, California 95192 J. M. Bove (293, 385), Laboratoire de Biologic Cellulaire et Moleculaire, Institut National de Recherche Agronomique, F-33883 Villenave d'Ornon, France Janet M. Bradbury (93, 361), Department of Veterinary Pathology, University of Liverpool, Leahurst, Neston, South Wirral L64 7TE, United Kingdom M. B. Brown (93), Department of Infectious Diseases, College of Veterinary Medicine, University of Florida, Gainesville, Florida 32611 Gail H. Cassell (123, 237, 327), Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294 A. J. DaMassa (265), Department of Population Health and Reproduction, School of Veterinary Medicine, University of California at Davis, Davis, California 95616 J. L. Danet (385), Laboratoire de Biologic Cellulaire et Moleculaire, Institut National de Recherche Agronomique, F-33883 Villenave d'Ornon, France J. K. Davis (93), Department of Comparative Medicine, University of Florida, Gainesville, Florida 32611 Bertille de Barbeyrac (61, 65), Laboratoire de Bacteriologie, Universite de Bordeaux II, F-33076 Bordeaux, France Richard A. Del Giudice (411, 439), Mycoplasma Laboratory, SAIC Frederick, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702 Lynn Duffy (123), Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294 X. Foissac (385), Laboratoire de Biologic Cellulaire et Moleculaire, Institut National de Recherche Agronomique, F-33883 Villenave d'Ornon, France Mark H. Forsyth (81), Department of Pathobiology, University of Connecticut, Storrs, Connecticut 06269 Patricia M. Furr (337), MRC Sexually Transmitted Diseases Research Group, Department of Genitourinary Medicine, St. Mary's Hospital Medical School, London W2 INY, United Kingdom Ginger Gambill (123), Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294

XI i

Contributors

Roberta S. Gardella (439), Mycoplasma Laboratory, SAIC Frederick, NCI-Frederick Cancer Research and Development Center, Frederick, Maryland 21702 Monique Gamier (137), Laboratoire de Biologic Cellulaire et Moleculaire, Institut National de Recherche Agronomique, F-33883 Villenave d'Ornon, France Steven J. Geary (81), Department of Pathobiology, University of Connecticut, Storrs, Connecticut 06269 Odile Grau (53), Department du SIDA et des Retrovirus, Institut Pasteur, Unite d'Oncologie Virale, 75724 Paris, France Kevin J. Hackett (313), Insect Biocontrol Laboratory, Plant Sciences Institute, United States Department of Agriculture, Agricultural Research Service, BARC, Beltsville, Maryland 20705 C. Hahn (211), Department of Biochemistry, University of Adelaide, Adelaide, South Australia 5000, Australia Ryo Harasawa (75), Animal Center for Biomedical Research, Faculty of Medicine, University of Tokyo, Hongo, Tokyo 113, Japan R. J. Harris (211), School of Pharmacy and Medical Sciences, University of South Australia, Adelaide, South Australia 5000, Australia Karl-Erik Johansson (29), Laboratory of Bacteriology, Research and Development, The National Veterinary Institute, S-750 07 Uppsala, Sweden George E. Kenny (185), Department of Pathobiology, School of Public Health and Community Medicine, University of Washington, Seattle, Washington 98195 B. C. Kirkpatrick (299), Department of Plant Pathology, University of California at Davis, Davis, California 95616 Stanley H. Kleven (283), Department of Avian Medicine, University of Georgia School of Veterinary Medicine, Athens, Georgia 30602 Marylene Kobisch (371), Centre National d'Etudes Veterinaires et Alimentaires, Station de Pathologic Porcine, F-22440 Ploufragan, France Remi Kovacic (53), Department du SIDA et des Retrovirus, Institut Pasteur, Unite d'Oncologie Virale, 75724 Paris, France Sharon Levisohn (283, 361), Department of Poultry Diseases, Ministry of Agriculture Veterinary Services and Animal Health, Kimron Veterinary Institute, Beit-Dagan 50250, Israel Shyh-Ching Lo (115, 247, 399), Department of Infectious and Parasitic Disease Pathology, Division of Molecular Pathology, American Registry of Pathology, Armed Forces Institute of Pathology, Washington, District of Columbia 20306 B. P. Marmion (211), Department of Pathology, Institute of Medical and Veterinary Science, University of Adelaide, Adelaide, South Australia 5000, Australia J. L. Martel (105), Laboratoire de Reference de I'OIE pour la PPCB, CNEVA-LPB, F-69342 Lyon, France Gerald K. Masover (419), Department of Quality Control, Genentech, Inc., South San Francisco, California 94080 Laurie H. May (431), Genentech, Inc., South San Francisco, California 94080 Jon Mowles (445), Biochem ImmunoSystems Ltd., Woking GU21 5JY, United Kingdom J. Nicolet (105), Institute of Veterinary Bacteriology, University of Berne, CH-3001 Berne, Switzerland

Contributors

xiii

Bernard L. Precious (151), Division of Cell and Molecular Biology, School of Biological and Medical Sciences, University of St. Andrews, St. Andrews, Fife KY16 9AL, Scotland Alexander H. Purcell (391), Department of Environmental Science, Policy, and Management, University of California at Berkeley, Berkeley, California 94720 Shmuel Razin (25, 47, 431), Department of Membrane and Ultrastructure Research, The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel Janet A. Robertson (189), Department of Medical Microbiology and Infectious Diseases, University of Alberta, Edmonton, Alberta, Canada T6G 2H7 R. F. Rosenbusch (311), Veterinary Medical Research Institute, College of Veterinary Medicine, Iowa State University, Ames, Iowa 50011 Richard F. Ross (275, 371), College of Veterinary Medicine, Iowa State University, Ames, Iowa 50011 H. Louise Ruhnke (255, 377), Mycoplasma Laboratory, Veterinary Laboratory Services, Ontario Ministry of Agriculture and Food, Guelph, Ontario, Canada NIH 6R8 C. Saillard (293, 385), Laboratoire de Biologic Cellulaire et Moleculaire, Institut National de Recherche Agronomique, F-33883 Villenave d'Ornon, France E. Seemueller (299), Biologische Bundesanstalt fur Land- und Forstwirtschaft, Institut fur Pflanzenschutz im Obstbau, D-69216 Dossenheim, Germany Ann Smith (445), Cantab Pharmaceutical and Research Ltd., Cambridge CB4 4GN, England Gerald W. Stemke (275), Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9 David Taylor-Robinson (61, 147, 199, 225, 337), MRC Sexually Transmitted Diseases Research Group, Department of Genitourinary Medicine, St. Mary's Hospital Medical School, Paddington, London W2 INY, United Kingdom Ed A. ter Laak (255), Department of Bacteriology, DLO Institute for Animal Science and Health, 8200 AB Lelystad, The Netherlands David Thirkell (151), Division of Cell and Molecular Biology, School of Biological and Medical Sciences, University of St. Andrews, St. Andrews, Fife KYI6 9AL, Scotland Chester B. Thomas (137), Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin, Madison, Wisconsin 53706 Joseph G. Tully (1, 89, 169, 207, 325, 407, 411), Mycoplasma Section, Laboratory of Molecular Microbiology, National Institute of Allergy and Infectious Diseases, Frederick Cancer Research and Development Center, Frederick, Maryland 21702 Connie Veilleux (431), Genentech, Inc., South San Francisco, California 94080 Ken B. Waites (237), Departments of Pathology, Microbiology, and Rehabilitation Medicine, Division of Laboratory Medicine, University of Alabama at Birmingham, Birmingham, Alabama 35233 Leigh Rice Washburn (349), Department of Microbiology, University of South Dakota School of Medicine, Vermillion, South Dakota 57069 R. F. Whitcomb (293, 313, 385), Insect Biocontrol Laboratory, Plant Sciences Institute, United States Department of Agriculture, Agricultural Research Service, BARC, Beltsville, Maryland 20705 J. Williamson (211), Department of Microbiology, Royal Hobart Hospital, Hobart, Tasmania 7000, Australia

XIV

Contributors

A. Yancey (327), Department of Microbiology, University of Alabama at Birmingham, Birmingham, Alabama 35294 David Yogev (47), Department of Membrane and Ultrastructure Research, The Hebrew University-Hadassah Medical School, Jerusalem 91120, Israel Richard Yuan-Hu Wang (115), Department of Infectious and Parasitic Disease Pathology, Division of Molecular Pathology, American Registry of Pathology, Armed Forces Institute of Pathology, Washington, District of Columbia 20306

Preface

The two volumes of Methods in Mycoplasmology published by Academic Press in 1983 have gained wide recognition as the most comprehensive and authoritative treatise on mycoplasma methodology, and are highly cited in the mycoplasma literature. These volumes have provided researchers and laboratory workers with well-tried and standardized procedures for the recovery, identification, and characterization of mycoplasmas. The developments in mycoplasmology which have taken place since the publication of these volumes have been outstanding due mainly to the application of molecular genetic methodology to mycoplasmas. Introduction of this methodology has had a significant impact on our understanding of the cell structure, genetics, metabolism, taxonomy, and phylogeny of mycoplasmas, as well as of the mechanisms of pathogenicity and the interaction of mycoplasmas with the immune system. These advances have found expression in the development of new diagnostic procedures, including those based on DNA probes and DNA amplification. As could be expected, significant developments have also taken place in the more "classical" procedures, those dealing with the cultivation, serological characterization, and pathogenicity testing of mycoplasmas. The two volumes of Molecular and Diagnostic Procedures in Mycoplasmology focus on the new procedures developed during the past decade, particularly those based on the new molecular methodology. This volume deals with the new genetic and immunological tools applied to the diagnosis of mycoplasma infections of humans, animals, plants, insects, and cell cultures. Volume I outlines the approaches, techniques, and procedures applied to cell and molecular biology studies of mycoplasmas. We are well aware that techniques outlined for rapidly moving subdisciplines may soon become dated. Yet experience gained through the use of the Methods in Mycoplasmology volumes confirms that the majority of methods detailed in the new volumes will continue to be useful for years to come. We thank our colleagues who were most helpful at the initial stages of selecting the topics and procedures to be covered and in developing the volume outlines. Considering the large number of chapters and contributors, keeping to the deadlines set by the publisher can be considered an outstanding achievement. Obviously, this could not have been accomplished without the cooperation of the many contributors. We express our gratitude and appreciation for their friendly cooperation in this endeavor. Shmuel Razin Joseph G. Tully

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Contents of Volume I Molecular Characterization

Molecular Properties of Moliicutes: A Synopsis Shmuel Razin SECTION

A

CULTIVATION AND MORPHOLOGY A1 Introductory Remarks Joseph C. Tully A2 Culture Medium Formulation for Primary Isolation and Maintenance of Moliicutes Joseph G. Tully A3 Cultivation of Spiroplasmas in Undefined and Defined Media Kevin J. Hackett and Robert F. Whitcomb A4 Insect Cell Culture Approaches in Cultivating Spiroplasmas Kevin j. Hackett and Dwight E. Lynn A5 Measurement of Mollicute Growth by ATP-Dependent Luminometry Janet A. Robertson and Gerald W. Stemke A6 Intracellular Location of Mycoplasmas David Taylor-Robinson A7 Localization of Mycoplasmas in Tissues Douglas j. Wear and Shyh-Ching Lo A8 Localization of Antigens on Mycoplasma Cell Surface and Tip Structures Duncan C. Krause and Maria K. Stevens

SECTION

B

GENOME CHARACTERIZATION AND GENETICS B1 Introductory Remarks Shmuel Razin B2 Isolation of Mycoplasma-like Organism DMA from Plant and Insect Hosts Bruce C. Kirkpatrick, Nigel A. Harrison, Ing-Ming Lee, Harold Neimark, and Barbara B. Sears B3 Mollicute Chromosome Size Determination and Characterization of Chromosomes from Uncultured Moliicutes Harold Neimark and Patricia Garle

Contents of Volume I

XVIII

B4 Physical and Genetic Mapping Thomas Proft and Richard Herrmann B5 Characterization of Virus Genomes and Extrachromosomal Elements Kevin Dybvig B6 Plasmid and Viral Vectors for Gene Cloning and Expression in Spiroplasma citri ). Renaudin and J. M. Bove B7 Artificial Transformation of Mollicutes via Polyethylene Glycol- and Electroporation-Mediated Methods Kevin Dybvig, Gail E. Gasparich, and Kendall W. King B8 DNA Methylation Analysis Aharon Razin and Paul Renbaum B9 Identification and Characterization of Genome Rearrangements Bindu Bhugra and Kevin Dybvig B10 Expression of Mycoplasmal Genes in Escherichia coli Paul Renbaum and Aharon Razin

SECTION C

MEMBRANE CHARACTERIZATION CI C2

C3 C4 C5 C6

SECTION D

Introductory Remarks Shmuel Razin Posttranslational Modification of Membrane Proteins Ake Wieslander, Susanne Nystrom, and Anders Dahlqvist Variant Membrane Proteins Kim S. Wise, Mary F. Kim, and Robyn Watson-McKown Membrane Fusion Shiomo Rottem and Mark Tarshis Mycoplasma Membrane Potentials Ulrich Schummer and Hans Gerd Schiefer Ion Flow and Cell Volume Shiomo Rottem

CELL METABOLISM D1 Introductory Remarks Shmuel Razin D2 Methods for Testing Metabolic Activities in Mollicutes j. Dennis Pollack

Contents of Volume I D3 Rapid Microcalorimetric and Ele troanalytical Measurements of Metabolic Activities R. j. Miles D4 Characterization of Heat Shock Proteins Christopher C. Dascher and jack Maniloff D5 Nucleolytic Activities of Mycoplasmas F. Chris Minion and Karalee j. jarvill-Taylor D6 Proteolytic Activities Tsuguo Watanabe and Ken-ichiro Shibata D7 Phospholipase Activity in Mycoplasmas Shiomo Rottem and Michael Salman

SECTION E

TAXONOMY AND PHYLOGENY El Introductory Remarks Shmuel Razin E2 Minimal Standards for Description of New Species of the Class Mollicutes Joseph C. Tully and Robert F. Whitcomb E3 Ribosomal RNA Sequencing and Construction of Mycoplasma Phytogenies Williann C. Weisburg E4 Restriction Endonuclease Analysis Shmuel Razin and David Yogev E5 Southern Blot Analysis and Ribotyping David Yogev and Shmuel Razin E6 Phylogenetic Classification of Plant Pathogenic Mycoplasma-like Organisms or Phytoplasmas Bernd Schneider, Frich Seemueller, Christine D. Smart, and Bruce C. Kirkpatrick E7 Determination of Cholesterol and Polyoxyethylene Sorbitan Growth Requirements of Mollicutes Joseph C. Tully

SECTION F

PATHOGENICITY F1 Introductory Remarks Shmuel Razin F2 Mycoplasma Adherence to Host Cells: Methods of Quantifying Adherence Itzhak Kahane and Fnno Jacobs F3 Mycoplasma Adherence to Host Cells: Epitope Mapping of Adhesins Fnno Jacobs

XIX

XX

Contents of Volume I F4 Oxidative Damage Induced by Mycoplasmas Itzhak Kahane F5 Activation of Macrophages and Monocytes by Mycoplasmas Ruth Gallily, Ann Avron, Gerlinde jahns-Streubel, and Peter F. Muhlradt F6 Identification, Characterization, and Purification of Mycoplasmal Superantigens Barry C. Cole and Curtis L Atkin F7 Mycoplasmal B-Cell Mitogens Yehudith Naot F8 Modulation of Expression of Major Histocompatibility Complex Molecules by Mycoplasmas P. Michael Stuart and Jerold G. Woodward F9 Interaction of Mycoplasmas with Natural Killer Cells Wayne C. Lai and Michael Bennett Index

MOLLICUTE-HOST INTERRELATIONSHIPS: CURRENT CONCEPTS AND DIAGNOSTIC IMPLICATIONS Joseph G. Tully

Introduction For almost four decades after their discovery, mollicutes (then called pleuropneumonia or pleuropneumonia-like organisms) were considered to be associated only with animal hosts as pathogens or commensals. The development of such a concept was reasonable when one examines the historical reports from the time of the cultivation of the first mollicute in 1898, the agent of contagious bovine pleuropneumonia, to the discovery of other mollicutes in other animal hosts (sheep, goats, rodents, dogs, and poultry) in the era between 1920 and 1937. As would have been expected, the report of a mollicute in the lower genital tract of a human female (Dienes and Edsall, 1937; Dienes, 1940) had a major impact on the ideas of host relationships of these organisms. However, the presence of competitive bacterial flora in humans and in many of the animal tissues being examined at the time was a major obstacle to further isolation and characterization of the mollicute flora of such hosts. The development of improved culture media, containing bacterial inhibitors for both gram-positive and gram-negative bacteria (Edward, 1947), considerably advanced the ability to cultivate and identify mollicutes from primary host tissues (Smith and Morton, 1951). This major technical accomplishment, coupled with the development of modified culture medium formulations and cultivation attempts on a variety of

Molecular and Diagnostic Procedures in Mycoplasmology, Vol. II

Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

2

Joseph G. Tully

different hosts and tissues, including plants and invertebrates, has resulted in a continual expansion in the numbers of known mollicute species. At this writing, more than 158 mollicute species have been formally described, with possibly a dozen or more candidates in waiting. The known host range of mollicutes currently includes humans, nonhuman primates, a variety of domestic and wild animals and birds, and a large and expanding number of plant and insect species. The individual host relationships extend from a prominent role as important primary or opportunistic pathogens to that of commensals or epiphytes, and some mollicutes might also exist as saprophytes. This introductory chapter attempts to provide a concise review of the known distribution of mollicutes, in which some current concepts on various levels of mollicute-host interrelationships and application of this information to current approaches to laboratory diagnosis of mollicute infections are discussed. More extensive coverage and documentation of historical developments and the epidemiological data discussed here can be found in the five-volume series of The Mycoplasmas (Barile and Razin, 1979; Tully and Whitcomb, 1979; Whitcomb and Tully, 1979, 1989; Razin and Barile, 1985), two earlier mycoplasma methodology volumes (Razin and Tully, 1983; Tully and Razin, 1983), and selective reviews, symposia, or books (Tully, 1985; Razin, 1991; Tully and Whitcomb, 1991; Maniloff er a/., 1992; Kahane and Adoni, 1993; Cassell, 1993; Whitford et aL, 1994; Blanchard and Montagnier, 1994).

Acquisition and Transmission Mechanisms of Mollicutes As with many other biological entities, acquisition of mollicutes can be associated with primary transmission involving direct contact of host with host or transfer mediated through secondary means, such as aerosols or fomites, food and/or water, insect vectors or other carriers, and nosocomial acquisition (e.g., organ or tissue transplants). This discussion considers only broad areas of mollicute acquisition since more detailed information on the means by which mollicute species might be acquired and disseminated are covered later or in other sections of the volume. Many mollicutes are inhabitants of the mucous membranes of the respiratory, urogenital, or gastrointestinal tracts of vertebrates, so that direct host to host transmission of organisms in man and in many animals occurs through oral to oral, genital to genital, or oral to genital contact. Certain mollicutes that are part of the normal flora of the oropharynx and lower genital tract are most likely acquired by oral to oral or genital to genital contact. However, changes in sexual mores have resulted in apparent alterations in the host tissue location of mollicutes, so that some mollicutes commonly found in the oropharynx can occur in the urogenital tract and vice versa (See below). In some predatory insects, the

Mollicute-Host Interrelationships

3

organisms are acquired directly by eating other colonized or infected hosts. Direct host to host acquisition of mollicutes, particularly Ureaplasma urealyticum, has also been documented in neonates through in utero infection (see Chapter D4, this volume). Secondary transmissions of mollicutes are probably acquired most frequently through respiratory aerosols or fomites, as evidenced by the wide occurrence of mollicutes as etiologic agents of the many acute respiratory diseases of man and animals. Some of the normal commensal mollicute flora of the oropharynx of man and animals also may be acquired by this means. Food (and possibly water) obtained through feeding excursions plays a major role in the acquisition of mollicutes by insects, where the organisms are present in plant sap or nectar, or where such materials are contaminated with other insect-derived excretions. Food also plays a major role in dissemination, colonization, and transfer of nonpathogenic or infectious animal mollicutes from adult to the young through mammary (milk) transmission. In the transmission of mollicute diseases (citrus stubborn, com stunt, and phytoplasma infections) from plant to plant, insect vectors play a predominant role. These transmissions usually involve a clearly defined biological cycle within the insect, concerning acquisition, multiplication in various tissues (adipose tissue, salivary gland, etc.), and eventual reinoculation into susceptible plants. Secondary transmission of mollicutes can also occur through healthy carriers of the organisms or through convalescent carriers recovering from acute infections, as apparently occurs in some Mycoplasma pneumoniae respiratory infections in humans and probably in other acute and chronic respiratory diseases in domestic animals. Infected carriers also play an important role in the transmission of Spiroplasma melliferum and S. apis infections from foraging bees to their hives. Also, mollicute transmission after plant grafting of infected tissue to healthy rootstock, or through human organ or tissue transplantation, has been described. The presence of mollicutes in semen is important in sexually transmitted diseases in a variety of hosts and has become a critical complication in artificial insemination programs in humans and bovines (see Chapters D3, D6, and E7, this volume). Contamination of semen has had important consequences, including transmission of mollicutes and mollicute infections to other hosts, direct detrimental effects on successful host fertilization, and introduction of both pathogenic and nonpathogenic mollicutes to other geographic areas.

Host Colonization versus Host Infection with Mollicutes Since many mollicutes possess the ability to adhere to epithelial surfaces, any mechanism that promotes the direct or indirect exposure of the organism to susceptible tissue sites (e.g., respiratory, urogenital, and gastrointestinal tissues)

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Joseph G. Tully

readily results in colonization. Adherence mechanisms are well developed in many of the pathogenic mollicutes with organized attachment structures and with specific adhesin proteins on their exterior membrane surface (see the introductory chapter and Chapters F2 and F3 in Vol. I). However, other mollicutes, particularly those considered to be commensals in the oropharynx and urogenital tracts of humans and other animals, also possess mechanisms for cell adherence without well-defined tip structures or established adhesin proteins. The cytadherence mechanism for such mollicutes appears to involve the presence of specific glycolipid and/or glycoprotein receptors on the eukaryotic host cell surface (Razin and Jacobs, 1992). Early studies have shown that the human upper respiratory and urogenital tracts of human newborns are first colonized with mollicutes acquired during passage through the female birth canal. However, as noted earlier, there is now substantial evidence that, on a rare occasion, mollicute colonization can occur in utero, again possibly related to transmission or acquisition of mollicutes (M. hominis and U. urealyticum) in the lower female urogenital tract at the time of fertilization or during early pregnancy. In the usual situation, neonatal colonization with both human oral and genital mollicutes generally does not persist, and the number and type of mollicutes decrease with age. However, welldocumented studies have shown that subsequent reacquisition of a urogenital mollicute flora in humans is directly related to sexual contacts. Likewise, it is assumed that oropharyngeal commensals are reacquired later through oral to oral transmission. Less information is available on the mechanisms and timing of mollicute colonization of newborn vertebrates, including birds, and insects or other arthropods. In vertebrates, acquisition from the maternal genital tract, and in utero transmission (including egg transmission in the case of birds), apparently represents the initial mechanism. Colonization of arthropod and insect hosts probably occurs most frequently during feeding excursions on plant surfaces, flowers, and on nectar (Hackett and Clark, 1989). However, transovarian transmission of spiroplasmas has been well established in Drosophila species (Williamson and Poulson, 1979). The normal persistent mollicute colonization of major anatomical sites in humans and numerous animal hosts has created major complications in efforts to define a relationship between organism and disease. To avoid such complications in experimental studies, investigators have utilized gnotobiotic or specific pathogen-free (SPF) hosts that are defined as free of mollicutes (see chapters in Section E, this volume). However, while experimental infections in a variety of large and small animal hosts have helped to establish the pathogenicity of many host-restricted mollicutes for the respiratory tract, difficulties have not been overcome in producing and maintaining large gnotobiotic or SPF animals free of urogenital tract mollicutes.

Mollicute-Host Interrelationships

5

As with many other microbe-host interrelationships, mollicutes can be found on the mucosal surface as part of the normal flora or the host may acquire a pathogenic species able to colonize, invade host tissue, and eventually produce tissue damage. Some of the mechanisms that are thought to be involved in mollicute pathogenicity are outlined in the introductory chapter in Vol. I. Host factors also play an important and complex role in determining whether an acquired mollicute behaves as a pathogen or is avirulent. Although most of these factors are not well defined or understood at this time, it is clear that endogenous mollicutes can become pathogenic when the human host immune system has been compromised or when surgical intervention or trauma allows normal mollicute commensals to enter the circulation or other host tissues. In these occurrences, such common commensals as M. hominis, M. salivarium, and U. urealyticum (Furr et al., 1994; Gelfand, 1993; Meyer and Clough, 1993) have been shown to induce septicemia and overt mollicute invasion of various host tissues and organs, with joint localization being especially prominent.

Host Specificity of Mollicutes One of the hallmarks of the mollicute-host relationship recognized by early workers was the remarkable host specificity of the organisms. The earliest isolates found in bovine pleuropneumonia had not at the time been isolated from goats or sheep, rodents, or from other animal or human hosts examined shortly thereafter. Although this striking host specificity has for the most part remained a characteristic feature of mollicutes, exceptions have been discovered as new mollicutes were isolated and the known host range expanded. Strains of M. arginini, M. canis, and many established Acholeplasma species have been found to occur in a wide variety of different animal hosts. Only a few examples of some recent unusual host relationships will be noted here since the earlier references and textbooks mentioned can provide additional background. Several other reports further support the apparent transmission of animal mollicutes to humans. The few earlier reports of the possible transmission of M. arthritidis and M. canis to humans have been reviewed (Armstrong et al, 1971; Tully, 1993). Of more immediate concern are instances where serious clinical infections in humans have occurred, including those with a fatal outcome, from apparently animal-derived mollicutes. McCabe and associates (1987) described a mycoplasmal infection in the hand of a veterinarian acquired through a cat bite. The infection resulted in severe soft tissue cellulitis with tissue destruction sufficient to require a tendon graft. The organism (strain M7806) was identified at the time as an unclassified, glucose-fermenting Mycoplasma species. Although other serologically related strains were later isolated from the oropharynx of felines, the

6

Joseph G. Tully

organism remains unclassified. Yechouron and colleagues (1992) presented observations on a fatal human infection with M. arginini. This organism was repeatedly isolated from the blood and bronchial washings of the patient during the course of the infection. The fact that the patient had advanced Hodgkin's disease and a marked immunodeficiency obviously contributed to the invasion of the organism and to the inability to control the infection with appropriate chemotherapy. Although the source of the infection was not determined through epidemiologic or occupational studies, the most likely acquisition was thought to have involved aerosol transmission during patient employment in a large animal slaughterhouse. Examples of the possible reverse transfer of mollicutes of human origin to animal hosts, and the somewhat less unusual cross transfer of mollicutes among certain animal hosts, form another part of the picture of changes in host specificity of mollicutes. M. salivarium, a species usually confined to humans or other primates, was repeatedly isolated from nasal and pharyngeal secretions of swine (Erickson et al., 1988). Mycoplasmas usually found in avian and bovine hosts have been reported to occur in swine (Taylor-Robinson and Dinter, 1968), and two mollicutes primarily of rodent origin, M. arthritidis andM. collis, have been isolated from synovial fluids of arthritic swine (Binder et al., 1990). Furthermore, the demonstration of a large number of isolates of a canine mycoplasma from the respiratory tract of calves also appears to indicate some changes in the usual host relationships. More than 40 mycoplasma strains were isolated from the lungs of calves in 19 herds in The Netherlands (ter Laak et al., 1993). Initially, these strains were thought to be new species since extensive serologic testing did not indicate that they were related to any established mollicute. However, subsequent investigations with other serologic techniques (agar plate immunofluorescence tests) and a comparison of restriction endonuclease patterns to mollicutes with identical DNA base composition (G + C) values indicated the likelihood that these strains were closely related to M. canis. The failure of initial tests to detect the serologic relationship was thought to be due to the extensive serologic heterogeneity among M. canis strains, first described more than 40 years earlier. Retrospectively, it was also determined that M. canis strains had been identified in the respiratory tract of calves in Canada as early as 1974 (H. L. Ruhnke and J. G. Tully, unpublished studies). The presence of mollicutes in plants and insects provides an opportunity for their possible transfer to human or other animal hosts and for the reverse transmission to occur. The isolation and identification of an avian mollicute (M. iowae, strain PPAV) from apple seeds has become not only an example of a mollicute in association with an unusual host transfer, but a classic demonstration of the application of 16S rDNA gene sequence analysis in mollicute identification. Details of the isolation and earlier difficulties in identification of the organism, including an extensive serological analysis, have been described (Grau et al, 1991; Tully, 1989). Again, the initial failure to detect the apparent

Mollicute-Host Interrelationships

7

serologic relationship was thought to relate to serologic heterogeneity among strains of M. iowae. In efforts to explore possible genetic relationships of PPAV to other mollicutes, the full 16S rDNA was sequenced. A search of gene data banks showed a difference of only five bases in the total 16S rDNA of PPAV and the type strain (695) of M. iowae. DNA/DNA hybridization, genome size comparisons, and other molecular and phenotypic comparisons later confirmed the identification of PPAV as M. iowae. An explanation of how this avian moUicute apparently occurred in seeds removed from an apple under aseptic isolation techniques is not readily apparent. Another example of possible transmission from animal host to plant was uncovered in the case of the plant-derived F5 strain (J. C. Vignault, C. Saillard, P. Carle, J. M. Bove, D. L. Rose, and J. G. Tully, unpubUshed studies; Tully, 1989). This nonhelical, sterol-requiring organism was isolated from a fieldcollected wild lettuce plant near Bordeaux, France in 1981. It was characterized extensively in comparative studies with other plant/insect mollicutes and appeared to be distinct from all other mollicutes in that habitat. However, tests to define an optimal growth temperature indicated the strain grew best at 37°C. Subsequent reciprocal serologic tests (growth inhibition and epi-immunofluorescence) with antisera to then current Mycoplasma species indicated that strain F5 was closely related to the T37 strain of M. equigenitalium. A comparison of genomic properties and biochemical activities of strains F5 and T37 (G + C of 31.0 mol%, genome of 880 kbp, positive glucose fermentation, negative arginine hydrolysis, positive film and spot reaction) provided additional confirmation of this identification. Since M. equigenitalium is a normal inhabitant of the equine genital tract, it was speculated that the most likely source for plant contamination by this mollicute was equine urine or other urogenital-derived material.

Habitat and Ecology of Mollicutes Distribution in Humans and Nonhuman Primates

Mycoplasmas, ureaplasmas, and a few acholeplasmas have been isolated from the oropharynx and lower urogenital tract of most humans (Table I) (Tully, 1993). The first five species listed in the table are considered to be representative of the normal human oropharyngeal flora. M. salivarium appears to be the most frequent mollicute in the oral cavity, with incidences of 60-80% in various adult populations, whereas M. orale can usually be found in 30-60% of adult throats. The other human oropharyngeal mollicutes are isolated only infrequently. The occurrence of M. salivarium in the human genital tract has been reported twice, and more than 20 isolates of M. pneumoniae were cultivated from cervical

Joseph G. Tully

TABLE I PRIMARY AND SECONDARY HUMAN TISSUE SITES OR ORGANS COLONIZED OR INFECTED WITH MOLLICUTES"

Species

Primary site

Mycoplasma salivarium M. orale

Oropharynx Oropharynx

M. buccale M. faucium M. lipophilum M. pneumoniae

Oropharynx Oropharynx Oropharynx Oropharynx; lung; pleural fluid; bronchoalveolar lavage fluid

M. genitalium

Urogenital tract; oropharynx Lower genital tract; oropharynx

M. hominis

M. fermentans

Lower genital tract; respiratory tract

M. primatum M. spermatophilum M. pirum

Female urethra Cervix/sperm Peripheral blood cells? ?

M. penetrans Ureaplasma urealyticum

Acholeplasma laidlawii A. oculi

Genital tract; oropharynx; bronchoalveolar lavage fluid; placenta 7 7

Secondary site Cervix/vagina; arthritic joint Leukemic bone marrow; lymph node and skin (in sarcoidosis)

Arthritic joints; skin lesions (in StevensJohnson syndrome; middle ear fluid; cerebrospinal fluid; tuboovarian abscess; cervix and vagina; pericardial fluid; heart blood, kidney, and brain at autopsy Synovial fluid in arthritic joint Lung and pleural effusion; blood in postpartum septicemia; prostheses infections; organ and tissue transplant infections; trauma/surgical site infections; malignancies; cerebrospinal fluid; peritoneum; synovial fluid (arthritis); skin; pericardium; amniotic fluid; neonatal septicemia Leukemic bone marrow; arthritic joints; peripheral blood lymphocytes in acquired immunodeficiency syndrome (AIDS); urine in AIDS Umbilicus Peripheral blood lymphocytes Urine (male) in AIDS Lung and lower respiratory tract (in pneumonia); blood in neonatal septicemia; cerebrospinal fluid; transplants; surgical sites, arthritic sites; renal calculi; amniotic fluid; postpartum septicemia; neonatal lung and brain Oropharynx; bum infections; vagina Amniotic fluid

"Modified from Tully, J. G. (1993). Current status of the mollicute flora of humans. Clin. Infect. Dis. 17 (Suppl. 1), S2-S9, by permission of the University of Chicago. Copyright © 1993 The University of Chicago Press.

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9

specimens of women attending various gynecological clinics in Canada (Goulet et al., 1995). The implications of these reports is that transmission had occurred through oral-genital contact. M. hominis and U. urealyticum are the most frequently encountered mollicutes in the lower urogenital tract of humans, and their recurrence and incidence after puberty are correlated with increasing sexual contact, especially to the number of sexual partners (McCormack et al, 1973). Rates of M. hominis colonization are lower in men than in women; in a survey of women attending a sexual disease clinic, a colonization rate as high as 94% was recorded (TuUy et al., 1983). The clinical roles that M. hominis and U. urealyticum play in human opportunistic infections, urogenital disease, and neonatal infections, and their laboratory detection, are discussed in Chapters D3 and D4 of this volume. The other major pathogens in the group, or those currently considered to have an association with human disease, include M. pneumoniae, M. fermentans, M. genitalium, and M. penetrans (for a discussion of individual species, see Chapters D2 through D5, this volume). M. pirum, which was first found as an in vitro cell culture contaminant (see chapters in Section F, this volume), has been isolated from human peripheral blood cells and is now tentatively considered to be of human origin (Blanchard and Montagnier, 1994) (see also Chapter D5, this volume). Although few isolates of M. primatum or M. spermatophilum have been reported, they appear to be part of the human urogenital flora. The few acholeplasmas found in humans and nonhuman primates may represent the occasional transmission from other animal hosts since some acholeplasmas have been isolated regularly from domestic animals and birds and others from plants and insects (see Waites et al., 1987 and later discussion). The mollicute flora of a considerable number of nonhuman primates has been explored, primarily from the standpoint of their use as experimental models for pathogenicity studies on microbial agents, including mycoplasmas of human origin. An extensive listing of mollicute species identified within various nonhuman primates (Somerson and Cole, 1979) included M. hominis, M. fermentans, M. salivarium, M. orale, M. buccale, M. faucium, M. primatum, M. lipophilum, M. moatsii, M. canis, M. arthritidis, Acholeplasma laidlawii, and some ureaplasmas. Most of the isolates were from the oropharynx, but M. moatsii and some M. salivarium strains occurred in the urogenital tract (see also Hill, 1983). A new species described as M. indiense has been isolated from the throats of a rhesus monkey and a baboon (Hill, 1993). Distribution in Large Domesticated Animals BOVINES

Twenty-three species of mollicutes occur regularly in cattle, including 14 Mycoplasma, 3 Acholeplasma, 1 Ureaplasma, and 5 AnaeroplasmalAsteroleplasma species (Gourlay and Howard, 1983; Robinson and Freundt, 1987; see

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Joseph G. Tully

also Chapter D6, this volume). Most isolates are found in the upper respiratory tract, ocular sites, or the genital tract. Species isolated from mammary sites also are frequently found in respiratory or urogenital tracts. The obligate anaerobic anaeroplasmas and asteroleplasmas have been found only in the bovine rumen (Robinson and Freundt, 1987). The list of species found in bovines has changed only slightly since an earlier review (Gourlay and Howard, 1983) and, as noted at that time, several equine, feline, or avian mollicutes (M. equirhinis, M. galeae, M. gallisepticum, and M. gallinarum) also have been recovered from bovines (see also Lauerman, 1994; Jasper, 1994; Ruhnke, 1994). EQUINES

The equine mollicute flora is generally considered to consist of 11 species from urogenital or respiratory tracts. (M. equigenitalium, M. equirhinis, M. fastidiosum, M.felis, M. subdolum, A. equifetale, A. hippikon, A. laidlawii, A. modicum, A. multilocale, A. oculi and A. parvum), with occasional isolates of M. arginini, M. feliminutum, M. pulmonis, M. salivarium, A. axanthum, and A. granularum also being made from these sites in horses (Hill et aL, 1992; Whitford and Lingsweiler, 1994). CAPRINES/OVINES

Twenty mollicute species occur regularly in goats and sheep, including 16 Mycoplasma and 4 Acholeplasma species, with another 2 unclassified groups (strain 2D and related mycoplasmas, and a ureaplasma cluster) representing putative species. A list of species, including biologic characteristics and isolation sites, is given in Chapter D7 of this volume (see also Rosendal, 1994). Species of the obligately anaerobic genera Anaeroplasma or Asteroleplasma also occur in the rumen of sheep (Robinson and Freundt, 1987). PORCINES

The principal mollicute flora of swine include M. hyorhinis, M. hyopneumoniae, M. hyopharyngis, M. hyosynoviae, and M. flocculare, with occasional isolations of M. arginini, M. bovigenitalium, M. buccale, M. gallinarum, M. iners, M. mycoides subsp. mycoides, M. salivarium, M. sualvi, A. axanthum, A. granularum, A. laidlawii, and A. oculi, and some unclassified ureaplasmas or obligately anaerobic mollicutes (see Chapter D8, this volume; Ross and Whittlestone, 1983; Binder and Kirchhoff, 1988; Armstrong, 1994). Distribution in Small Domesticated Animals and Birds CANINES/FELINES

The canine mollicute flora consists of at least 14 species, including primarily M. canis, M. cynos, M. edwardii, M. maculosum, M. molare, M. opalescens.

Mollicute-Host Interrelationships

11

M. spumans, and U. canigenitalium, with occasional isolates of M. arginini, M. bovigenitalium, M. felis, M. feliminutum, M. galeae, A. laidlawii, an unclassified isolate (HRC 689), and several serologically distinct groups of ureaplasmas (Ogata, 1983; Whitford and Lingsweiler, 1994). Smaller numbers of mollicute species have been isolated from cats, with the principal flora consisting of M. felis, M. feliminutum, M. galeae, Ureaplasmafelinum, and U. call, and a currently unclassified group of mycoplasmas (M7806) (H. L. Ruhnke, personal communication). Other reported isolations include M. arginini, M. arlhrilidis, M. galliseplicum, and M. pulmonis (Ogata, 1983). SMALL LABORATORY ANIMALS

Mice and rats generally share a characteristic mollicute flora, consisting of M. arlhrilidis, M. collis, and M. pulmonis. Although mice also have both M. neurolylicum and M. muris as part of their normal flora, these two mollicutes so far have not been identified in rats (Cassell el ai, 1983; Davidson el al., 1994). Most of the mollicutes are maintained in the respiratory or urogenital tracts, but many also can be isolated from conjunctivae, middle ear, and brain. Guinea pigs harbor strains of M. caviae, M. cavipharyngis, A. cavigenilalium, A. granularum, and A. laidlawii (Hill, 1983), with most of the species occurring in the urogenital tract. Chinese hamsters are reported to have both M. criceluli and M. oxoniensis in the conjuctivae (Hill, 1991). Few mollicutes have been identified in rabbits, although earlier isolation of M. pulmonis was reported from rabbits housed near rodent colonies, and a new species (A. mullilocale) has been isolated from rabbit feces (Hill, 1992). AVIAN

The widespread occurrence and distribution of mollicutes in avian hosts is reviewed in Chapter D9 (this volume). Tv^tniy-i^o Mycoplasma species and U. gallorale have been identified in various birds, with many of the more recent isolates from birds of prey. Additional information on tissue locations of these mollicutes can be found in other reviews (Jordan, 1983; Kleven, 1994). Distribution in Wild Animals and Aquatic Hosts The occurrence of mollicutes in many types of domestic animals prompted questions about the possible distribution of the organisms in wild animals or aquatic hosts. Thus, a considerable mollicute flora has now been defined in a variety of wild animals, particularly those housed in zoos, as well as in several aquatic hosts. Some references to earlier isolations can be found in reports of the International Research Programme on Comparative Mycoplasmology (1990) or in a review (Razin, 1992), including M. cilelli from the trachea, lung, andhver of ground squirrels (Rose el al., 1978); M. mobile from a fresh water trench, and

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Joseph G. Tully

M. phocarhinis and M. phocacerebrale from the upper respiratory tract and tissues of harbor seals (Giebel et al., 1991). Another harbor seal isolate from lung and trachea also was described as M. phocidae (Ruhnke and Madoff, 1992). M. testudinis was isolated from the cloaca of a pet turtle (Hill, 1985) and M. mustelae from the lungs of mink (Salih et al, 1983). Previously unknown mollicute species have been found in many wild felines, including M. felifaucium from the respiratory tract of a puma (Hill, 1986), M. leopharyngis and M. simbae from the lion pharynx, and M. leocaptivus from the throats of both a lion and a leopard (Hill, 1992). Two additional moUicutes, apparently distinct from other established species, are currently being described as new species: a urogenital isolate from an elephant (H. Kirchhoff, personal communication) and several related strains isolated from the desert tortoise (M. Brown, personal communication). Distribution in Arthropods^ Including Insects Since the initial discovery of moUicutes in insect-transmitted phytoplasma plant diseases in 1967 and the isolation and characterization of the first helical mollicute {Spiroplasma citri) in citrus stubborn disease, insects and other arthropods have been a rich source of moUicutes. The organisms include numerous species and group representatives now assigned to the genus Spiroplasma and to an expanding number of species in the newly established genera of Entomoplasma and Mesoplasma (Tully et al., 1993). Since the information on this topic is also extensive, key references to work prior to 1991 can be found in earlier books and reviews (Whitcomb and Tully, 1979; Razin and Tully, 1983; Tully and Razin, 1983; Tully et al, 1987; Tully, 1989; Whitcomb and Tully, 1989; Williamson et al, 1989; Hackett et al, 1990; Tully et al, 1990; Tully and Whitcomb, 1991). The Appendix in this volume contains a list of currently recognized species and type strains of arthropod-associated moUicutes. In the case of spiroplasmas, the list includes group representatives that have not yet received species names. Spiroplasmas are found most frequently in the insect gut, less frequently in hemolymph, and occasionally in salivary glands. The organisms are generally acquired by natural feeding, from plant tissue, nectar, or insect excretions, or by ingestion of other insects. As noted earlier, at least one example of transovarial transmission of spiroplasmas (in Drosophila) is known. Most spiroplasmas are found in six evolutionarily advanced insect orders: Hymenoptera, bees and wasps; Coleoptera, plant- and flower-feeding beetles, firefly beetles, etc.; Diptera, flower-feeding flies, tabanids (horseflies), mosquitoes, fruit flies, etc.; Lepidoptera, butterflies; Homoptera, leafhoppers; and Hemiptera, green-leaf bugs. A serologically distinct spiroplasma (PALS-1) has been identified in the gut of a dragonfly, at this time the single representative from a primitive insect order (Odonata) found to harbor spiroplasmas. Two species {Spiroplasma mirum

Mollicute-Host Interrelationships

13

and S. ixodetis) have been isolated from tick genera (Haemaphysalts and Ixodes, respectively), but tissue localization within ticks and conditions involved in acquisition and maintenance are not well known. Some insect hosts of spiroplasmas function as important vectors in the transmission of plant or insect diseases. Leafhoppers infected with S. citri are the principal vectors for the transmission of citrus stubborn disease, not only among citrus plants but also among other infected plants or weeds and citrus. Likewise, leafhoppers infected with S. kunkelii are the principal vectors for the transmission of com stunt disease. Honeybees infected with either S. melliferum or S. apis are the major carriers and vectors for the transmission of lethal spiroplasma diseases to other honeybees or to their hives. Techniques for diagnosis of spiroplasmal infections in insects and experimental infections in such hosts are presented in Chapters DIO, D12, and E8 (this volume). Most nonhelical mollicutes of the genera Entomoplasma or Mesoplasma have been found as inhabitants of the insect gut. This situation may reflect an emphasis on the ecology of insect mollicutes. However, some entomoplasmas and mesoplasmas have now been identified also on plant surfaces (see later), and it is expected that most species in either group are acquired from various plant surfaces or flowers through insect visitation and feeding excursions. E. ellychniae was found in the hemolymph of a firefly, whereas E. somnilux, E. lucivorax, and E. luminosum appear to be gut inhabitants of other firefly species (Williamson et al., 1990; Tully et al., 1993). In contrast, E. melaleucae was first isolated from several species of subtropical flowers in 1979 and was only later found in a bee. The current distribution of Mesoplasma species, which were initially described as acholeplasmas (Tully et al., 1993), has been summarized (Tully et al., 1994b). Again, most isolates have been made from gut fluids of insect hosts, with a few isolations also from plant surfaces. The first species described in the group {Me. florum) was found on flower surfaces, but subsequent strains have been identified in the gut and hemolymph of beetles. Me. entomophilum is widely distributed among insects, occurring as part of the gut flora of more than nine different insect genera. The only occurrence of Me. seijfertii strains in insects has been reported in isolations made from the gut contents of Aedes mosquitoes and a Chrysops fly in France (Chastel et al., 1994). With the exception of Me. lactucae, the balance of the currently recognized Mesoplasma species (see Table IV in the Appendix) are inhabitants of a variety of insects (fireflies, horseflies, other beetles, bees, butterflies, etc.). The initial reports describing non-sterol-requiring mollicutes in insects as Acholeplasma species were eventually corrected when it was established that these mollicutes (now termed mesoplasmas) were phylogenetically distinct from classic acholeplasmas (Tully et al., 1993). Currently, conventional acholeplasmas have not been identified as part of the insect or arthropod flora. However, since at least four or five Acholeplasma species have been found on a

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Joseph G. Tully

variety of plant surfaces (see later), it is more than likely that these mollicutes also are part of the insect flora. Finally, phloem-feeding insects (mainly leafhoppers) are the primary hosts and vectors of a large collection of plant pathogenic mollicutes, including a few spiroplasmas and many phytoplasmas (see McCoy et aL, 1989; Chapter E6, in Vol. I; and Chapter E9, this volume).

Distribution in Plants

The largest group of mollicutes inhabiting plants is the currently uncultivable cluster of plant-pathogenic organisms now called phytoplasmas. These mollicutes are transmitted primarily by phloem-feeding leafhoppers and are the causative agents of more than 300 different plant "yellows" diseases. Genetic and taxonomic studies have indicated that these organisms have a close evolutionary relationship to the acholeplasmas and that DNA from more than 51 representative phytoplasmas can be placed into 12 distinct groups (see Chapter E6, Vol. I; Chapters DU and E9, this volume; and McCoy et al, 1989). Spiroplasma occurrence in plant hosts is based either on invasion of the plant sieve tubes in the course of a biological cycle involving the feeding activities of homopterous insects (leafhoppers) or as external contamination of floral parts from deposition by flower-visiting insects. Only spiroplasma invasion into plant sieve tubes results in disease, as is seen in various citrus plant diseases with S. citri, in com stunt disease with S. kunkelii, and in the so-called periwinkle disease due to infections with S. phoeniceum (see Chapters DIO and E8, this volume). Numerous other Spiroplasma species (or groups) have been isolated from flower surfaces and nectar, but no evidence is currently available that these organisms also occur within internal plant tissues (see Table V in Appendix). However, some normal flower-inhabitating spiroplasmas acquired by specific insects function as pathogens, as in the case of S. apis strains acquired by honeybees. The only isolations of Entomoplasma species from plants so far have come from tree flowers; these include flowers of two species of the genus Melaleuca and the silk oak (Grevillea sp.) in Florida. Tropical flowers (grapefruit, lemon, and powder puff plant) have also been the source of three strains of Me. florum. This organism and Me. entomophilum occur frequently in a variety of insects, which explains their repeated isolation from various wild plants (aster, sunflower, gumweed, goldenrod, etc.) in Colorado and Nebraska. The remaining mesoplasmas have not been found often in plant hosts, which also appears to be reflected in a limited number of isolations of these organisms from insects. The reported isolations include Me. seiffertii from flowers of a sweet orange tree in Morocco and wild angelica in France, Me. lactucae from lettuce, and Me. coleopterae from flowers of the vera dulca tree in Texas.

Mollicute-Host Interrelationships

15

The occurrence of classic acholeplasmas on plant surfaces is somewhat enigmatic since none of the acholeplasmas identified so far has been isolated from insects. A. axanthum has been identified on vegetable plants, on wild plants (mesquite, Prunus sp., creosote bush) in the southwestern United States, and on crown tissues of coconut palms with lethal yellowing disease (phytoplasma) in Jamaica. Both A. oculi and A. palmae have been isolated from similar coconut palm crown tissues in Jamaica, and A. laidlawii and A. brassicae occur as surface contaminants on vegetables (Tully et al., 1994a).

Habitat Consideration and Diagnostic Laboratory Approaches One of the most interesting undercurrents running through the foregoing discussion is the possibility that mollicutes might be able to cross species barriers much more frequently than had been expected from earlier experiences. Whether such breaks represent actual changes in host relationships or the recognition of a few mollicutes that might be less host-specific than others is still uncertain. However, experiences with unusual host transfers suggest that it would now seem prudent to develop broader approaches for laboratory diagnostic techniques for mollicutes that would emphasize rapid differentiation to the genus level. After a preliminary screening and separation, other conventional techniques (serological, etc.) could then be employed for a more critical differentiation to the species level. A forward step in this direction has been outlined in a discussion of diagnostic procedures for arthropod mollicutes (Chapter D12, this volume). This approach is based on the description of new taxonomic groups of mollicutes identified in plant and insect hosts (Tully et al., 1993; see also Chapter E2, Volume I). The following discussion expands this idea to mollicutes of human and animal origin and details practical techniques required for final identification. Since the obligate anaerobic mollicutes (members of the genus Anaeroplasma and genus Asteroleplasma) require specialized equipment for isolation and growth, and the phytoplasmas are presently uncultivable, these organisms are not considered here. A revision of recommended techniques for describing new mollicutes will provide additional references for methods discussed next (International Committee on Systematic Bacteriology, 1995). A schematic approach to the differentiation of mollicute genera is presented in Table II. A preliminary requirement is that the organism to be identified can be cultivated in some type of broth and agar media. Growth in liquid medium will vary from heavy to light, but large amounts of sediment should raise suspicions of a bacterial agent. Several mollicute species require anaerobic conditions for growth in liquid medium, so initial attempts at cultivation should include this environment. Classic fried egg-type agar colonies are frequently not observed

16

Joseph G. Tully TABLE II SCHEMATIC APPROACH TO LABORATORY DIFFERENTIATION OF MAJOR GENERA OF CLASS MOLLICUTES

A. Preliminary characteristics to be determined Growth on hquid and solid medium (preliminary biochemical properties) Purification by filtration cloning (3 X) (450-nm membrane filters) Filterable through 450-, 300-, 220-, and 100-nm membrane filters Growth in presence of penicillin and failure to revert to bacterial-type growth when grown in absence of antibiotics B. Morphology (dark-field microscopy or negative stain with electron microscopy) Helical > Genus Spiroplasma Nonhelical > Other moUicute C. Growth in serum-free medium < ^ > Genus Acholeplasma Positive Negative ^ Other moUicute D. Growth in serum-free medium containing 0.04% Tween 80 « —" Positive > Genus Mesoplasma Negative —» Other moUicute E. Optimal growth temperature