Preparedness Against Bioterrorism And Re-Emerging Infectious Diseases (Nato Science Series I:Life and Behavioural Sciences)

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PREPAREDNESS AGAINST BIOTERRORISM AND RE-EMERGING INFECTIOUS DISEASES

NATO Science Series A series presenting the results of scientific meetings supported under the NATO Science Programme. The series is published by IOS Press and Kluwer Academic Publishers in conjunction with the NATO Scientific Affairs Division. Sub-Series I. II. III. IV. V.

Life and Behavioural Sciences Mathematics, Physics and Chemistry Computer and Systems Sciences Earth and Environmental Sciences Science and Technology Policy

IOS Press Kluwer Academic Publishers IOS Press Kluwer Academic Publishers IOS Press

The NATO Science Series continues the series of books published formerly as the NATO ASI Series. The NATO Science Programme offers support for collaboration in civil science between scientists of countries of the Euro-Atlantic Partnership Council. The types of scientific meeting generally supported are "Advanced Study Institutes" and "Advanced Research Workshops", although other types of meeting are supported from time to time. The NATO Science Series collects together the results of these meetings. The meetings are co-organized by scientists from NATO countries and scientists from NATO's Partner countries - countries of the CIS and Central and Eastern Europe. Advanced Study Institutes are high-level tutorial courses offering in-depth study of latest advances in a field. Advanced Research Workshops are expert meetings aimed at critical assessment of a field, and identification of directions for future action. As a consequence of the restructuring of the NATO Science Programme in 1999, the NATO Science Series has been re-organized and there are currently five sub-series as noted above. Please consult the following web sites for information on previous volumes published in the series, as well as details of earlier sub-series: http://www.nato.int/science http://www.wkap.nl http://www.iospress.nl http://www.wtv-books.de/nato_pco.htm

Series I: Life and Behavioural Sciences - Vol. 357

ISSN: 1566-7693

Preparedness Against Bioterrorism and Re-Emerging Infectious Diseases Edited by Janusz Kocik Military Institute of Hygiene and Epidemiology, Warsaw, Poland

Marek K. Janiak Military Institute of Hygiene and Epidemiology, Warsaw, Poland

and Marian Negut Cantacuzzino Institute, Bucarest, Romania

IOS

Press Amsterdam • Berlin • Oxford • Tokyo • Washington, DC Published in cooperation with NATO Scientific Affairs Division

Proceedings of the NATO Advanced Research Workshop on Preparedness Against Bioterrorism and Re-Emerging Infectious Diseases 15-18 January 2003 Warsaw, Poland

© 2004, IOS Press All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, without prior written permission from the publisher. ISBN 1 58603 4170 Library of Congress Control Number: 2004103094

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LEGAL NOTICE The publisher is not responsible for the use which might be made of the following information. PRINTED IN THE NETHERLANDS

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Editorial: Building Integrated Preparedness against Bio-Terrorism 1

Janusz KOCIK1, Marek K. JANIAK1, Marian NEGUT2 Military Institute of Hygiene and Epidemiology, Warsaw, Poland Cantacuzzino Institute, Bucarest, Romania

NATO Advanced Research Workshop (ARW) entitled "Preparedness against BioTerrorism and Re-Emerging Infectious Diseases - Regional Capabilities, Needs, and Expectations in Central and Eastern European Countries" was held on 15-18 January 2003 in Warsaw, Poland. The meeting was organized by the Military Institute of Hygiene and Epidemiology, Warsaw Poland, in co-operation with the Cantacuzzino Institute, Bucarest, Romania. The primary objective of the ARW was to provide countries in which bio-defence systems are currently under development with the experience and expertise of those that are more advanced and/or have already been exposed and responded to a bio-terrorist attack. Such an approach was taken to allow avoiding mistakes and to properly allocate limited resources in building preparedness against intentional and natural outbreaks of infectious diseases. The ARW also aimed at increasing awareness of the problem at the political level in the Region. Yet another objective was to consolidate the community of experts in the Region and to reinforce connections with NATO and other international organisations in this particular area. This forum allowed for integration of thinking, elaboration of common approach to the problem, and exchange of the experience. One of the main lessons learned from the ARW is that, in most of the countries, the existing systems of the management of biological threats are convoluted and fragmented. There is also no single international system that would provide all the information needed to rapidly assess each particular situation and help bring a biological crisis under control. Several international bodies only fragmentally deal with parts of the problem that are within their competence. Some of these efforts were presented during the Workshop (Cosivi, Niedrig, Kyncl). Projects for the informal experts' networks have been demonstrated (Price et al, Woodal). Also, national approaches to the bio-defense and disease surveillance were presented (Plochev, Faludi, Niedrig et al., Asokliene, Chomiczewski, Kapustiri). Moreover, as a result of the attendance of specialists from such relevant fields as epidemiology, infectious diseases, NBC threats, medical planning, vaccinology, detection and identification of biological agents, physical protection and decontamination it was possible to examine most of the avenues a country should ideally explore in order to build its preparedness and capacity to respond to a biological attack. Development of the integrated system of response against bio-terrorism is a very complex endeavor requiring integration of the activities of the relevant institutions at the national level. In this regard, a multidisciplinary approach is crucial. Physicians should be trained to entertain suspicions facing unusual profiles of the disease (Pavlin). Other medical personnel and first responders should be also familiar and adequately trained to cope with signs and symptoms presented by victims of a biological attack (Stopa). New laboratory systems and approaches can revolutionize disease surveillance. Integration of the real time epidemiological data collection, analysis, and dissemination with the timely laboratory

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support based on the advanced technology platforms (Morse, Hanson et al, Niemeyer) will accelerate diagnosis and increase awareness of the situation. Even though rapid progress is being made in the field of the remote sensing (Niemeyer), biosensors (Donlori), proteomics (delVecchio), and other relevant methodologies (Bartoszcze), there is currently no "silver bullet" for application in the real time biological detection and identification. Since a variety of approaches and technologies are evolving in the biological threat detection, prophylaxis, and therapeutics, a flexible and rapidly responsive bio-manufacturing infrastructure is an essential part of the effective overall strategy for bio-terrorism preparedness (O'Connell). Consequence management should be planned in advance (Ahrens), preferably based on the effective risk assessment? (Bellenkes). Innovations in medical countermeasures and prophylaxis (Oyston) as well as in the decontamination procedures (Pivovarov et al., Kartel et al.) are still necessary and being introduced. In view of the fact that infectious diseases do not respect boundaries preparedness against outbreaks should be built from the scratch using the "thinking outside-of-the-box" approach with consideration for the international interdependence. This requires unification of the epidemiological and laboratory procedures as well as of the public health planning in countries of the Region. Taking into account the differences and gaps in the existing health systems of these countries, the very complicated goal of leveling the approaches certainly requires firm and sustained regional co-operation. We hope to witness it in the nearest future. Hopefully, the present NATO ARW will help to pave the way for such co-operation. The Editors: Dr. Janusz Kocik, M.D., Ph.D. Dr. MarekK. Janiak, M.D., Ph.D. Prof. Marian Negut, M.D., D.Sc.

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Acknowledgements The meeting was sponsored by the NATO Scientific and Environmental Affairs Division, the U.S. Army Research Office and the U.S. Army Soldier, Biological and Chemical Command; the US. Naval Research International Field Office, as well as Baxter Vaccines. We take this opportunity to express our thanks to the sponsors. We also are grateful to Dr. Deborah Niemeyer, Kevin O'Connel, Mildred Donlon, and Adam Wilczynski for their efforts to spread the news about the ARW in the scientific community and help attract the outstanding key speakers to the event. Finally, the hard and dedicated work of Mrs. Magdalena Baranowska, Dr. Dr. Emil Lisiak, Marek Brytan, and Mr. Hubert Radziejewski of the Military Institute of Hygiene and Epidemiology is highly appreciated. Indeed, without their organizational and technical support this meeting would not have gone as smoothly as it did. Dr. Marek K. Janiak, M.D.,Ph.D. - Co-Director Prof. Marian Negut, M.D., D.Sc. - Co-Director Dr. Janusz Kocik, M.D., Ph.D. - Head of the Organizing Committee

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KEY SPEAKERS Theodore Ahrens US European Command Feldbergstrasse 102 71134Aidlingen, GERMANY e-mail: [email protected] LTC Gerard P. Andrews, Ph.D. Chief, Bacteriology Division USAMRIID, Ft. Detrick, MD, 21701, USA Dr. Loreta Asokliene Public Health Specialist Centre for Communicable Diseases Prevention and Control Kalvariju str. 153, LT-2042 Vilnius, LITHUANIA e-mail: [email protected] tel: +370 5 277 86 61 fax: +370 5 277 87 61 Dr hab. n. med. Michal Bartoszcze Military Institute of Hygiene & Epidemiology 2 Lubelska St., 24-100Pulawy, POLAND CDR Dr. Andrew H. Bellenkes MSC USN, School of Aviation Safety Naval Postgraduate School (Code 10) 1588 Cunningham Rd., Monterey, California 93943-5002, USA tel. US (country code) + 831.656.2581 e-mail: [email protected] Prof. Krzysztof Chomiczewski Military Institute of Hygiene & Epidemiology 4 Kozielska St., 01-163 Warsaw, POLAND

Ditta Ciganikowa Bio Weapons Prevention Project C/o PSIS 132, Rue de Lausanne 1211, Geneva 21, SWITZERLAND tel: +41 22 908 57 34 fax: + 41 22 738 35 82 e-mail: [email protected], www.bwpp.org Dr. Ottorino Cosivi World Health Organization Department of Communicable Disease Surveillance and Response 20, Avenue Appia CH-1211 Geneva 27, SWITZERLAND tel:+41 22 791 25 31 fax: +41 22 791 48 93 e-mail: [email protected] Helene van Cuyck Expert in Biology Weapons of Mass Destruction Centre, AB 310 NATO Bd. Leopold III BE. IIIo. Brussels, BELGIUM tel: 00322 707 1905 e-mail: [email protected] Prof. Vito DelVecchio Institute of Molecular Biology and Medicine University of Scranton Scranton, PA 18510-4625, USA Dr. Mildred Donlon DARPA/SPO 3701 North Fairfax Drive Arlington, VA 22203-1714, USA

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Dr. Phil Elzer Louisiana State University Dept. of Veterinary Science 111 Dalrymple Building Baton Rouge, LA 70803, USA Col. Dr. Gabor Faludi Hungarian Defence Forces Institute of Health Protection General Director 1555 Budapest Pf. 68, HUNGARY e-mail: [email protected] Major Eric Hanson Office of the Air Force Surgeon General Directorate, Expeditionary Medical Operations, Science & Technology 5201 Leesburg Pike, Suite 1401 Falls Church, VA, 22041, USA tel:(703)9243172wk tel: (703) 216 0839 cell e-mail: [email protected] Nick Kanellopoulos Membranes & Materials for Environmental Applications Laboratory National Center for Scientific Research "Demokritos" 15310 Agia Paraskevi, Athens, GREECE tel: +30 210 65 35 294 fax:+30 210 65 11766 e-mail: [email protected] Dr. Alexander Kapustin Chemical and Biological Weapons Conventional Problems Sector Ministry of Foreign Affairs of Ukraine 01018, Mykhaylivska sqr.l, Kyiv, UKRAINE tel: (380) 44 238 17 26 fax:(380)442381653 e-mail: [email protected]

Nikolai T. Kartel, Prof, Dr. Sci. (Chem.) Institute for Sorption & Problems of Endoecology National Academy of Sciences of Ukraine 13 General Naumov str. Kiev 03164, UKRAINE tel: +380 44 452 93 25 fax: +380 44 452 93 27 e-mail: [email protected] [email protected] Dr. Janusz Kocik Military Institute of Hygiene & Epidemiology 4 Kozielska St., 01-163 Warsaw, POLAND tel: +48 22 681 61 06, mobile (preferred) + 48 607 461 970 e-mail: [email protected] Dr. Mzia Kutateladze G. Eliava Institute of Bacteriophages, Microbiology and Virology, Georgian Academy of Sciences Tbilisi.3, Gotua str., Tbilisi 380060, GEORGIA tel/fax.: + 99 532 91 1836 e-mail: [email protected] Dr. Jan Kyncl Dept. of Epidemiology Srobarova 48, 100 42 Prague 10, CZECH REPUBLIC tel: +420 267 082 891 fax: +420 272 741 433 e-mail: [email protected] Dr. Christian Loucq Acambis plc Peterhouse Technology Park 100, Fulbourn Road Cambridge CB1 9PT, UK tel: +44 1223 275 300 mobile:+44 7881 81 75 41 fax: +44 1223 416 300 e-mail: [email protected]

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Dr. Stephen Morse Associate Director for Science Bioterrorism Preparedness and Response Program, Centers for Disease Control and Prevention 1600 Clifton Rd., Atlanta, GA 30333, USA Prof. Marian Negut MD, PhD Cantacuzzino Institute 103, Splaiul Independentei sect. 5 Bucharest 70 100, ROMANIA tel: +402 14 10 73 30 fax:+402 14 11 56 72 e-mail: [email protected] Dr. Mattias Niedrig Secretary of the European Network for Diagnosis of "Imported" Viral Disease (ENIVD) Robert-Koch-Institut Nordufer 20 13353 Berlin, GERMANY tel: +49 30 45 47 23 70 fax: +49 30 45 47 26 25 e-mail: [email protected] Dr. Kevin O'Connell AMSSB-RRT-BMResearch and Technology Diorectorate Edgewood Chemical Biological Center Aberdeen Proving Ground, MD 210105424, USA Dr. Petra Oyston Microbiology B07A, CBS Porton Down Salisbury, Wiltshire SP4 OJQ, UK

LTC Julie Pavlin DoD Global Emerging Infections System 503 Robert Grant Ave. Silver Spring, MD 20910-7500 USA tel: 301-319-9346 fax: 301-319-9104 [email protected] Prof. Alexander Pivovarov Department of Equipment and Technology of Food Industry Ukrainian State University of Chemical Engineering 8 Gagarina Av., Dnepropetrovsk 49005, UKRAINE tel: +38 0562 47 05 55 fax: +38 0562 36 68 37 e-mail: [email protected] a.pi vo varov@ua. fm home tel:+38 056 772 1050 Dr. Kamen Plochev Military Medical Academy G. Sofiiski 3 1606 Sofia, BULGARIA Home address: 97' Erlogi Georgiev Blvd. Sofia 1000, BULGARIA tel: +35 92 922 53 (i) 952 53 50 mobile: +359 98 48 98 27 Barbara B.S. Price, PhD Senior Scientist Biodefense Systems Battelle Eastern Regional Technology Center (BERTC) 1204 Technology Drive Aberdeen, MD 21001 USA tel (main): 410-306-8500 fax: 410-306-8420 direct: 410-306-8580 cell: 207-831-3398 [email protected]

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Col. Richard M. Price, USAF Ret. Applied Science and Analysis, Inc. PO Box 1144, Aberdeen, MD 21001 USA Tel 410-638-9480 Fax 410-638-9481 Web: http://www.asanltr.com Dr. Peter J. Stopa 5183Blackhawk Rd BldgE3549 AMSSB-REN-E-MC US Army Edgewood Chemical Biological Center Aberdeen Proving Ground, MD 210105424, USA Tel: 410 343 22 78 Fax: 410 612 50 83 e-mail: [email protected] private email: [email protected] Marek Tomaszewski Chief Sanitary Inspectorate (GIS) Epidemic Dept. 38/40 Dluga str., Warsaw, POLAND tel: +48 22 635 45 81 (i) 107 e-mail: [email protected] Dr. David Trudil New Horizons Diagnostic Corporation 9110 Red Branch Road Columbia, MD 21045-2014, USA tel: +410 992 93 57 ext. 222 fax: +410 992 03 28 e-mail: [email protected]

Prof. Tomasz Twardowski Kierownik Zespolu Biosyntezy Bialka Instytut Chemii Bioorganicznej PAN Noskowskiego 12/14, 61-704Poznan, POLAND Dr. Adam Wilczynski Permanent Mission of Poland to the UN Office at Geneva 15 1'Ancienne Route 1218 Grand Saconnex (CH), SWITZERLAND tel: +41 22 7109 710 fax: +41 22 7109 799 e-mail: [email protected] Dr. Jack Woodall Director, Nucleus for Investigating Emerging Infectious Diseases, Dept. of Medical Biochemistry, Institute of Biomedical Sciences, Federal University of Rio de Janeiro Cidade Universitaria Rio de Janeiro - RJ - 21941-590, BRAZIL tel: +5524 2225 1395 e-mail: [email protected] Doc. dr hab. Andrzej Zielinski Zaklad Epidemiologii Panstwowy Zaklad Higieny ul. Chocimska 24 00-791 Warszawa, POLAND

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OTHER PARTICIPANTS AND OBSERVERS Dr. Pawel Abramczyk Department of Defence Affairs / Ministry of Health Team of Reaction on Emergency Situations Miodowa str. 15, 04-950 Warsaw, POLAND e-mail: [email protected] fax: +48 22 634 94 24 Dr. Vladimir Bundin Russian Agency for Munitions Department for BTWC Shepkina 42, 107996 Moscow, RUSSIA tel: 007 095 206 43 64 fax: 007 095 206 35 93 Eric Bush-Petersen Baxter Vaccines Industriestrasse 67 A-1220 Vienna, AUSTRIA [email protected] Prof. Dumitru Carstina Infectious Diseases Clinic 3400 Cluj-Napoca Str.Iuliu Moldovan, 23, ROMANIA tel/fax:+40264193 105 e-mail: [email protected] Agnieszka Chycak-Kryczka Glowny Inspektorat Sanitarny, Departament Przeciwepidemiczny 38/40 Dtuga str., Warsaw, POLAND tel: +48 22 635 45 81 w. 109/107

Zbigniew Ciolek Dept. of International Cooperation Polish Ministry of Defense 1 Krolewska Str. 00-909 Warsaw, POLAND tel: +48 22 68 73 106 fax:+48 22 68 73 182 e-mail: [email protected] Flavio Del Ponte Humanitarian Aid / SDC Federal Department of Foreign Affairs 130 Freiburgstrasse 3003 Bern, SWITZERLAND tel:+4122 73162 04 e-mail: [email protected] Dr. Antonio della Guardia Presidenca Del Consiquo Dei Ministri Via XX Settembre, 8 00100 Roma, ITALY tel:+39 06 61174 612 mob: +39 347 039 63 66 e-mail: [email protected] Dr. Marek K. Janiak Military Institute of Hygiene & Epidemiology Kozielska 4 01-163 Warszawa, POLAND tel:+48 22 685 3101 m.j [email protected]

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Dr. Tomasz Szkoda Respiratory Virus Laboratory Department of Virology National Institute of Hygiene Chocimska 24, 00-791 Warszawa, POLAND tel. +48 (22) 54 21 230 tel./fax+48(22)5421385 NIH oper.: tel +48 (22) 54 21 400 e-mail: [email protected] http://www.pzh.gov.pl/ Major Dr. Corina Taubner Chief laboratory Central Military Emmergency Clinic Hospital in Bucharest 134, Calea Plevnei, 77103 Bucharest 1, ROMANIA tel:+40 21224 94 06/160 fax:+40 2141156 72 Prof. Sadi Osman Yenen General Manager KANSAS AS Blood and Health Service Co. Ataturk Bulvary, 219/14 Kavaklydere06680- Ankara, TURKEY

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Contents Editorial: Building Integrated Preparedness against Bio-Terrorism Janusz Kocik, Marek K. Janiak and Marian Negut Key Speakers Other Participants and Observers

v ix xiii

Part 1. Contemporary Epidemiology With Laboratory Support as a Biological Attack Identification Tool Epidemiology of Bioterrorism J.A. Pavlin

3

Health Preparedness for the Deliberate Use of Biological Agents to Cause Harm: WHO's Activities 9 O. Cosivi Epidemic Outbreak Systems (EOS) - Microarray Incorporation for Pathogen Identification E. Hanson, R. Roweley, B. Agan, C. Tibbetts and D. Niemeyer

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Preventing Emerging Infectious Diseases: Epidemiology and Laboratory Capacity Support S.A. Morse

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The Laboratory Response Network S.A. Morse, R. Kellogg, S.R. Perry, R.F. Meyer, D. Bray, D. Nichelson and M.J. Miller

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Imported Viral Diseases, Surveillance and Control in Europe - European Network for the Diagnostics of "Imported" Viral Diseases (ENIVD) M. Niedrig

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Surveillance of Acute Respiratory Infections in the Czech Republic and in Europe Example of an Early Warning System J. Kyncl and B. Kriz

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Part 2. National Approaches to Biodefence in Central and Eastern Europe Countries Organization of Military Medical Response to Bioterroristic Attacks K. Plochev and E. Penkov

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Georgian Approach to Biodefense M. Kutateladze

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National Approach of Germany to Bioterrorism and Bio-Warfare M. Niedrig, R. Fock and E. Finke

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National Approach of Hungary to Bioterrorism and Biowarfare G. Faludi, A. Csohan and G.Berencsi

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Emerging Infectious Diseases and Their Surveillance in Lithuania L. Asokliene

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Biological Recognition Teams in the Polish Army K. Chomiczewski

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National Approach of Ukraine to Bioterrorism and Biowarfare A. Kapustin

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Part 3. Risk Assessment, Crisis Management and NBC Training Effective Risk Management in the Human Factors Assessment of Chemical/Biological Threats A.H. Bellenkes

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Crisis Management: Expediting Information and Resource Flow via the Global Incident Analysis and Alerting System (GIAAS) for CBRN R. Price, J. Woodall and S. Netesov

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Civilian Relief after Release of Weapons of Mass Destruction: Need for a New Task Force 'Scorpio' J. Woodall

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Consequence Management of a Bioterrorist Incident (CMBI) J. T. Ahrens

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GM Modified Food/Feed as Biowarfare T. Twardowski

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Antibiotic Resistance Bacteria - A Potential Threat M. Negut

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Effective Training for First Responders P.J. Stopa, K. Quinn-Doggett and R.A. Vigus

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Part 4. Applied Research Brucellosis-A Biowarfare Threat and Public Health Concern P.H. Elzer

161

Medical Countermeasures Research at Dstl Porton Down, UK P.C.F. Oyston

164

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Current Problems Regarding Detection and Identification of Biological Threats M. Bartoszcze

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Rediscovering Remote Sensing: Improving Infectious Disease Surveillance D.M. Niemeyer

179

The Joint Biological Agent Identification and Diagnostic System (JBAIDS) Debra M. Niemeyer

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Biosensors - The Tool for Fast Detection M.A. Donlon

191

A Flexible Approach to Biomanufacturing K.P. O'Connell, P.E. Anderson, D.C. Lukens, M.H. Kim, A.S. Khan, R.G. Thompson, J.T. Park and J.J. Valdes

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Development of Rapid Fingerprinting and Detection Assays for Biological Agents of Mass Destruction V.G. DelVecchio

207

Decontamination of Drinking Water and Liquid Media by Cold Plasma in the Special Periods A.A. Pivovarov

215

Antibacterial Adsorption-Filtering Materials for Individual Protection Means of Organism - Antibacterial Action of Metal-Containing Carbon Adsorbents N. Kartel, A. Grigoriev, D. Shvets and V. Strelko

226

Antibacterial Adsorption-Filtering Materials for Individual Protection Means of Organism - Fine Filtering Materials on the Base of Polypropylene Microfibres and Their Antibacterial Property M. Tsebrenko, V. Rezanova, I. Tsebrenko, M. Mayboroda and N. Kartel

232

Antibacterial Adsorption-Filtering Materials for Individual Protection Means of Organism - Composite Materials Based on Polyurethanes and Active Carbon Yu. Savelyev, L. Robota, O. Savelyeva, N. Kartel and V. Strelko

239

List of Tables and Figures Author Index

251 255


Part 1

Contemporary Epidemiology With Laboratory Support as a Biological Attack Identification Tool


Preparedness Against Bioterrorism and Re-Emerging Infectious Diseases J. Kocik et al. (Eds.) IOS Press, 2004

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Epidemiology of Bioterrorism* Julie A. PAVLIN Walter Reed Army Institute of Research, Silver Spring, MD USA * Adapted from Emerg Inf Dis 1999 Jul-Aug;5(4):528-30. Abstract. The risk of a bioterrorist attack occurring anywhere in the world has increased with the increased willingness of terrorists to inflict mass casualties. A sound epidemiologic investigation of a disease outbreak, whether natural or humanengineered, will assist medical personnel in identifying the pathogen, as well as instituting the appropriate medical interventions. Documenting who is affected, possible routes of exposure, signs and symptoms of disease, and the rapid identification of the causative agents will greatly increase the ability to plan an appropriate medical and public health response. Good epidemiologic information will allow the appropriate follow-up of those potentially exposed, as well as assist in risk communication and responses to the media. To minimize the effects of a biological terrorist attack, health care professionals and public health authorities must be aware of the threat of biological warfare and terrorism and have an increased index of suspicion that such an attack can occur. They must have some understanding of the classes of agents that have been and can be weaponized and their effects after inhalation and other dissemination methods. They need to be trained to recognize and treat casualties of biological warfare or terrorism and they must be able to apply appropriate preventive measures rationally and without unnecessary panic or alarm. Well before any event, public health authorities must implement surveillance systems so they can recognize patterns of nonspecific syndromes that could indicate the early manifestations of a biological warfare attack. The system must be timely, sensitive, specific, and practical. To recognize any unusual changes in disease occurrence, surveillance of background disease activity should be ongoing, and any variation should be promptly followed up with a directed examination of the facts regarding the change.

The use of Bacillus anthracis spores as an agent of terror in the United States in October 2001 has demonstrated the need for increased vigilance and preparedness for attacks using biological agents. It is impossible to monitor every nation, group and individual who may be considering the use of biological terrorism, so in addition to increased law enforcement vigilance, we must improve our public health monitoring. Even as this will improve our ability to detect and react to biological attacks, it will improve basic public health capacity to watch for new naturally occurring disease trends and outbreaks and to institute appropriate preventive measures. Today more than ever we must be on the alert for new emerging infections and bioterrorism, and we need to be able to rapidly investigate them and prevent further cases. The awareness of the threat of biological agents as weapons is not new. Since the discovery of Iraq's biological weapons program, today's military leaders and policy makers have had concerns regarding the threat of biological warfare [1]. However, the increase in terrorist acts both domestically and internationally demonstrates the need to protect civilian populations as well as military forces against this possibility. Increases in the acquisition of, and alleged, or actual use of biological organisms as weapons of terrorism had been claiming

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J.A. Pavlin /Epidemiology ofBioterrorism

front-page status in our newspapers even before the anthrax letter attacks. The threat of bioterrorism is a reality, and to effectively counter the potentially devastating effects of an attack, we need to first understand basic epidemiologic principles of biological agents used as weapons. Best-selling fiction novels about bioterrorism attacks in the United States tend to portray a biological agent as highly contagious, genetically engineered organisms resistant to all known vaccines. While this scenario is not impossible, experiences to date have not proven it to be true. Besides the attacks with non-communicable anthrax that was susceptible to commonly used antibiotics, alleged attacks by the Aum Shinrikyo did not result in a single illness from a biological agent [2], and the successful 1984 attack contaminating salad bars in The Dalles, Oregon by a religious cult utilized a common salmonella strain that was not lethal, not contagious, and susceptible to antibiotics [3]. These examples are not intended to imply that a bioterrorism attack could not have a devastating impact, but only to point out that our level of suspicion and diligence in identifying and reacting to a biological attack must remain high, since it will likely not appear as popular literature describes. Furthermore, a small outbreak of illness could be an early warning of a more serious attack, and recognition and prompt institution of preventive measures such as effective vaccines and antibiotics could save thousands of lives. To facilitate the rapid identification of a bioterrorist attack, all health care providers and public health personnel should have basic epidemiologic skills and knowledge of what to expect in such a setting.

1. Differential Diagnosis Any outbreak of disease, in small or large numbers, should be evaluated as a potential bioterrorist attack. This initial investigation does not have to be time consuming or involve law enforcement. A simple look at the facts surrounding the outbreak to determine if anything seems unusual or indicative of a man-made epidemic should suffice. Since a disease outbreak can be of natural or unnatural origin, the differential diagnosis of an outbreak should first be considered. The possibilities include a spontaneous outbreak of a known endemic disease, a spontaneous outbreak of a new or reemerging disease in that area, a laboratory accident, or an intentional attack with a biological agent. Epidemiological tools can assist in differentiating between these possibilities. It may be very difficult to determine the cause of a disease cluster, or even that something unusual is occurring, especially if the initial cases are small in number. Not only should unusually high rates of illness trigger an investigation, but also any unusual disease event should signal a warning. For example, even one case of inhalation anthrax should cause immediate concern and action. To further complicate awareness of an attack, unlike chemical terrorism, biological terrorism will not be immediately obvious, but will most likely appear insidiously, with primary care providers witnessing the first waves of casualties. However, it may not even be emergency room personnel who first realize there is a problem. The first to notice could be a hospital laboratory seeing unusual strains of organisms, or the county epidemiologist keeping track of hospital admissions, or even pharmacists distributing more antibiotics than usual, 911 operators noticing an increase in respiratory distress calls, or funeral directors with increased business. All potential avenues of epidemiologic data need to be tracked and aggressively followed to ensure the most rapid recognition and response.

J.A. Pavlin /Epidemiology of Bioterrorism

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2. Epidemiologic Approach The basic epidemiologic approach in the evaluation of a potential bioterrorist or biowarfare attack is not different from any standard epidemiologic investigation. The first step is to confirm that an alleged disease or event actually has occurred using laboratory and clinical findings. A case definition should be constructed and used to determine the actual number of cases, and consequently the approximate attack rate. The use of objective criteria in the development of a case definition is very important in determining an accurate case number, as both additional cases may be found, and some may be excluded, especially as the potential exists for hysteria to be confused with actual disease. Once an estimated rate of illness is calculated, a comparison of rates during previous years should be performed to determine if this event constitutes a deviation from the norm. Once the case definition and attack rate have been determined, one can now characterize the outbreak via the conventional time, place and person. This data will provide crucial information in determining the potential source of the outbreak, and consequently, whether it appears to be of natural or man-made origin.

3. Epidemic Curve Using data gathered on cases over time, an epidemic curve can be calculated. The disease pattern is an important factor in differentiating between a natural outbreak and an intentional attack. In most naturally occurring outbreaks, numbers of cases gradually increase as a progressively larger number of people come in contact with other patients, fomites and vectors that can spread disease. Eventually, most of the population has been exposed, and is immune to further disease, and the number of cases, or epidemic curve, gradually decreases. Conversely, a bioterrorism attack is most likely to be caused by a point source, with everyone coming in contact with the agent at approximately the same time. The epidemic curve in this case would be compressed, with a peak in a matter of days or even hours, even with physiological and exposure differences. If the biological agent is contagious, it is possible to see a second curve peak after the first, as original cases expose originally unexposed people to the agent. The steep epidemic curve expected in a bioterrorism attack is similar to what would be seen with other point source exposures, such as food-borne outbreaks. Therefore, the compressed epidemic curve is still not pathognomonic for an intentional bioterrorism attack. If a specific group of people have become victims, the epidemic curve may give some suggestion on when they were exposed. From this information, a possible incubation period can be calculated. The incubation period can assist in determining the potential cause of illness, as well as suggesting a possible intentional attack if the incubation period is shorter than usual due to an unusually high inoculum or more effective exposure route than what is seen normally. Calculating the incubation period may also help in determining if the disease is contagious via person-to-person spread. The implications of a contagious disease are obviously of extreme importance to effective disease control measures.

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4. Epidemiologic Clues As steep epidemic curves can be seen in natural point-source exposures, additional characteristics of the outbreak should be investigated in determining the potential for it being man-made [4,5]. None of the following clues alone constitute proof of intentional use of a biological agent, but together can assist greatly in determining if further investigation is warranted. 1 - The presence of a large epidemic, with greater case loads than expected, especially in a discrete population. 2 - More severe disease than would normally be expected for a given pathogen, as well as unusual routes of exposure, such as a preponderance of inhalational disease as was seen in Sverdlovsk after the accidental release of aerosolized B. anthracis spores [6]. 3 - A disease that is unusual for a given geographic area or presents outside of the normal transmission season, or is impossible to transmit naturally in the absence of the normal vector for transmission. 4 - Multiple simultaneous epidemics of different diseases. 5 - A disease outbreak with zoonotic as well as human consequences, as many of the potential threat agents are pathogenic to animals. 6 - Unusual strains or variants of organisms, or antimicrobial resistance patterns disparate from those currently circulating. 7 - Higher attack rates in those exposed in certain areas; such as inside of a building if it was released indoors, or lower rates in those inside a sealed building if an aerosol was released outdoors. 8 - Intelligence that an adversary has access to a particular agent or agents. 9 - Claims by a terrorist of the release of a biologic agent. 10 - Direct evidence of the release of an agent, with findings of equipment, munitions or tampering. Even with the presence of more than one of the above indicators, it may not be easy to determine that an attack occurred through nefarious means. For example, the outbreak of salmonellosis in Oregon took months to determine that it was caused by an intentional contamination of salad bars [3]. Other naturally occurring incidents have been suspected of being caused by unnatural means, such as the hantavirus outbreak in the Four Corners area of the United States [7] and the West Nile virus epidemic in New York City [8]. There are no definite answers to this dilemma, but most importantly, even if no conclusive answer can be derived quickly, the means employed in reaching for that answer will still provide medical personnel with information that will help them prevent further morbidity and mortality. 5. Anthrax Example The anthrax letter attacks demonstrated many of the epidemiologic characteristics previously described. The cases were of a disease unusual for the geographic location, of exceptional severity and an unusual exposure route (inhalational) and warning was given by the perpetrator through the letters. In other ways, this outbreak would have been very difficult to detect or confirm as bioterrorism should another agent have been used. Only 22 cases occurred, they were spread out over a six week time frame, the cases were geographically widespread, and the strain of bacteria was not unusual and had normal antibiotic susceptibilities [9]. This attack has also provided more insight into how a bioterrorist attack might present. The presentation of an attack very much depends on the population targeted, the characteristics of the agent and the mode of transmission [10]. That a terrorist might use an unsuspected dissemination device (i.e., a letter) must be factored into any investigation. The attack may even have consequences unexpected by the perpetrator as it was unknown at that time the ability of anthrax spores to disseminate from letters through the mechanical action of mail


1

sorters. The infinite number of variables present in an attack with a biological agent (target, dissemination method and device, differences in population immunity and susceptibility, type of agent, wind speed and direction if an aerosol, etc.) means that the medical and response community must always stay vigilant in detecting and responding to a bioterrorist attack. It is impossible to accurately predict all the epidemiologic features for the next occurrence. 6. Recommendations for Preparedness Recommendations to improve awareness and readiness should a bioterrorism attack occur include education of all medical personnel, especially those primary care providers and emergency personnel who will probably be the first to see victims of a biological attack. Training should include the basic epidemiologic principles outlined here as well as clinical information on diagnosing and treating the highest threat agents. Training should be refreshed periodically to ensure that skills remain current. Improved surveillance efforts should be instituted with as close to real time data gathering as possible. Robust surveillance systems are essential to detect any emerging or reemerging disease, whether natural or man-made. Quick recognition of any change in disease patterns will facilitate determining the source and preventing further exposure, which should be the key driving force behind any epidemiologic investigation. Many new types of disease surveillance systems have been developed for the purpose of rapid detection of disease outbreaks and bioterrorism [11-17]. Some of the systems use medical data sources that are routinely collected for other purposes, (e.g., emergency room logs), some collect new data at the point of patient encounter, and some use non-clinical data (e.g., pharmacy sales, school absenteeism) to trigger an alert. Many use new statistical methods to detect aberrations. Since the terrorist attacks of September 2001 and the subsequent anthrax mailings, there has been a proliferation of these systems developed and in use by city, county and state public health personnel, as well as by academia and the military [18,19]. Public health departments can use the expertise of those working in this area to assist in setting up additional real-time surveillance. Through strong epidemiologic training, a close attention to disease patterns, and a healthy respect for the threat of biological terrorism, potential problems can be discovered rapidly, and actions can be taken to decrease the impact of disease, regardless of its origin. References [1] [2] [3] [4] [5] [6] [7] [8]

Proliferation: threat and response. Office of the Secretary of Defense; November 1997. Broad WJ, Miller J. The threat of germ weapons is rising. Fear, too. New York Times 1998 Dec 27. Torok TJ, Tauxe RV, Wise RP, Livengood JR, Sokolow R, Mauvais S, et al. A large community outbreak of salmonellosis caused by intentional contamination of restaurant salad bars. JAMA 1997;278:389-95. Weiner SL. Strategies of biowarfare defense. Milit Med 1987; 152:25-28. Noah DL, Sobel AL, Ostroff SM, Kildew JA. Biological warfare training: infectious disease outbreak differentiation criteria. Milit Med 1998; 163:198-201. Meselson M, Guillemin J, Hugh-Jones M, Langmuir A, Popova I, Shelokov A, et al. The Sverdlovsk anthrax outbreak of 1979. Science 1994;266:1202-1208. Horgan J. Were four corners victims biowar casualties? Sci Am 1993;269(5):16. Fine A, Layton M. Lessons from the West Nile viral encephalitis outbreak in New York City, 1999: implications for bioterrorism preparedness. Clin Infect Dis 2001;32:277-82.

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[9] [10] [11] [12] [ 13] [ 14] [15] [16] [17] [18] [19]


Centers for Disease Control and Prevention. Update: investigation of anthrax associated with intentional exposure and interim public health guidelines, October 2001. MMWR 2001;50:889-93. Jernigan DB, Raghunathan PL, Bell BP, et al. Investigation of bioterrorism-related anthrax, United States, 2001: epidemiologic findings. Emerg Inf Dis 2002;8:1019-28. Miller JR, Mikol Y. Surveillance for diarrheal disease in New York City. J Urban Health 1999;76:38890. Pavlin JA, Kelley PW, Mostashari F, et al. Innovative surveillance methods for monitoring dangerous pathogens. In: Institute of Medicine (US). Biological threats and terrorism: assessing the science and response capabilities. Washington, DC: National Academy of Sciences; 2002. p. 185-196. Espino JU, Tsui F-C, Wagner M. Realtime outbreak detection system (RODS). Available from https://www.health.pitt.edu/rods/rods.htm. Accessed on 11 Feb 2002. Lazarus R, Kleinman KP, Dashevsky I, DeMaria A, Platt R. Using automated medical records for rapid identification of illness syndromes (syndromic surveillance); the example of lower respiratory infection. BMC Public Health 2001; 1:9. New Mexico Department of Health. Rapid Syndrome Validation Project (RSVP) Project Description. Available from http://epi.health.state.nm.us/rsvpdesc/default.asp. Accessed on 11 Feb 2002. Lober WB, Karras BT, Wagner MM, et al. Roundtable on bioterrorism detection: information systembased surveillance. J Am Med Inform Assoc 2002 Mar-Apr;9(2):105-15. Green MS, Kaufman Z. Surveillance for early detection and monitoring of infectious disease outbreaks associated with bioterrorism. Isr Med Assoc J 2002 Jul;4(7):503-6. Centers for Disease Control and Prevention. Syndromic surveillance for bioterrorism following the attacks on the World Trade Center - New York City, 2001. MMWR 2002;51:13-5. Lewis MD, Pavlin JA, Mansfield, JL, et al. Disease outbreak detection system using syndromic data in the greater Washington, DC area. Am J Prev Med 2002;23(3): 180-6.


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Health preparedness for the deliberate use of biological agents to cause harm: WHO'S activities Dr Ottorino COSIVI Project Leader, Preparedness for Deliberate Epidemics, Global Alert and Response Team Department of Communicable Disease Surveillance and Response World Health Organization, Geneva In most of the world, the public health infrastructure is already stretched to its limits in coping with natural health hazards. Against such a background, the additional threat to public health services posed by the deliberate use of biological or chemical agents could be considered as little more than a slight addition to the existing burden. The deliberate use of such agents, however, could be of such a scale or of such a nature that health-care systems would be unable to cope. For deliberate releases or threats of release of such agents, a spectrum of outcomes can be envisaged that ranges between two extremes: relative insignificance at one end, and mass destruction of life or mass casualties at the other. Widespread panic and fear are expected to follow any use or threat of use such agents, whatever the actual number of casualties it may provoke, leading to increased demand for medical and other emergency services. Remedies or countermeasures may be beyond the resources of many countries and therefore only available, if at all, through international cooperation. In 1970, technical guidance was made available to Member States by WHO in the publication Health aspects of biological and chemical -weapons. The WHO report was instrumental in achieving international consensus on the Biological and Toxins Weapons Convention [1] and the Chemical Weapons Convention [2]. The events of 11 September 2001 in New York and Washington in the United States of America and the dissemination of anthrax spores though the United States Postal Service in the fall of 2001 highlighted the need for public health preparedness for the possible use of biological and chemical agents to cause harm. Health organizations in both developing and industrialized countries were overwhelmed by requests for information and guidance on various aspects of chemical and biological weapons (CBW). Through the World Health Assembly resolution WHA55.16 of 18 May 2002 [3], WHO Member States requested the Director General to strengthen activities on global public health preparedness and response to deliberate use of biological and chemical agents or radionuclear material that affect health. WHO focuses exclusively on the public health aspects of preparedness and response of such threats and its actions are implemented through the existing framework provided by the Global Health Security: epidemic alert and response strategy as per WHA resolution WHA54.14 of 21 May 2001. WHO activities relevant to resolution WHA55.16 include four main areas of work: (a) international preparedness; (b) national capacity strengthening on preparedness and response; (c) public health preparedness for diseases associated with biological warfare, and (d) global outbreak alert and response. Ongoing activities within these four areas of work summarized below. In addition, a WHO

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O. Cosivi / Health Preparedness for the Deliberate Use of Biological Agents

Headquarters working group on Biological, Chemical Radiological Threats (BCR Working Group) has been established to exchange information and co-ordinate activities within the Organization. International preparedness The objective of this area of work is to provide international guidance and to monitor closely international developments on public health aspects of biological weapons, to avoid duplication and explore possibilities for new collaborations. Activities in this area of work include the fmalization of the second edition of the Public health response to biological and chemical weapons: WHO guidance, which is expected to be published by June 2003. Plans have been developed to publish further technical guidance material in the form of Supplements to the second edition. A WHO CBW Advisory Group is being established as a permanent resource for WHO and its Member States. WHO also continues monitoring international developments on various aspects of biological weapons to explore further possibilities for collaboration. Such activities particularly include the follow-up process of the Biological Weapons Convention, as agreed during its Fifth Review Conference in November 2002.

National capacity strengthening on preparedness and response The objective of this area of work is to respond to the increased number of requests for technical assistance by Member States for assessment of their national CBW preparedness and response programmes and training of staff. Field missions to advise Ministries of Health have been conducted in 2002 and 2003. WHO is developing and field testing guidelines to assess national health CBW preparedness and response programmes as these remain a high priority for reinforcing country capacities. WHO is strengthening laboratory and epidemiology country capacities for epidemic prone diseases, including possible deliberate diseases, through a programme targeting microbiologists and epidemiologists from several countries of the African, Eastern Mediterranean and European regions. This includes, in-depth review of surveillance system leading to national plan of actions for strengthening surveillance and early warning systems for both deliberate and naturally occurring epidemic diseases [4]. An Intercountry Meeting on Emergency Preparedness Strategies, was held in Bangkok, Thailand, 17— 20 March 2003, including countries of the WHO Regional Office for South-East Asia. WHO is also working with the United Nation Disaster Management Programme [5] (UN DTMP) on the development of a training module on the management of preparedness and response programmes on chemical, biological and radio-nuclear incidents for policy makers.

Public health preparedness for diseases associated with biological warfare selected diseases The objective of this area of work is to contribute to international preparedness on diseases associated with biological warfare, by (a) establishing global networks of experts and laboratories; (b) establishing standard and procedures, and disseminating information, and (c) setting up and implementing training. WHO is strengthening selected disease-specific networks, starting with anthrax. Activities on plague and smallpox are also being carried out.

O. Cosivi / Health Preparedness for the Deliberate Use of Biological Agents

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Other priority diseases — identified by a WHO risk assessment — include tularaemia, brucellosis, glanders, melioidosis, Q fever, typhus fever, coccidioidomycosis, and Venezuelan equine encephalomyelitis. Work in ongoing for the setting up a global database of anthrax experts and laboratories capabilities. Priorities of this project include the establishment of quality control mechanisms for laboratories of the anthrax laboratory network, the development of training materials as well as publication of the fourth edition of Guidelines for the surveillance and control of anthrax in humans and animals. In addition, the development of a plan of work for tularaemia —similar to that developed for anthrax — for implementation in late 2003 onwards is being considered.

Global alert and response The objective of this area of work is to provide the global public health community with timely information on "public health emergencies of international concern" and to provide support to Member States in their response activities, when required. Alert and response operations includes disease intelligence, verification, response and follow up. The Global Outbreak Alert and Response Network includes some 110 partners has proven its capacity to provide Member States with technical assistance in case of outbreaks of epidemic origin and intoxication. This mechanisms is being strengthen to address the challenge posed by CBW. The International Health Regulations of 1969 are being revised to provide the legal framework of the alert and response operations. The revised IHR will require Member States to report all "public health emergencies of international concern". The revised IHR would include provision for the provision of assistance to Member States for response and an option of confidential/provisional notification. Any "public health emergencies of international concern" would include events related to possible use or threat of use of CBW. However some key issues should be considered with regard the possible inclusion of deliberate epidemics in the revised IHR: (a) the need for WHO to maintain neutrality and focus its action on the public health component of response only; and (b) WHO has no mandate to assess the deliberate nature of a possible CBW use or threat of use. Should Biological Weapons be involved in such events, the responsibility to investigate such reports in order o ascertain the possible deliberate nature remain with the UN; and for Chemical Weapons with the Organization for the Prohibition of Chemical Weapons (OPCW). However, the UN may request WHO technical assistance in case of such investigations. References [1] [2] [3] [4] [5]

Convention on the prohibition of the development, production and stockpiling of bacteriological (biological) and toxin weapons and on their destruction, which came into force in 1975. Convention on the prohibition of the development, production, stockpiling and use of chemical weapons and on their destruction, which came into force in 1997. Global public health response to natural occurrence, accidental release or deliberate use of biological and chemical agents or radionuclear material that affect health, WHA55.16, 18 May 2002. http://www.who.int/cosr http://www.undmtp. rg/

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Epidemic Outbreak Systems (EOS) Microarray Incorporation for Pathogen Identification* Eric HANSON1, Robb ROWELY1, Brian AGAN2, Clark TIBBETTS2, Debra NIEMEYER3 Expeditionary Operations, Science & Technology Directorate, Office of the Air Force Surgeon General, Falls Church, VA 22041, 2Infectious Disease Department, WilfordHall USAF Medical Center, Lackland AFB, TX78236, 3Joint Program Office for Chemical and Biological Defense, Falls Church, VA 22041 Originally published in the Society of Armed Forces Medical Laboratory Scientists (SAFMLS) Newsletter, Society Scope Fall 2002; 5(3): 1. Reprint coordinated through Editor; modified for the NATO Conference Series Book

Abstract. A model is proposed for validating the use of oligonucleotide arrays (microarrays) in the rapid detection and identification of biological agents. Random samples will be obtained from a study population of 76,000 active duty military personnel requiring approximately 30,000 evaluations for respiratory infections per year. This high incidence density of infectious disease in a controlled military setting creates a real-world opportunity for implementing and validating genomics technologies for the rapid detection of disease causing pathogens. Program processes include rapid biologic agent identification (target time for detection is less than two hours); diagnostic correlation with specific syndromic indicators for outbreak and surveillance purposes; longitudinal tracking of affected personnel; and utilization of hybrid information system technologies for relationship, association and predictive modeling. Program outcomes include assessments of 1) clinical accuracy of respiratory pathogen diagnosis and therapeutics utilizing genotypic-phenotypic information correlation; 2) cross platform comparison with existing polymerase chain reaction technologies and 3) predictive modeling techniques for outbreak investigations and disease surveillance. Future research applications based on this test program will include microarray assessments of other biologic, chemical and physical exposures (i.e., ecogenomics) and incorporating a genomics module into an electronic surveillance system, the Lightweight Epidemiology Advanced Detection and Emergency Response System (LEADERS) for nationwide surveillance information.

Disclaimer. The conclusions and opinions expressed in this document are those of the authors. They do not reflect the official position of the United States Government, Department of Defense, Joint Program Executive Office for Chemical & Biological Defense, United States Army or Air Force.

E. Hanson et al. / Epidemic Outbreak Systems (EOS)

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1. Introduction The primary goal of the Epidemic Outbreak Systems (EOS) project is the rapid detection and identification of respiratory pathogens using genotypic specific information. The EOS project will serve as a model for microarray identification of organisms in an outbreak investigation. Success with this methodology will be a first step in revisiting the current methodology used in outbreak investigation protocols. This project also provides a real-world model for pathogen identification, which is required for early detection purposes in a scenario of a biologic agent release. Early pathogen detection and identification is key to disease prevention and maintenance of airpower projection. Case in point: 57% of personnel deployed in Desert Shield had diarrheal disease and 20% of the affected personnel were incapable of carrying out their duties [1]. Early detection of the diarrhea-associated pathogen can limit disease. This was demonstrated in December 1999 when a laboratory team deployed in Southwest Asia using new testing technology (real-time PCR) rapidly identified a food borne outbreak, thereby limiting the effects to only 3% of the base population with minimal mission impact [2,3]. Real-time PCR screening of a variety of environmental samples was key to alerting public health officials to improper food-handling practices. Subsequent molecular assessment of the food borne pathogen afforded determination of the contamination source4. The utility of PCR was again demonstrated when the technology was successfully applied to aid agent identification and clear sample backlogs following intentional release of anthrax October 2001 [5,6]. Worldwide, laboratories currently utilize culture for pathogen detection. This results in delays with specific pathogen identification, feedback to health care providers (HCPs) and proper implementation of public health interventions. Furthermore, this technology is difficult to use in the deployed environment. The end result is inadequate epidemiologic information for HCPs and other decision-makers to promptly stop disease transmission. Additional delays result from a combination of disjointed record-keeping often inherent in a mobile patient population, "shoe leather" epidemiologic techniques, inadequate real-time disease surveillance data and inability to access filtered or analyzed data. Advances in the fields of genomics and information systems have produced breakthroughs that will eliminate many of the current outbreak investigation problems.

2. The Approach To accomplish rapid pathogen identification, EOS will innovate current technology in microarrays. The EOS project will use low-density, high-throughput oligonucleotide-based microarrays for the rapid detection of respiratory pathogens in a controlled population. Basically, microarrays are miniature DNA-based testing platforms upon which a test sample is applied. Short pieces of single stranded DNA on the array corresponding to DNA of various pathogens will adhere to complementary DNA in the test sample. A visible signal is generated with DNA strand matching or hybridization (Figure 1) [7-9]. Innovations will include the combination of an advanced microarray substrate and matrix, sequence-specific identification of pathogens, and advanced signal detection methodologies. A project goal is the identification of a viral respiratory pathogen from clinical sample to identification in less than two hours. This represents outbreak investigation timesavings of over 500 hours based on current viral culture methods.

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E. Hanson et al. /Epidemic Outbreak Systems (EOS)

Figure 1. Microarray Technology a. A High Throughput Oligonucleotide Microarray [9] Custom arrays offering up to 500,000 features

106 -107 copies of each DNA probe per feature

b. Sample Preparation and Testing [7,8]

Clinical and epidemiological surveillance for infectious disease outbreaks in a large training population is critical to military mission accomplishment. This has been true throughout the history of warfare and the threat of infectious disease remains an omnipresent threat for the Department of Defense (DoD) to confront. Controlled populations in the DoD, Veterans Administration (VA) and civilian communities are numerous [10]. They include special operations training, deployed sites, military academies, prisons, nursing homes, childcare and even the dining facilities at the Pentagon. Time savings in pathogen identification in any one of these settings will significantly decrease to mission accomplishment and save significant amounts of time, rescued productivity, money and resources by decreasing morbidity and mortality in the event of a biological disease outbreak. EOS will demonstrate rapid pathogen detection capabilities in a controlled population and realworld setting. Likelihood of technology transition to civilian industry and other governmental


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agencies will be favorable when total cost-effectiveness evaluations are modeled for outbreak scenarios. Decreased microarray costs with increased production will follow the initial pilot studies proposed here. Data generated from the microarrays will be fed into a genomics module surveillance system to include the Lightweight Epidemiology Advanced Detection and Emergency Response System (LEADERS). LEADERS is currently being developed by the US Air Force and tested by DoD as a nationwide surveillance information system to provide early warning of aberrant events, particularly the presence of environmental hazards [11-13]. The goal of phase I is to standardize production of disease-specific diagnostic microarrays to improve the speed and sensitivity of respiratory pathogen detection in the Basic Military Training (BMT) population. Young recruits residing in close quarters are physically and mentally stressed during this training and this can increase their susceptible to a variety of communicable diseases. Close monitoring with improved pathogen identification will limit disease spread, diminishing morbidity and mortality. Phase II goals are performing comparisons with the current "gold standard" of viral cell cultures, and real-time polymerase chain reaction (PCR) technology. An additional application consideration for future development is a field-capable handheld microarray processing and reader unit. The assembled resources and team of USAF personnel partnering with other DoD institutions, civilian industry and academic leaders will provide the research setting and expertise to create this model system. This system will demonstrate the effectiveness of microarray technologies in pathogen identification. EOS will start with detection of viral respiratory pathogen-specific sequences, but the system proposed can be expanded to evaluate bacterial, fungal, parasitic and chimeric nucleotide sequences. Development efforts for EOS have emphasized future capabilities that allow not only direct hybridization and re-sequencing capabilities, but will evaluation for simultaneous assessment of biochemical reactions and proteomics capabilities.

3. Conclusion Challenges for the future microarray applications to overcome will be decreasing the expense of microarray production. It is not cost-effective to use microarrays to diagnose every respiratory pathogen seen in the HCPs office. However, this model will demonstrate utility of microarray use in a controlled population to rapidly diagnose militarily important disease processes in an operationally relevant model. Performing additional molecular assessments of biological, chemical, physical, and social exposures (i.e., ecogenomics) will be the logical progression in this developmental process. Continued collaborations within DoD, with other governmental agencies, industry and academia will be necessary to make this happen. References [1]

[2]

[3]

Hyms, KC, AL Bourgeois, BR Merrell, et.al. Diarrheal Disease During Desert Shield, NEJM, Vol 325:1423-1428, No. 20, November 1991; abstract: http://content.nejm.org/cgi/content/abstract/325/20/1423?maxtoshow=&HITS=10&hits=10&RESULTF ORMAT=&searchid=1015709380815 1301 l&stored search=&FIRSTINDEX=0&volume=325&firstpa ge= 1423&iournalcode=nejm. USAF Force Protection Battlelab News Release: Rapid Biological Agent Identification Initiative Enhances Force Protection through Early Foodborne Outbreak Detection, 20 June 2000: http://www.idahotec.com/rapid/success.htm. Niemeyer, D, M Corkern, W Mobley, M Eitutis, D Dubois, W Barnes, W Hamilton, K Lohman

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[4]

[5] [6] [7] [8] [9] [10] [11]

[12] [13]


R. McBride. Real-time PCR Speeds Laboratory Diagnosis to Rapidly Identify a Salmonella Outbreak at a Deployed Location. 24th Annual Meeting of Society of Armed Forces Medical Laboratory Scientists (SAFMLS) Proceedings, p 34, March 2000. Niemeyer, D, R Watson, N Wertz, A Salmen, F McCleskey. Salmonella Surveillance Using Molecular Genotyping-Part of a Prevention Oriented Process Approach to Food Safety in the Deployed Environment. Presented at the International Conference on Emerging Infectious Diseases, Atlanta, GA, Jul 00. Jernigan, J, et. al. Bioterrorism-Related Inhalational Anthrax: The First 10 Cases reported in the United States. Emerging Infectious Diseases, Nov-Dec 01;Vol.7, No. 6, Nov 01, Center for Disease Control: http://www.cdc.gov/ncidod/eid/vol7no6/jernigan.htm. Air Force Link. Air Force sends teams to help anthrax assessment. 22 October 2001: http://www.af.mil/news/Oct2001/n20011022_1502.shtml. Brazma, A, H Parkinson, T Schlitt, M. Shojatalab. A quick introduction to elements of biology—cells, molecules, genes, functional genomics, microarrays. October 2001. EMBL European Bioinformatics Institute: http://www.ebi.ac.uk/microarrav/biologv intro.htm. Brown, PO, D Botstein. Exploring the new world of the genome with DNA microarrays. Nature Genetics, Vol 21, Supplemental pp 33-37, 1999: http://www.nature.com/cgi-taf/DvnaPage.taf?file=/ng/journal/v21/nls/ruU/ng0199supp 33.html. Agan, B, E Walter. Presentation entitled, "Genomics and Bioinformatics: Revolutionizing Medicine through Integrated Technologies." USAF Force Protection Battlelab Meeting, 11 December 2002. Hanson, E., B Agan, L Folio, R Rowley, D Niemeyer. Evaluation of Potential Methods for Rapid Identification of Central Nervous System Pathogens Using Microarray Technology. Submitted for publication, Mil Med April 2003. Niemeyer, D, E Hanson, R. Rowley, R Munson, K Schafer, Real-time Medical Surveillance for Early Warning and Mitigation of Environmental Hazards. Proceedings of the International Conference on Protection Against Biological Threats, Sponsored by DARPA and the General Karol Kaczkowski Military Institute of Hygiene and Epidemiology, Warsaw, Poland, June 2001. Jackson, J. On the edge. Washington Technology, Vol 16, No. 20, 21 January 2002: http://www.washingtontechnology.com/news/16 20/emergingtech/17706-1.html. Graham-Rowe, D. Hospital internet system could spot bioterrorist attack. 17 December 2001: http://web.mit.edu/hst.921/www/IEEE.htm.

Preparedness Against Bioterrorism and Re-Emerging Infectious Diseases J. Kocik et al (Eds.) IOS Press, 2004

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Preventing Emerging Infectious Diseases: Epidemiology and Laboratory Capacity Support Stephen A. MORSE Bioterrorism Preparedness and Response Program, National Center for Infectious Diseases, Centers for Disease Control and Prevention Atlanta, Georgia 30333, USA Abstract. Epidemiology and laboratories have been vital components of the public health system. The realization that emerging infectious diseases were a major threat to the U.S. provided the stimulus for limited resources to enhance these capacities. However, the concern about the growing threat of bioterrorism has emphasized to need to further enhance the public health system including many of the same components and activities needed to respond to naturally occurring outbreaks of infectious diseases. A strengthened disease surveillance system will be important for detecting covert bioterrorist attacks. New laboratory systems and approaches can revolutionize disease surveillance by providing real-time data to epidemiologists.

1. Introduction By the middle of the twentieth century, it was widely believed that humans were winning the centuries-long war against infectious microorganisms [1]. However, this optimism was premature. Infectious diseases continue to be a menace to all people, regardless of age, gender, lifestyle, ethnic background, and socioeconomic status. They cause suffering and death, and impose an enormous financial burden on society. While some diseases have been conquered by improvements in urban sanitation and water quality, antibiotics and vaccines, new ones are constantly emerging (e.g., AIDS and hantavirus pulmonary syndrome), and others reemerge in drug-resistant forms (e.g., tuberculosis and malaria). In the early 1990s, the growing concern about the threat of emerging infectious diseases was highlighted in a report issued by the Institute of Medicine of the National Academy of Sciences [2]. The report emphasized the intimate links between U.S. health and international health and concluded that emerging infectious diseases were a major threat to U.S. health. There have been a number of events over the past few years that have focused attention on the growing threat of bioterrorism in the U.S. [3]. There is no guarantee that a terrorist will announce an attack. Therefore, without such an announcement, there will be no recognition that a biological attack is occurring until enough cases, perhaps including a number of fatalities, are observed and reported to allow recognition of an epidemic of an unusual disease. Many biological agents can cause illness in humans, but not all are capable of impacting public health and medical infrastructure on a large scale [4]. Historically, the military has been primarily concerned with an aerosol release of a biological agent on the battlefield [5], In

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contrast, public health preparedness activities have had to focus on a number of ways that a biological agent could be introduced into a civilian population. Thus, a bioterrorist may choose from among a number of different agents and dispersal mechanisms, e.g., aerosol, food, water, and infected animal vectors. Intentional release of pathogenic microorganisms could result in an outbreak similar to a naturally occurring one. Thus, with few exceptions (e.g., smallpox), an epidemiologic investigation would be needed to determine whether the outbreak was naturally occurring or due to the intentional release of an infectious microorganism. It has been recognized that there is also a need to improve the public health infrastructure to respond to bioterrorism [6], which would involve enhancing many of the same components and activities needed to respond to naturally occurring outbreaks of infectious diseases [7]. Public health has been a cornerstone of health protection and public safety, yet it has often lacked resources commensurate with its role, resulting in erosion in the capability of public health agencies to do their job [8].

1.1 U.S. Public Health Infrastructure In 1994, the Centers for Disease Control and Prevention (CDC) launched the first phase of a nationwide effort to revitalize national capacity to protect the public from infectious diseases [9]. This effort focused on four goals: improving disease surveillance and outbreak response; supporting research to understand and combat emerging infectious disease threats; preventing infectious diseases by implementing disease control programs communicating public health information; and rebuilding the infectious disease-control component of the public health infrastructure. An updated plan, which was organized under the same four goals, but in a different order, was published in 1998 [10]. The new document describes the second phase of CDC's strategy, taking into account new discoveries and challenges, and building on the experience, success, and knowledge gained from implementing the 1994 plan. In Goal I: Surveillance and Response, the objectives called for strengthening infectious disease surveillance and response in the U.S. and internationally, as well as improving methods for gathering and evaluating surveillance data. In Goal II: Applied Research, the objectives included improving tools for identifying and understanding emerging infectious diseases; determining risk factors for infectious diseases; and conducting research to develop and evaluate prevention and control strategies. The public health infrastructure is the underlying foundation that supports the response to emerging infections. In Goal III: Infrastructure and Training, the objectives and activities focused on enhancing epidemiologic and laboratory capacity in the U.S. as well as internationally. In the U.S., this required improving CDC's ability to communicate electronically with its partners as well as strengthening it's capacity to serve as a reference center for infectious diseases and drug-resistance testing. The objectives and activities of Goal III also addressed the need to enhance the nation's capacity to respond to outbreaks, including those caused by bioterrorism. All of the efforts described in the plan [10] are ultimately directed at the national and international implementation of Goal IV: Prevention and Control. 1.2 Global Infectious Disease Strategy It has been recognized that it is not possible to adequately protect the health of the U.S. without addressing infectious disease problems that occur elsewhere in the world [2]. In an age of

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expanding air travel and international trade, infectious microorganisms are transported across borders, carried by infected people, animals and insects, and contained within commercial shipments of contaminated food. Diseases can emerge in one region and spread throughout the world and foreign governments have come to rely on CDC to provide outbreak assistance and public health information whenever a new or reemerging disease threat is detected anywhere on the globe. This outbreak assistance would also be required if an intentionally caused outbreak occurred in the U.S. or abroad. CDC in consultation with public and private partners, both domestic and international, developed a document to further define it's global mission and to improve the capacity to detect, control, and prevent infectious diseases [11]. CDC's ongoing efforts to strengthen the U.S. public health infrastructure are critical to the success of its international efforts. The global infectious disease strategy defines priorities in six areas: international outbreak assistance; a global approach to disease surveillance; applied research on diseases of global importance; the application of proven public health tools; global initiatives for disease control; and public health training and capacity building. The priorities are described in Table 1. Table 1. Priorities for CDC's global infectious disease strategy, 2001-2002 (modified from [11]).

Area

Priority

International outbreak assistance

Dedicate specific resources (e.g., epidemiologic, diagnostic, and logistic) to international outbreak investigations.

Global approach to disease surveillance

Work with WHO and other partners to provide technical assistance to regional networks in Africa, Asia, and Latin America that can fill gaps in global disease surveillance and become components of a global network of networks.

Applied research on diseases of global Importance

Establish two or more long-term, on-site research collaborations in developing countries to test new strategies for disease control and prevention.

Application of proven public health tools

Work with developing country partner to launch a demonstration project that employs three or more proven public health tools to prevent and control infectious diseases, depending on local priorities.

Global initiatives for disease control

Work with foreign ministries and WHO on specific programs (e.g., eradication of polio and guinea worm disease, HIV/AIDS control).

Public health training and capacity building

Establish the first International Emerging Infections Program as a partnership among a ministry of health, CDC, a Field Epidemiology training Program, and one or more local universities or medical research institutes.

This article will address the enhancement of epidemiology and laboratory capacity and it's role in identifying and responding to a bioterrorism attack.

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2. Epidemiology and laboratory capacity (ELC) program The purpose of the Epidemiology and Laboratory Capacity for Infectious Diseases (ELC) program was to implement the CDC plan [9,10] by assisting state and eligible local public health agencies in strengthening their basic epidemiologic and laboratory capacity to address infectious disease threats. The ELC program has focused on notifiable diseases [10], food-, water-, and vector-borne diseases, vaccine-preventable diseases, and drug-resistant infections. The ELC program has supported activities that enhance the ability of a program to: identify and monitor the occurrence of infectious diseases of public health importance in a community; characterize disease determinants; identify and respond to disease outbreaks and other infectious disease emergencies; use public health data for priority setting and policy development, and; assess the effectiveness of activities. Funding for the ELC program began in fiscal year 1995 with ten awards and has grown steadily (Figure 2). By the end of fiscal year 2002, all states, six local health departments, and Puerto Rico had been funded for the ELC program. The average award per grantee was just over $900,000. Selected examples of how ELC program resources were used by states and large public health agencies to improve infectious disease surveillance and outbreak response are shown in Figure 3.

Figure 2. Epidemiology and Laboratory Capacity (ELC) program, fiscal years 1995 through 2000 (Courtesy of Debbie Deppe, CDC).


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Figure 3. Selected examples of how ELC program resources were used by states and large public health agencies to improve infectious disease surveillance and outbreak response (Courtesy of Debbie Deppe, CDC).

Strengthening collaboration between laboratory and epidemiology practice has been a crucial component of this plan. This collaboration is exemplified by the role of PulseNetTM, which was initially funded through the ELC program, in food-borne disease surveillance. Food has been used as a vehicle to intentionally spread infectious microorganisms among civilians [12,13]. Thus, PulseNet™, or a similar system, may be useful in detecting a bioterrorism event involving food. PulseNet™ is a national laboratory network, which includes state, local and federal public health laboratories. The laboratories perform standardized molecular typing of foodborne, disease-causing bacteria by pulsed-field gel electrophoresis (PFGE) using a rapid one-day protocol [14]. The PFGE patterns are shared electronically by participants via the Internet and are compared to a dynamic database of PFGE patterns maintained at CDC. PulseNet™ functions as a "cluster" detection tool. Clusters, which are identified by PulseNet™ are investigated by epidemiologists. If epidemiologic links are found between cases, the "cluster" is classified as an outbreak. Thus, PulseNet™ can both facilitate the early identification of common source outbreaks as well as assist in rapidly identifying the source of the microorganism. PulseNetTM began in 1996 with a standardized PFGE protocol for Escherichia coli O157:H7 and 10 participating laboratories. By 2002, it had grown to 65 participating laboratories with standardized protocols for additional important food-borne pathogens including Salmonella serotypes, Shigella spp., Listeria monocytogenes, and Clostridium perfringens. A similar network has been developed in Canada. Participating laboratories receive training and are certified. Laboratories also participate in proficiency testing. These


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networks have revolutionized food-borne disease surveillance in North America by providing real-time data to epidemiologists. An example of the impact that PulseNetTM on an outbreak of E. coli O157:H7 due to contaminated meat is shown in Figure 4. It has been estimated that the outbreak would have been detected about a week earlier and that the number of cases would have been reduced by 68%.

Figure 4.

Estimated impact of PulseNetTM if the 1993 multistate food-borne outbreak of Escherichia coli O157:H7 had occurred in 1998 (Courtesy of Balasubra Swaminathan, CDC).

3. Epidemiology and laboratory capacity for detecting a bioterrorism event An evaluation of the vulnerability of the U.S. to an attack on civilians with biological agents has focused on the role public health would have in detecting and managing a covert bioterrorist incident [15]. It was earlier noted that the local, state, and federal public health infrastructure was already stretched thin as a result of other public health problems and lack of resources. Laboratories are a vital component of the public health system. Local, state, and federal public health laboratories have been protecting the health of the U. S. population for more than a century [16]. They detect and report infectious diseases and work in concert with epidemiologists to control outbreaks, including those resulting from a covert bioterrorist incident. Laboratory reports form the basis of active surveillance systems [17], which will be needed for early detection and to determine the extent of a covert incident. The epidemiological skills, surveillance methods, diagnostic techniques, and resources required to identify and respond to an attack with a biological agent are similar to those required to detect and investigate unusual or unknown diseases. Although additional resources were needed to rebuild the public health infrastructure and reduce U. S. vulnerability to biological terrorism, the new resources would also strengthen U. S. capacity to respond to any outbreak of infectious disease. CDC's strategic plan for biological and chemical terrorism preparedness and response


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[15] was implemented in 1999 through funding of cooperative agreements to states and several large municipalities. Awards were based on up to five focus areas, with each area integrating training and research. These focus areas were: preparedness and planning; detection and surveillance; diagnosis and characterization of biological and chemical agents; response; and communication. After three years of funding, there were substantial improvements (see article on the Laboratory Response Network); however, additional preparedness efforts were needed. In the aftermath of the incidents involving the World Trade Center disaster and the dissemination of spores of Bacillus anthracis via the mail [18,19], a supplemental allocation of approximately $915 million went to all state health departments and select major metropolitan cities and territories for bioterrorism preparedness. In addition to the original focus areas, two new focus areas were added: risk communication and health information dissemination; and, education and training. Of these funds, $183 million went toward epidemiology and surveillance activities and $119 million went for laboratory capacity. There was also awareness that the issue is broader than bioterrorism. These funds support state and local health departments as they greatly enhance their surveillance, epidemiology, and laboratory capacity for bioterrorism, other infectious disease outbreaks, and other public health threats and emergencies. CDC is working to integrate surveillance for illness resulting from biological terrorism into a strengthened U.S. disease surveillance system. As part of this program, there is increased accountability on the part of the awardees to ensure that specific goals are met, such as increasing the number of epidemiologists in a given population area and strengthening ties with the public health laboratory.

4. Surveillance Systems for Emerging Infections There are a number of surveillance systems that have been developed that are useful in detecting outbreaks of emerging infectious diseases or illness due to the covert release of infectious microorganisms. Some examples are described below. The Emerging Infections Network (EIN), which functions as a sentinel system to monitor new or resurgent infectious diseases, was developed by the Infectious Diseases Society of America (IDSA) in cooperation with CDC [20]. Members of the EIN include adult and pediatric infectious disease consultants. Its membership represents a ready source of infectious disease expertise for CDC and state health departments to draw on during outbreaks or when unusual illnesses occur. EIN is a bridge between the infectious diseases and public health communities. GeoSentinel is a global surveillance network consisting of 22 travel/tropical medicine clinics located in the U.S. and elsewhere. It was begun in 1995 by the International Society of Travel Medicine and is based on the concept that these clinics are ideally situated to effectively detect geographic and temporal trends in morbidity (specific etiology or as syndromes) in travelers. The U.S.-Mexico Border Infectious Diseases Surveillance (BIDS) Project was designed to coordinate syndromic surveillance for infectious diseases (e.g., syndromes consistent with hepatitis and febrile-rash illness) along the 2,000-mile U.S.-Mexican border. EMERGEncy ID NET is a network of academically affiliated emergency medicine centers that operate emergency departments at 11 hospitals in large U.S. cities [21]. The network monitors a number of syndromes, including bloody diarrhea, illnesses that follow exposure to animals, and illnesses in immigrants. Laboratory-based surveillance for emerging antimicrobial resistance is also a critical element in preventing emerging infectious diseases. A recent example is the emergence of vancomycin-resistant Staphylococcus aureus in the U.S. and the necessity for laboratory testing [22,23].

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5. Summary Preparedness for bioterrorism will require certain specialized programs and policies; however, many aspects of bioterrorism preparedness will use strategies that are built on existing systems that are used routinely for naturally occurring infectious disease threats. Epidemiology and laboratories have been vital components of public health. Strengthening the public health infrastructure for surveillance and outbreak response, including on-the-ground epidemiologic expertise and enhanced laboratory capability will have a dual use in protecting the U.S. against emerging infectious diseases and covert bioterrorism attacks. References [1] [2] [3] [4] [5] [6] [7]

[8] [9] [10] [11] [12] [13] [14] [15]

Burnet, M., and D. O. White. 1972. Natural History of Infectious Diseases. Cambridge University Press, London, U. K. Institute of Medicine, National Academy of Sciences. 1992. Emerging Infections. Microbial Threats to Health in the United States. National Academy Press, Washington, D. C. Tucker, J. B. 1999. Historical trends related to bioterrorism: an empirical analyis. Emerg. Infect. Dis. 5:498-504. Rotz, L. D., A. S. Khan, S. R. Lillibridge, S. M. Ostroff, and J. M. Hughes. 2002. Public health assessment of potential bioterrorism agents. Emerg. Infect. Dis. 8:225-230. Eitzen, E. M., Jr. 1997. Use of biological weapons, p. 437-450. In Sidell, F. R., E. T. Takafuji, and D. R. Franz (ed.), Medical Aspects of Chemical and Biological Warfare, Textbook of Military Medicine, Part 1. Office of the Surgeon General, Washington, D. C. Institute of Medicine, National Academy of Sciences. 1999. Chemical and Biological Terrorism, National Academy Press, Washington, D. C. LeDuc, J. W., S. M. Ostroff, J. E. McDade, S. Lillibridge, and J. M. Hughes. 1999. The role of the public health community in detecting and responding to domestic terrorism involving infectious agents, p. 219230. In W. M. Scheld, W. A. Craig, and J. M. Hughes (ed.), Emerging Infections 3. ASM Press, Washington, D. C. Hamburg, M. A. 2002. Public health preparedness. Science 295:1425. Centers for Disease Control and Prevention. 1994. Addressing emerging infectious disease threats: a prevention strategy for the United States. Public Health Service, U. S. Department of Health and Human Services, Atlanta, Ga. Centers for Disease Control and Prevention. 1998. Preventing emerging infectious diseases: a strategy for the 21st century. Public Health Service, U. S. Department of Health and Human Services, Atlanta, Ga. Centers for Disease Control and Prevention. 2002. Protecting the nation's health in an era of globilization: CDC's global infectious disease strategy. Public Health Service, U. S. Department of Health and Human Services, Atlanta, Ga. Torok, T. J. R. V. Tauxe, R. P. Wise, et al. 1997. A large community outbreak of salmonellosis caused by intentional contamination of restaurant salad bars. J. A. M. A. 278:389-395. Kolavic, S. A., A. Kimura, S. L. Simons, L. Slutsker, S. Barth, and C. E. Haley. 1997. An outbreak of Shigella dysenteriae type 2 among laboratory workers due to intentional food contamination. J. A. M. A. 278:396-398. T. J. Barrett, H. Lior, J. H. Green, et al. 1994. Laboratory investigation of a multistate food-borne outbreak of Escherichia coli O157:H7 by using pulsed-field gel electrophoresis and phage typing. J. Clin. Microbiol. 32:3033-3037. Centers for Disease Control and Prevention. 2000. Biological and chemical terrorism: strategic planning, preparedness and response. Recommendations of the CDC strategic planning workgroup. Morbid. Mortal. Wkly. Rep. 49(RR04):1-14.

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[16] [17] [ 18] [19] [20] [21] [22] [23]

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Anderson, G., A. DiSalvo, W. Hausler, J. Liddle, J. McDade, and J. Sampson. 1993. Task force report on the public health laboratory: a critical national resource. Association of Public Health Laboratories, Washington, D. C. Roush, S., G. Birkhead, D. Koo, A. Cobb, and D. Flemming. 1999. Mandatory reporting of diseases and conditions by health care professionals and laboratories J. A. M. A. 282:164-170. Centers for Disease Control and Prevention. 2001. Recognition of illness associated with the intentional release of a biologic agent. Morbid. Mortal. Wkly. Rep. 50:893-897. Jernigan, J. J., P. L. Raghunathan, B. B. Bell, et al. 2002. Investigation of bioterrorism-related anthrax, United States, 2001: epidemiological findings. Emerg. Infect. Dis. 8:1019-1028. Executive Committee of the IDSA Emerg. Infect. Network. 1997. The emerging infections network: a new venture for the Infectious Diseases Society of America. Clin. Infect. Dis. 25:34-36. Talan, D. A., G. J. Moran, W. R. Mower, et al. 1998. EMERGEncy ID NET: an emergency departmentbased emerging infections sentinel network. Ann. Emerg. Med. 32:703-711. Centers for Disease Control and Prevention. 2002. Staphylococcus aureus resistant to vancomycin— United States, 2002. Morbid. Mortal. Wkly. Rep. 51:565-567. Centers for Disease Control and Prevention. 2002. Public health dispatch: vancomycin-resistant Staphylococcus aureus—Pennsylvania, 2002. Morbid. Mortal. Wkly. Rep. 51:902.

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The Laboratory Response Network Stephen A. MORSE, Richard KELLOGG, Samuel R. PERRY, Richard F. MEYER, David BRAY, David NICHELSON, Michael J. MILLER Bioterrorism Preparedness and Response Program, National Center for Infectious Diseases, Centers for Disease Control and Prevention Atlanta, Georgia 30333, USA Abstract. The Laboratory Response Network is an international, unified consortium of laboratories that was established in response to the threat of bio-terrorism. The Laboratory Response Network is fully capable of responding quickly and appropriately to national or local public health emergencies. It has the capacity to respond to both bioterrorism events and naturally occurring outbreaks of infectious disease.

1. Introduction There have been a number of events over the past few years that have focused attention on the growing threat of bioterrorism in the United States [1]. From the public health perspective, bioterrorism is defined as the deliberate release of pathogens or their toxins into a population for the purpose of causing illness or death. Although some authorities had initially felt that the threat of bioterrorism was exaggerated [2], the recent incident using spores of Bacillus anthracis has made bioterrorism a reality in the United States [3], and has focused attention on national preparedness should another crisis occur. The Centers for Disease Control and Prevention (CDC) was designated by the Department of Health and Human Services to prepare the nation's public health system to respond to a bioterrorism event [4]. To enhance state and local preparedness, CDC funded cooperative agreements with every state and several large municipalities that focused on preparedness efforts [5]. Five critical areas were emphasized during the first three years of this program that began in 1999: preparedness planning and readiness assessment; surveillance and epidemiology capacity; biologic laboratory capacity; chemical laboratory capacity; and health alert network and information technology [4]. Based on lessons learned from the recent anthrax attack [3], additional resources and focus areas have been added: communicating health risks and health information dissemination; and, education and training. Bioterrorist attacks can occur as one of two scenarios, i.e., covert and overt. Because we currently lack the ability to conduct real-time monitoring for the release of a biological agent in U. S. cities, an unannounced (i.e., covert) release of a biological agent would likely go unnoticed for some time, with those exposed leaving the area long before the act of terrorism becomes evident. Due to an incubation period, the first signs that a biological agent has been released may not become apparent until days or weeks later, when individuals become ill and seek medical care. Thus, the "first responders" to a covert bioterrorism attack will likely be the astute clinician, laboratorian, or public health worker who recognizes the index case or identifies the responsible agent. Because of their terrorism training, traditional "first

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responders" (e.g., firefighters, hazmat or law enforcement personnel) are the most likely to respond to an announced (i.e., overt) release of a biological agent or, more likely, to a hoax. Thus, the initial recognition of a bioterrorism event (or hoax) in the U. S., whether announced or unannounced, would be at the local and state level. A comprehensive public health response to bioterrorism (or for that matter, to any outbreak of infectious disease) will involve epidemiologic investigation, medical treatment and prophylaxis for affected persons, and the initiation of disease prevention activities. The success of these activities is dependent, to a large extent, upon a rapid and accurate identification of the threat agent. 2. Agents Many biological agents can cause illness in humans, but not all are capable of impacting public health and medical infrastructures on a large scale [6]. In order to bring focus to public health preparedness activities, CDC convened a meeting of national experts in 1999 to review the criteria for selecting the biological agents that posed the greatest threat to civilians and to help develop a prioritized list of agents [6]. This list of "Critical Agents" (Table 2) was prioritized based on considerations such as: the ability of the agent to cause mass casualties; the ability of the agent to be widely disseminated either by aerosol or other means; the ability of the agent to be transmitted from person to person; the public's perception, correctly or incorrectly, associated with the intentional release of the agent; and special public health preparedness needs (e.g., vaccines, therapeutics, enhanced surveillance, or diagnostics). Table 2. Critical biological agents for public health preparedness (modified from [5]) Agent

Disease

Category A Variola major Filoviruses (e.g., Ebola and Marburg) Arenaviruses (e.g., Lassa and Junin) Bacillus anthracis Yersinia pestis Francisella tularensis Clostridium botulinum neurotoxin Category B

Smallpox Hemorrhagic fever Lassa fever, Argentine hemorrhagic fever Anthrax Plague Tularemia Botulism

Alphaviruses (e.g., Venezuelan, Eastern, and Encephalomyelitis Western encephalomyelitis viruses) Q fever Coxiella burnetti Brucella spp. Brucellosis Burkholderia mallei Glanders Staphylococcal enterotoxin B Staphylococcal food poisoning Ricin from Ricinus communis Ricin intoxication Clostridium perfringens epsilon toxin Food- and water-borne agents including (but not limited to): Salmonellosis Salmonella species Bacillary dysentery Shigella dysenteriae Escherichia coli O1 57: H7 Hemolytic uremic syndrome

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S.A. Morse et al. / The Laboratory Response Network Vibrio cholerae Cryptosporidium parvum Category C

Cholera Cryptosporidiosis

Multidrug-resistant Mycobacterium tuberculosis Yellow fever virus Tickborne encephalitis complex (flavi) viruses Tickborne hemorrhagic fever viruses Nipah and Hendra Complex viruses Hantaviruses

Tuberculosis Yellow fever Encephalitis Hemorrhagic fever Hantavirus pulmonary syndrome

As currently defined, Category A, which includes some of the classic biowarfare agents, are high priority agents that are most likely to cause mass casualties if deliberately disseminated and require broad-based public health preparedness efforts. Natural infections caused by agents in Category A are uncommon in the U. S. or nonexistent. For example, prior to the bioterrorist attack with B. anthracis in 2001, the last case of inhalational anthrax in the U. S. was in 1976 [7]. Furthermore, the World Health Organization declared smallpox eradicated in 1977 [8]. Category B agents also have some potential for large-scale dissemination, but generally cause less illness and death than those in Category A. Many of these agents have been weaponized in the past, or are being considered as weapons by some state-sponsored programs [9]. Some of the Category B agents could be used to contaminate food or water sources. In addition, many of these agents are relatively easy to obtain, and are thus more likely to be used in the setting of a biocrime [10]. Biological agents that are not currently believed to present a high bioterrorism risk to public health, but which could emerge as future threats were placed in Category C. Some of these agents are associated with emerging infections or have characteristics that, in the future, could be exploited for deliberate dissemination. The Critical Agent list has been used by the National Institutes of Health (NIH) in establishing priorities for bioterrorism-related research [11]. In the U. S., both clinical and laboratory experience is limited for the recognition and confirmation of the Category A agents and for many of the agents in Category B. The low numbers of human infections in the U. S. caused by Category A agents (as well as for many of those in Category B) has been given as a reason why there has been a general lack of interest by the commercial sector in spending money for the development, manufacture, and FDA approval of diagnostic tests. This situation has created the need for the development and restricted distribution of biodetection assays and specialized reagents, which would not otherwise be available to support the public health infrastructure and national security interests of the U. S. [12].

3. Laboratory Response Network Some individuals had suggested that a single high-throughput laboratory or perhaps a few regional laboratories would be sufficient to detect and respond to a bioterrorist attack in the U.S. However, an effective public health response would have to be rapid since there is only a small window of opportunity during which prophylaxis or other control measures could be implemented to reduce the morbidity and mortality associated with a bioterrorism event [13]. In order to facilitate the rapid identification of threat agents, the Laboratory Response Network (LRN) was created. The LRN was initially established as a national system designed to link


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state and local public health laboratories with advanced capacity clinical, military, veterinary, agricultural, water- and food-testing laboratories. It is a critical component of CDC's mission to lead the effort in strengthening the public health infrastructure, and consequently enhancing readiness to detect and respond to bioterrorism at the federal, state and local levels. Operational in August 1999, the LRN was established in recognition that the existing national infrastructure of diagnostic testing laboratories competent to deal with biological (or chemical) terrorism was extremely limited. The LRN was developed by the CDC in concert with the Association of Public Health Laboratories (APHL) and with collaboration from the Federal Bureau of Investigation (FBI) and the United States Army Medical Research Institute of Infectious Diseases (USAMRIID). The LRN is the first example of a public health-law enforcement partnership. It was conceived to build on the existing interaction of nationwide public health laboratories, which participate in routine disease surveillance activities [14]. The LRN has a dual function in that it has the ability to detect and respond to agents released by a bioterrorist as well as to those that occur naturally. This capacity is particularly important, since it will generally not be known at the time of detection whether the outbreak was intentional or natural. The LRN collaborative partnership operates as a national network of laboratories designated level A through level D. Because there were marked differences in capabilities and infrastructure, each member laboratory initially provided an agent-specific designation of level A, B, or C. Each of these levels represented progressively stringent levels of safety, containment, and technical proficiency necessary to perform the essential tests to rule-out, rulein, as well as perform the referral functions that are required for agent identification and confirmation (Figure 5A). Thus, a laboratory could be level C for botulinum toxin testing and level B for anthrax.

Figure 5. Conceptual diagram of the Laboratory Response Network as it was conceived in (A) 1999; and, a proposed diagram to reflect the expansion of the Laboratory Response Network to include other types of sentinel laboratories (2002)

Level A laboratories are, for the most part, hospital and other community clinical laboratories. The reasoning behind this decision is that in the aftermath of a covert bioterrorism

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attack, patients will seek care at many widely dispersed hospitals where these laboratories exist [14]. These laboratories participate in the LRN by ruling out or referring critical agents (Table 2) that they encounter in their routine work to the nearest LRN level B or C laboratory. To make this process as rapid as possible, protocols and algorithms have been developed in collaboration with the American Society for Microbiology (ASM) that can be implemented by the clinical laboratory in its routine day-to-day operation. The algorithm for Bacillus anthracis is shown in Figure 6.

Bacillus anthracls: Level A laboratory flowchart Morphology: Large aerobic, gram positive rods (1 to 1.5 by 3 to 5 (in.) Smears/blood/CSF: Short chains of 2-4 Cells that appearencapsulated Sheep blood agar (amble nt atmosphere): Oval, central-to-sub terminal spores which do not cause significant swelling of cell; often In long chains Growth on sheep blood agar. 2-5 mm, tenacious, nenhemolytic colonies after 15-24 h (flat/slightly convex, Irregularly round colonies with Irregular/wavy border and ground glass appearance) Perform all additional work In biosafety cabinet Hemolysis: Negative Catalase: Positive Motility: Nonmotile

NO

(features net present) Report:Bacillusspecles, NOT B. anthracls; continue Identification per laboratory procedures

Yes (features present) Report Bidllus spedes, sent to reference laboratory to rule out a anthracis

Figure 6. Algorithm developed for the rapid rule-out of Bacillus anthracis by clinical laboratories

This algorithm could be used to rapidly rule-out B. anthracis from blood cultures growing Gram positive rods. The algorithms for B. anthracis, Yersinia pestis, Francisella tularensis, Clostridium botulinum neurotoxin, and Brucella spp. can be accessed on the internet at either www.asmusa.org or www.bt.cdc.gov. In order to make the referral process work effectively, level A (i.e., clinical) laboratories must know the location of the nearest LRN level B or C laboratory. This has been facilitated through an educational process carried out at the state and local levels. At the beginning of the program, Level B laboratories were primarily state and local public health laboratories, with biosafety level 2 facilities where biosafety level 3 practices are observed. Level C laboratories were primarily public health laboratories with biosafety level 3 facilities, or those with certified animal facilities, which are necessary for performing the mouse toxicity assay for botulinum toxin. Level C laboratories could perform all of the Level B tests as well as additional tests that required biosafety level 3 containment (e.g., handling powders suspected of containing anthrax spores). Many Level B/C laboratories work closely with local FBI agents. This partnership was especially evident during the recent anthrax attack when FBI agents helped LRN laboratories triage specimens for analysis by performing threat assessments. There are currently 110 LRN level B and C laboratories in the U. S. and Canada,


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with new members being added quickly. All 50 states now have the capacity to perform presumptive and confirmatory laboratory identification methods for B. anthracis, Y. pestis, and F. tularensis as well as access to specialized level C laboratories for confirmation of Brucella spp. and C. botulinum neurotoxin. With the increased threat of smallpox and the plans to begin smallpox vaccination, all LRN level B and C laboratories have been given the capacity to test for Vaccinia virus and Varicella zoster; selected laboratories will have the capacity to test for Variola major. There are two Level D federal laboratories (CDC and USAMRJID) with biosafety level 4 facilities that can handle agents such as Ebola and Variola major, for which other laboratories have insufficient safety facilities or unvaccinated staff. Level D laboratories also have the capacity to perform all of the Level B and C procedures. In addition, they identify agents in specimens (i.e., human and animal) and samples (i.e., environmental, food, etc.) that have been referred by the Level B and C laboratories, identify recombinant microorganisms (e.g., chimeras) that may not be recognizable by conventional isolation and identification methods or by those used in the Level B/C laboratories. Level D laboratories also maintain extensive culture collections of critical agents against which the isolate(s) from a bioterrorist event may be compared using molecular methods to determine its likely origin. Over the past four years, the LRN has matured and clear distinctions between Level B and Level C have all but vanished because of the improvements that were made in every state as a result of the funds provided by the U. S. Congress to strengthen the public health infrastructure. Among the improvements were the construction of a number of biosafety 3 laboratories, hiring of additional personnel, and the purchase of state-of-the-art equipment for performing real-time nucleic acid amplification and time-resolved fluorescence antigen detection assays. As a result, a new description of the structure is currently being developed. The LRN is now more accurately described as a unified network of integrated laboratories functioning through a single operational plan and represented by sentinel laboratories of laboratory 1st responders (formerly Level A) and by confirmatory testing laboratories (formerly Levels B, C, and D) representing several disciplines (Figure 5B). The maturity of the LRN structure is commensurate with a parallel increase in technical capacity and response ability of the public health system. The basis of admission into the LRN is predicated by: (1) the laboratory's contribution to the public health needs of the state; (2) the ability of the laboratory to meet safety requirements for handling select agents [15]; (3) the ability of the laboratory to meet physical and personnel security requirements [16,17]; (4) successful completion of a checklist evaluating these and other items; and (5) participation in the secure communication of test results. For non-public health laboratories, such as clinical laboratories, to become members of the LRN in a confirmatory role, requires documentation from the state public health laboratory director that there is a need for the additional laboratory capacity and that the invited laboratory meets all requirements. CDC's role through the LRN is to support the public health infrastructure, which is defined by public health laboratory work. In this regard, CDC currently develops, produces, validates, packages, and ships all reagents used in the rapid screening tests developed by CDC laboratories and its federal partners for these agents. State funding for the LRN has come from the Federal cooperative agreements, which began in 1999. In the first year of this program, 43 project areas (41 states, New York City and Los Angeles County) received $8.7 million (range of award was $58,755-$566,906, with an average award of $203,000) to enhance public health laboratory capacity to respond to a bioterrorism event. In order to extend laboratory coverage throughout the country, states that were not initially funded were invited to join the LRN and were thus able to receive protocols,

32


reagents and training. By the beginning of the third year, and prior to 9/11/2001, the number of funded project areas increased to 54 (50 states, New York City, Los Angeles County, and Guam). During the first three years of this program, a total of $23.1 million was awarded to enhance laboratory capacity to respond not only to bioterrorism, but to any outbreak of infectious disease. In the aftermath of the anthrax attack, project areas received supplemental awards totaling $118 million for biologic laboratory capacity. The LRN has been strengthened through collaborations with the Department of Defense (Army, navy, Air Force), Department of Justice (FBI), Department of Energy (Lawrence Livermore and Los Alamos national Laboratories), Food and Drug Administration (Centers for Food Safety and Devices and Radiological Health), U. S. Department of Agriculture (National Veterinary Service Laboratory and Food Safety Inspection Service), Environmental Protection Agency (Office of Research and Development), and National Institutes of Health (Office of Research Service), as well as professional societies (American Society for Microbiology), and public health organizations (Association of Public Health Laboratories), thus leveraging not only highly developed expertise, but also galvanizing larger networks for emergency response and overall continuity of operations. Many of these collaborations have been incorporated into the new proposed LRN structure (Figure 5B). The LRN has cautiously begun to expand internationally. In 2000, two laboratories in Canada were added to the LRN. More recently, laboratories in the United Kingdom and Australia have become members. 3.1 Protocols, Reagents and Assays Confirmatory laboratories use standard protocols and reagents for the identification and confirmation of threat agents. Bioterrorism is a criminal act and specimens or cultures will be evidence in a criminal investigation. Thus, the protocols also have information concerning chain of custody requirements. Protocols for the Category A and B agents were written by subject matter experts at CDC, USAMRIID and the FBI and reviewed for accuracy and ease of use by laboratorians representing the LRN. The protocols, which are available to LRN members on a secure web site currently managed by CDC, contain the information for ordering the necessary reagents and control strains for performing the tests. Technologically, many U. S. public health laboratories have lagged behind those in the private sector. Thus, the original protocols relied on techniques (e.g., culture and staining with fluorescein-labeled antibodies [DFA]) that were already familiar to all public health laboratories to identify many of the critical Category A agents. Due to the lack of commercially available DFA reagents, the LRN relied on reagents produced at CDC or purchased from USAMRIID. Subsequently, protocols were updated as new, rapid assays were developed and validated [18]. The focal point for the advances in technology within the LRN was the Rapid Response and Advanced Technology (RRAT) laboratory at CDC, which was created in 1999. The RRAT laboratory is a vital component of CDC's bioterrorism preparedness effort and a partner to the LRN where it serves as a source of test methods, validation data, training, and proficiency testing for the member laboratories. The RRAT laboratory develops novel approaches to molecular screening for the Category A and B agents using real-time polymerase chain reaction (RT-PCR) assays, time-resolved fluorescence (TRF) immunoassays, and other technologies. LRN laboratories were given the option of choosing which commercially available thermocycler capable of performing real-time PCR was best suited for its laboratory. With the availability of supplemental funds, many laboratories


33

purchased additional instruments. The instruments for which RT-PCR assays were optimized by the RRAT laboratory were: LightCycler (Roche Diagnostics Corporation, Indianapolis, IN); SmartCycler (Cepheid, Sunnyvale, CA); and the GeneAmp 5700, Prism 7000 and 7700 (Applied Biosciences, Foster City, CA). Protocols for RT-PCR assays for the detection of nucleic acid from a number of Category A agents have been added to the LRN web site together with protocols for TRF assays for antigen or antibody detection. The Victor2 (Perkin Elmer-Wallach) instrument was selected for the TRF assays because many state public health laboratories were using a similar instrument for newborn screening assays. The new rapid assays have markedly reduced the amount of time it takes to make an identification of a critical agent. For example, culture may take 24 to 72 hours to grow and identify the agent; with DFA staining, some agents could be identified in 3 to 4 hours. Today, using technology available in LRN laboratories, an answer can be obtained in approximately 1 hour. 3.2 Training Training has always been considered critical in the overall effort to prepare the nation to respond to a bioterrorism attack. Initial efforts centered on increasing the awareness of the clinical laboratories to the threat of bioterrorism, the agents of concern, and the LRN. A 1-day course, cosponsored by CDC and the National Laboratory Training Network (NLTN), was presented during the summer of 1999 in several cities in the U. S. (Atlanta, GA; Boston, MA; Philadelphia, PA; Denver, CO; Chicago, IL; Dallas, TX; Phoenix, AZ; Seattle, WA). More than 600 individuals attended these courses. Since 1999, the NLTN has put on more than 115 awareness courses for clinical microbiologists involving 4,915 students. In an attempt to reach additional individuals who worked in clinical laboratories, talks were presented at national and regional meetings of professional societies (e.g., ASM, American Society for Clinical Pathology). The presentations at these meetings were publicly available on CDC's bioterrorism web site. The results of a survey of state public health laboratory capacity conducted by APHL during the summer of 1998, indicated that most respondents had limited experience in identifying the bacterial agents in Category A and B. In order to address this need, a one-week course consisting of lectures and laboratory training was developed and offered four times in the fall of 2000 at the Georgia State Public Health Laboratory. This course addressed the Level B protocols for B. anthracis, Y. pestis, F. tularensis, and Brucella spp. Additional topics included the role of the RRAT laboratory, handling hazardous evidence, and automated microbial identification systems. One individual representing each of the cooperative agreement sites as well as individuals from states that did not receive funding were invited to attend. The intent of the course was to train additional trainers. After completion of the course, these individuals were expected to return to their respective public health laboratories and train additional laboratorians in their respective jurisdictions. However, the results of a recent survey suggested that some of these "trainers" have not trained additional laboratorians [19]. Rapid nucleic acid amplification and antigen detection assays and protocols were developed and validated by the RRAT laboratory and placed on the LRN web site in October 2001. The bioterrorism-related anthrax attack caused a delay in the training necessary for performing these assays. Six one-week training courses for these RT-PCR and TRF were completed by the spring of 2002. Again, one individual representing each of the cooperative


34

agreement sites received training and was expected to return to their respective laboratory and train additional laboratorians.

3.3 LRN Response The LRN played a critical and successful role in the nation's response to the bioterrorismrelated anthrax incidents during October through November 2001. The LRN concept was validated when on October 3, 2001 the LRN laboratory in Jacksonville, FL rapidly confirmed that the Gram positive rod isolated from the cerebrospinal fluid and blood of the index case in Palm Beach County was B. anthracis [20]. Although the bioterrorism-related anthrax cases were limited to four states (Florida, New Jersey, New York, Connecticut) and the District of Columbia [21], the impact was felt nationwide. During this period, LRN laboratories tested over 125,000 clinical specimens and environmental samples involving approximately 1 million assays: 69% of the specimens were processed by public health LRN laboratories, 25% by Department of Defense LRN laboratories, and 6% by the CDC. Of particular interest was the observation that the majority of the environmental specimens were from states where there was no anthrax (Figure 7). Most of these were either hoaxes or specimens provided by frightened or concerned individuals.

Figure 7.

Environmental and clinical specimens processed by LRN laboratories, October - November 2001.The black portion of the graph represents specimens processed by laboratories in states where there were anthrax cases; the gray portion of the graph represents specimens processed in states where there were no anthrax cases.


35

3.4 Data Management and the LRN Coordination of the LRN's daily operations is entrusted to a dedicated help desk resource that acts as a clearinghouse and funnels tasks to the appropriate individuals. This eliminates confusion by providing a single point of triage for all ongoing membership activities from which the needs of laboratories and laboratorians can be reviewed and tracked. An added benefit has been the positive feedback from LRN member laboratories, who have been reassured by the presence of a committed help desk and point of contact with whom they could develop a relationship. A Data Management Team was established to address the IT needs of the LRN. For example, to support electronic reporting of proficiency testing results through the LRN website, a reporting database was designed and built, which permitted LRN member laboratories to compare the results they had submitted to the correct results along with a grade of each laboratory's performance. A database of LRN members eligible for the licensed anthrax vaccine was also developed. To track the number of laboratorians desiring the vaccine, each laboratory was contacted, information was collected, and subsequently conveyed upon request to a contractor who was administering the vaccine. Currently, near term and long term solutions are being developed for the secure, real-time reporting of laboratory data as well as the sharing of information that is critical to the public health response to bioterrorism.

4. Summary The LRN is an international, unified laboratory consortium that was established in response to the threat of bioterrorism that is fully capable of responding quickly and appropriately to national or local public health emergencies. Funding for the LRN has helped rebuild the country's public health laboratory capacity to respond to a bioterrorism event as well as to naturally occurring outbreaks .of infectious disease. By providing funding for infrastructure, state-of-the-art equipment, and personnel, LRN laboratories are in a better position to respond to infectious disease threats. A well-trained laboratory workforce, including American Board of Medical Microbiology (ABMM) or equivalently certified specialists, is essential for national security. However, this program was not designed to address the current nationwide shortage of laboratorians. This cannot be accomplished through short-term funding, but requires a longterm national strategy.

References [1] [2] [3] [4]

[5]

Tucker, J. B. 1999. Historical trends related to bioterrorism: an empirical analysis. Emerg. Infect. Dis. 5:498-504. Cohen, H. W., R. M. Gould, and V. W. Sidel. 1999. Bioterrorism initiatives: public health in reverse. Am. J. Pub. Health 89:1629-1631. Centers for Disease Control and Prevention. 2001. Update: investigation of anthrax associated with intentional exposure and interim public health guidelines, October 2001. MMWR, Morbid. Mortal. Wkly. Rep. 50: 889-893. Centers for Disease Control and Prevention. 2000. Biological and chemical terrorism: strategic plan for preparedness and response. Recommendations of the CDC Strategic Planning Workgroup. MMWR, Recomm. Rep. 49(RR-4):1-14. Khan, A. S., S. Morse, and S. Lillibridge. 2000. Public-health preparedness for biological terrorism in the USA. Lancet 356:1179-1182.

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[6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21]


Rotz, L. D., A. S. Khan, S. R. Lillibridge, S. M. Ostroff, and J. M. Hughes. 2002. Public health assessment of potential bioterrorism agents. Emerg. Infect. Dis. 8:225-230. Centers for Disease Control. 1976. Anthrax-California. MMWR, Morbid. Mortal. Wkly. Rep. 25:33-34. World Health Organization. 1980. The global eradication of smallpox: final report of the global commission for the certification of smallpox eradication. World Health Organization, Geneva, Switzerland. Miller, J., S. Engelberg, and W. Broad. 2001. Germs. Biological weapons and America's secret war. Simon & Schuster, New York. Kolavic, S. A., A. Kimura, S. L. Simons, L. Slutsker, S. Barth, and C. E. Haley. 1997. An outbreak of Shigella dysenteriae type 2 among laboratory workers due to intentional food contamination. JAMA 278:396-398. National Institute of Allergy and Infectious Diseases. 2002. The counter-bioterrorism research agenda of the National Institute of Allergy and Infectious Diseases (NIAID) for CDC Category A agents. National Institutes of Health, Bethesda, MD Meyer, R. F., and S. A. Morse. 2002. Bioterrorism preparedness for the public health and medical communities. Mayo Clin. Proc.77:619-621. Kauiman, A. F., M. I. Meltzer, and G. P. Schmid. 1997. The economic impact of a bioterrorist attack: are prevention and post-attack interventions justifiable? Emerg. Infect. Dis. 3:83-94. Gilchrist, M. J. R. 2000. A national laboratory network for bioterrorism: evolution from a prototype network of laboratories performing routine surveillance. Mil. Med. 165(suppl. 2):28-31. Department of Health and Human Services. 2002. 42 CFR Part 1003. Possession, use, and transfer of select agents and toxins. Fed. Reg. 240:76886-76905. Centers for Disease Control and Prevention, National Institutes of Health. 1999. Biosafety in microbiological and biomedical laboratories, 4th ed. U. S. Govt. Printing Office, Washington, D. C. Pub. L. No. 107-56. Uniting and Strengthening America by Providing Appropriate Tools Required to Intercept and Obstruct Terrorism ("USA PATRIOT") Act of 2001. Hoffmaster, A. R., R. F. Meyer, M. P. Bowen, et al. 2002. Evaluation and validation of a real-time polymerase chain reaction assay for rapid identification of Bacillus anthracis. Emerg. Infect. Dis. 8:1178-1181. Association of Public Health Laboratories. 2002. State public health laboratory bioterrorism capacity. Public health laboratory issues in brief, October 2002. Washington, D. C. Jernigan, J. J., D. S. Stephens, D. A. Ashford, et al. 2001. Bioterrorism-related inhalational anthrax: the first 10 cases reported in the United States. Emerg. Infect. Dis. 7:933-944. Jernigan, J. J., P. L. Raghunathan, B. B. Bell, et al. 2002. Investigation of bioterrorism-related anthrax, United States, 2001: epidemiological findings. Emerg. Infect. Dis. 8:1019-1028.


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Imported viral diseases, surveillance and control in Europe - European Network for the Diagnostics of "Imported" Viral Diseases (ENIVD) Mattias NIEDRIG Robert Koch-Institut, Berlin, Germany

In the last decades we became aware of the emergence of several viral disease outbreaks in humans which have their origin in non-human species. The most important of these, AIDS caused by HIV-1 and -2 virus probably entered the human population from the simian host more than 60 years ago and became pandemic since the 1980s. Viruses like Hendra and Nipah passed the species barrier just recently and cause fatal diseases in humans with close contact to infected horses or pigs. Others are known for many years as zoonotic viruses like Hanta and human Influenza and are a constant threat for the developed world, and yet others are only endemic in tropical areas but cause increasingly problems like Yellow Fever, Lassa and Dengue. There are several reasons for the emergence of viral threats: Increase of migration by tourism and of refugees; Increase of world population; Spread of vectors by microbial adaptation & climate change; Breakdown of public health systems. Through the imported Yellow Fever and Lassa cases to Europe we all became aware that these dangerous infections could be imported to Europe from endemic regions in a very short time. The surveillance of such diseases very much depends on good and reliable case definitions and good diagnostic tools. Just recently the EC has decided for setting up a network for epidemiological surveillance and control of communicable diseases the European community. There are also several viral diseases like Yellow Fever and all viral haemorrhagic fevers (Crimean Congo HF, Ebola, Lassa fever, etc.) on the list. To improve the diagnostic capacity and quality for "imported" viral infections scientists from nearly all European countries from laboratories working in the field of diagnostics of "imported" viral diseases have started to build up a network and have worked out the objectives to be addressed in this collaboration. In eleven meetings scientists from laboratories working in the field of diagnostics of "imported" viral diseases in the United Kingdom, Sweden, France, Greece, Spain, Denmark, Netherlands, Belgium, Portugal, Finland, Italy, Ireland, Slovenia, Lithuania, Switzerland, Bosnia-Herzegovina, Czech Republic, Slovakia, and Germany have started to build up a network to improve the diagnostics of "imported" viral infections and have worked out objectives to be addressed in this collaboration: 1. Build a network of European laboratories working on diagnostics of "imported", rare and emerging viral infections. Provide mutual help in the exchange of diagnostic samples, i.e. sera, viruses, methods, and information in order to improve diagnostics

M. Niedrig / Imported Viral Diseases, Surveillance and Control in Europe

38

2.

3. 4. 5. 6. 7. 8.

Identify those viral infections more likely to be imported and co-ordinate the objectives and identify laboratories, capable and willing to perform the rapid diagnostics (100) V

Unusual Risk to Emergency Responders and Civilians V

Crime Scene Evidence and Preservation

*

V

Major Interaction with Other Federal, State, and Local Agencies

*

V

Scene Communication Overload

*

V

Chaos

V

Resources Overwhelmed V

Secondary Device Designed to Kill Responders

*

V

Pre-Incident Indicators

V

Increased Time Required for Response

V

Dissemination Indoors

V

Major Psychological Impact V Long Term Health and * Restoration Concerns V * An asterisk denotes a characteristic that may have some degree of overlap between a classical HAZMAT incident and a WMD incident. However, there are differences. V The delta denotes a significant difference between a classical HAZMAT incident and an incident involving WMD materials.

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Let us further explore how a HAZMAT incident differs from a WMD terrorism incident, and explore the challenges and consequences presented by those differences. Deliberate attack: Many of you are already familiar with a HAZMAT response. HAZMAT incidents are generally accidents. A WMD terrorism incident is intentional, even if non-warfare gases, chemical, or biological materials are employed. Supertoxic material: Most of the typical military chemicals are significantly more toxic than industrial ones. For example, Sarin (GB) is close to 200 times more toxic than chlorine and is approximately 60 times more toxic than methylisocyanate. This is the chemical leaked in the HAZMAT incident in Bhopal, India, that caused 200,000 casualties; 10,000 were severely affected and 3,300 died. Biological agents have the potential to kill millions. Some of the biological agents are contagious. They not only infect the people initially exposed, but can travel through the infected people and infect people away from the initial release site. Small amounts of radiological material can contaminate large areas for years. Small quantities of WMD agents have the capability to produce large numbers of casualties (symptomatic and psychological) *Early hazard identification: Early identification of hazardous material is relatively easy. Many countries require placards on vehicles or shipping papers (manifests) that identify the material. Workers or the location of the incident can also provide information on the identification of the material involved in the incident. The WMD agents, on the other hand, will be difficult to identify (except by signs and symptoms, which will give you a clue) because none of the identifiers associated with an HMI will normally be present. Compounding the situation is the fact that while the signs and symptoms of most chemical releases will be evident immediately, those from a radiological release may be slightly delayed depending on the dose received, and those from most biological releases may be significantly delayed. *Mass casualties/many fatalities: In the vast majority of cases, a hazardous material incident does not result in mass casualties or fatalities. In the Tokyo incident, there were 5,500 casualties; all but 1,200 were psychological. However, a WMD terrorism incident has the potential to create mass casualties and many fatalities, particularly a biological attack. Some industrial incidents, such as the release in Bhopal, India of methyl isocyante, caused thousands of casualties; but this is atypical. Mass decontamination: A HAZMAT incident typically requires the decontamination of only a few people. Experience from the responders in our courses suggests that decontamination of 10 persons is typical while the decontamination of 45 people is perhaps the most that have had to be decontaminated after an incident. A WMD terrorism incident will require the decontamination of numerous individuals, perhaps hundreds or even thousands. Moreover, we may have to decontaminate large structures, buildings, and real estate in order to render area safe for human use again. Currently, there are no effective means to do this economically. Mass decontamination will require assigning some of the emergency responders to perform this very important task. Other units may be able to assist if they are properly trained and equipped. Please remember that another part of the HAZMAT team should be committed to establishing a separate decontamination corridor to provide technical decontamination for responders. Unusual risk to emergency responders and civilians: The WMD materials are extremely toxic; most are heavier than air and may be persistent. As a result, they pose a greater threat to responders than most hazardous materials for a longer period of time in the area where the responders must execute their mission.Because of their higher toxicity, the downwind hazard could be a greater risk than in a conventional hazardous material incident.

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*Crime scene and evidence preservation: Local laws dictate whether or not a HAZMAT incident may or may not be labeled as a crime scene. Factors such as environmental laws, criminal liability of the operators of vehicles, observance of regulations, etc., will dictate this. An act of WMD terrorism, even if it is a hoax, is a federal crime in many countries; therefore, the site of the incident mandates a crime scene designation and evidence preservation. These are key elements for apprehension and prosecution of the perpetrator(s). *Major interaction and coordination with local, state, and federal agencies: This may be required in a HAZMAT incident, but most definitely will be required during a WMD terrorism incident due to the complexity of the situation and the public interest. A WMD terrorism incident will require the Incident Commander to effectively use an Incident Command System and to assign areas of responsibility to other responding personnel. In a WMD terrorism incident, the Incident Commander will be faced with managing and coordinating multiple agencies - some requested, some not. In a WMD terrorism incident, one liaison officer and several assistants may be needed to serve on the Incident Commander's staff to assist in the management and coordination of all the responding agencies, and will be critical to the timely and efficient application of resources. The staging area(s) manager(s) will have to control a much larger number of resources than during a HAZMAT incident. Scene communications overload: Due to the large interaction among various agencies, communications overload will be the most significant challenge during and after a WMD terrorism incident. The Incident Commander must deal with communications overload immediately or he/she will soon become overwhelmed. While cellular phones may be useful during the early moments, cell sites could quickly become jammed by the media and others. Chaos: Because of the public's concern about the consequences of exposure to WMD materials, there will be more mass panic and hysteria in a WMD terrorism incident than in a HAZMAT incident. Fire companies and HAZMAT units alone will be unable to maintain scene safety and security at a WMD terrorism incident. The importance of proper and immediate employment of law enforcement to manage scene control in a WMD terrorism incident cannot be overemphasized. Public officials should also make a presence. This was very effective during the incidents in the U.S. during the Fall of 2001, and helped to minimize panic. Resources overwhelmed: In a HAZMAT incident, intervention can be handled methodically and deliberately by the HAZMAT team. Due to the number of victims, the catastrophic nature of injuries, and the speed with which WMD agents can kill, it is expected that the normally deployed assets, such as fire companies, HAZMAT teams, Emergency Medical Services (EMS) responders, Explosive Ordnance Disposal (EOD) units, and law enforcement units, will quickly be overwhelmed. Resources, which include hospitals, protective equipment, decontamination equipment, Pharmaceuticals, cover for victims after decontamination, and transportation for victims will likewise be overwhelmed. A WMD terrorism incident will demand immediate intervention on the part of the first responders if lives are to be saved. Secondary device designed to kill responders:Recent events suggest that emergency responders need to be aware of the threat of secondary devices or multiple WMD devices at or near the incident site. One should always assume in a terrorism incident that a secondary device might be present when responding to an incident scene. Secondary devices may be a conventional high explosive fill, or may be constructed to deliver additional WMD material. The threat of secondary devices presents unique tactical considerations. Pre-incident indicators: Before a terrorism incident, the law enforcement or intelligence community may have some indications and intelligence information about the

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possibility of a terrorism act. These indicators may take the form of phone calls to authorities describing the event or stating the reason for the attack, or might be the discovery of the dissemination device before it releases the agent into the atmosphere. There is also a possibility that a pre-incident call could be a hoax, or even a diversion, to tie up resources. Increased Time Required for Response: Due to the large numbers of casualties that may be involved, time in the hot zone may need to be increased for effective rescue and reconnaissance of the area. Also, the super toxic nature of the materials may require that additional time/steps may be needed for decontamination of responder personnel. All of these factors together result in an increased time that is needed in protective gear by the responder forces. Dissemination Indoors: To increase the potential for injury, terrorists may disseminate WMD materials indoors, causing a higher concentration and avoiding the effects of weather that may degrade and dissipate the agent. Most HAZMAT incidents take place outdoors. Major Psychological Impact: A WMD terrorism incident will create large numbers of psychological effects on casualties, thus magnifying the mass casualties situation. Long Term Health and Restoration Concerns: We are just becoming familiar with the long-term effects of exposure to conventional chemical warfare agents. There are still lingering effects in the population of people exposed to mustard agent some twenty-five years after the use of this agent during the Iran-Iraq War. Likewise, we still need to develop guidelines for how "clean is clean" since this will impact use of buildings or other assets after an attack. How low can we really detect chemical warfare agents? Are they still harmful? How clean is a room from anthrax? One spore? One thousand? And of course the use of radiological materials is of concern, although easy to detect.

3. Key Elements for Effective Training The increasing threat of the use of WMD materials in an act of terrorism places both civilian and military personnel at risk. Key components of the infrastructure, such as postal systems, water supplies, arenas, buildings, etc., can also be affected by such an attack. Regardless of what or who is attacked, the training approach described here provides a proven process for preparing both civilian and military responders to respond to asymmetric attacks involving WMD materials and for the mitigation of the impact of such an attack. The Anthrax attacks of 2001 in the U.S. confirmed that planning, training, exercising, and the use of appropriate resources could substantially reduce the impact of such an attack on both personnel and infrastructure. The target audience is the command staff, emergency responders (fire and rescue, HAZMAT, law enforcement, and security), medical responders (EMS and healthcare providers), and mutual aid responders. The program includes the following key elements: a. Command and Staff Workshop: This workshop is intended to develop an awareness within the command and staff of the implications of a WMD incident and to provide an overview of the WMD Training Program. The workshop is conducted in an interactive forum with lecture, discussion, case studies, and video clips. b. Baseline Exercise: The baseline assessment is accomplished with a facilitated tabletop exercise using a chemical agent scenario. The baseline will provide a focus for future preparedness efforts and allow the development of a strategy for enhancing preparedness. The baseline assessment will also include a review of any existing disaster preparedness plans and

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standing operating procedures (SOPs), and any previous vulnerability assessments that may have been conducted. c. Training: The training objective is to develop an awareness and understanding of the implications of a WMD incident to your installation and its mission. The training step will provide installation participants with a solid foundation and a much greater grasp of the unique considerations involved in responding to a WMD incident, prior to the planning step. The training includes six responder courses, covering the following topics: WMD signs and symptoms, the threat of WMD terrorism, detection, identification, sample collection, downwind hazard analysis, personal protection, decontamination, medical management of casualties, and consequence management tactics, techniques, and procedures. The training is conducted utilizing classroom lecture, videos, practical exercises, and tabletop exercises. The courses are described in detail in the next session. d. Planning Workshop: The planning phase involves either review and refinement of existing response plans/annexes by WMD experts, or joint development of response plans/annexes, if current plans do not exist. WMD planning assistance is conducted during a two-day facilitated workshop. The planning stage is critical in defining roles and responsibilities of each entity involved in your installation's response, and for conducting a comprehensive inventory of existing response assets and capabilities. The workshop provides an excellent mechanism for building an effective, executable Antiterrorism/Force Protection (AT/FP) plan that will be effective in responding to a WMD incident. Once the plan or draft plan is in place, the next step involves the conduct of a tabletop exercise to validate the plan and reinforce the training. e. Technical Assistance: The Technical Assistance effort complements the planning, training, and exercise phases in the WMD Installation Preparedness process by filling in any technical voids that exist. Technical Assistance ranges from vulnerability assessments of facilities to a WMD incident to equipment consultations. Technical Assistance is dependent upon the level of effort required. This effort can be conducted at any time during the process. f. Chemical/Biological (CB) Tabletop Exercises (TTXs): TTXs tend to focus on the command, control, and communication aspects of the response, and how various response functions combine into a total integrated system. The TTXs will assist installation participants and mutual aid partners in gaining a comprehensive understanding of an emergency response to a WMD incident, and specifically their role in that response. The TTXs also assist in focusing objectives for the field training exercise.



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Figure 28. Key elements of WMD preparedness

g. Chemical Field Training Exercise (FTX): A chemical incident functional or field training exercise should be conducted to test, to the maximum extent possible, all or some aspects of the AT/FP plan and its response to a WMD incident. The exercise will be tailored to meet the specific objectives of the exercise planning committee. The functional exercise provides a practical means to assess the plan's ability to be executed in an effective and timely manner. It provides insight into required changes to the plan, as well as areas of the response requiring additional work. h. Sustainment: The provider provides sustainment of training by leaving behind compact discs (CD) with all course, exercise, and planning materials. The materials can be used as part of an annual in-service training program. The training and exercises are continually updated to incorporate technical breakthroughs and improved response studies.

4. WMD Courses The WMD Training Courses are based on 26 performance objectives. These performance objectives were developed using various guidelines, established by the U.S. Government and professional organizations [1,2,3]. These courses are intended for personnel who have already been trained at one of the "standard" OSHA defined training levels: Awareness, Operations, Technician/Specialist, and Incident Commander. The WMD IP courses do not replace the OSHA training requirements, nor can they be substituted for credit. The WMD IP courses supplement the OSHA courses with additional, specialized WMD training.

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Figure 29. Elements of a successful WMD Training Program. The arrows on the slide show the WMD prerequisite training classes

a. For responders who are trained at the Awareness level, the Responder Awareness course provides 4 hours of WMD awareness instruction. The goal of this course is to enable students to recognize the signs and symptoms of chemical, biological, and nuclear/radiological materials exposure, recognize potential dissemination devices, to make proper notifications, and to take defensive actions to safeguard themselves. b. For responders who are trained at the Operations level, both the Responder Awareness and the Responder Operations are recommended. The Responder Operations course will provide the technical aspects of WMD incidents and the defensive actions required for responders to protect themselves. These courses provide 4 hours each, for a total of 8 hours of training. c. For responders who are trained at the Technician (or specialist) level, there are three courses offered:Technician-Emergency Medical Services (EMS): This course provides 8 hours of instruction on the technical aspects of WMD incidents and the defensive actions required by EMS responders to protect themselves as well as triage, treatment, and transport of victims. Technician-HAZMAT Level: This course provides 16 hours of instruction on the technical aspects of WMD incidents and the offensive actions required for FIAZMAT responders to protect themselves and their installation. This course covers the same subjects as the Responder Awareness and Responder Operations, but at a more advanced level. (3) Medical Facility Provider: This course provides 8 hours of instruction to emergency room physicians, nurses, and hospital safety administration personnel on the medical technical aspects of WMD incidents and the defensive actions required to protect themselves and their medical facilities as well as the proper medical management of victims. d. The Incident Command course provides 8 hours of instruction for incident commanders and key responders on the challenges, consequences, and special considerations of managing a WMD incident. This course requires one of four prerequisites: Responder Awareness, Responder Operations, Technician-EMS, Technician-HAZMAT, or Medical Facility Provider.



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e. In addition to the responder level training, we have prepared a 30-minute Employee Awareness video that provides basic WMD awareness to a diversified audience of employees at high-risk locations such as large, enclosed facilities with high concentrations of people. There is a separate module in the video that specifically targets 911 operators/dispatchers. 5. The Ultimate Goal of Training - Preparing the Response Plan Once the training is complete, the next step in the process is to develop an integrated response plan that covers all phases of an incident. The primary purpose of this plan is to safeguard lives, preserve health and safety, secure and eliminate the hazard, protect property, and mitigate the damage of the incident to the facility and the environment. Preparedness also maintains the confidence of the community in the responder element and also facilitates compliance with directives. All incidents have several phases to them. The first phase is Crisis Management. This phase consists of taking steps to prevent an attack, recognizing an attack, assessing the situation, and calling in the appropriate assets to confront the problem. At the local level, one can conduct a vulnerability assessment and determine response needs. The next phase is Consequence Management. It is this phase whereby one needs to develop response plans involving evacuation or sheltering in place, sampling and survey of the site, rescue and control of victims, decontamination of people and facilities, evidence collection, and medical intervention. The last phase, Mitigation and Restoration, is the phase whereby one returns the situation to normal. In a crisis the two most important phases are the first two: Crisis Management and Consequence Management. The relationship between these two phases can be summarized as per Figure 30 [4,5].

Figure 30. Relationship between Crisis Management and Consequence Management

As the figure shows, this effort requires considerable coordination among assets. Law enforcement plays the key role during the crisis management phase while the responder community plays the key role during the consequence management phase.

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Planning starts with the formation of a planning team. This team should consist of all of the stakeholders in the plan. The size of team will depend on operations, requirements, and resources. Examples of groups to include in the planning team include management, public affairs, legal, law enforcement, responders, and others.

Figure 31. Steps in an effective planning process

The planning team works together to perform a "front-end" analysis of the problem whereby potential vulnerabilities to various threats are determined. Response needs are then determined. It is this information that leads to the determination of appropriate, practical, and feasible response actions. These actions should then be documented to form the basis of the response plan. These actions are summarized in Figure 31. Once the plan has been developed and coordinated, it should be distributed for review and revised as needed. For a second review, conduct a tabletop exercise with management and personnel who have a key emergency management responsibility and have been trained on the plan. For example, in a conference room setting, describe an emergency scenario and have participants discuss their responsibilities and how they would react to the situation. Based on this discussion, identify areas of confusion and overlap, and modify the plan accordingly. Distribute the final plan to senior managers and key members of the emergency response element. Once the plan has been adopted, it should then be trained to all the stakeholders. Training builds competence and competence builds confidence. All players that might respond to a WMD incident should participate in established training and exercise programs to the extent possible, to understand each others' roles and responsibilities during and after the incident. If any shortfalls are found during the training exercises, the plan should be modified accordingly, and the new response should be implemented into the next cycle of training. Thus training and exercises becomes a continuous cycle of plan, train, exercise, and revise (Figure 32). An effective response to a WMD incident requires this [6].

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Figure 32. Effective elements of a WMD response - Plan, Train, Exercise, and Revise

6. Conclusions Prior to the initiation of this program, there was little, if any, consideration given to response against WMD agents. Through this process, which is a systematic analysis of concepts, plans, procedures and equipment, our first responders are able to enhance their effectiveness when dealing with chemical and biological terrorist incidents. This program showed that the training using this approach could provide emergency managers and first responders logical response approaches. These approaches can be used as a starting point to improve their overall preparedness for chemical and biological terrorism. This training proved invaluable during the anthrax attacks of the Fall of 2001. Many of the personnel involved in the response effort were trained under this program or actually served as trainers. Response plans were in hand and the professionals knew what their jobs would entail. The low death rate, minimal panic, and rapid response all proved that this program was effective.

References [1] [2] [3] [4] [5] [6]

Occupational Safety and Health Administration (OSHA) 29 Code of Federal Regulation (CFR) 1910.120 (Hazardous Waste Operations and Emergency Response). National Fire Protection Agency (NFPA) 472 (Professional Competence of Responders to Hazardous Materials Incidents) NFPA 473 (Competencies for Emergency Medical Services (EMS) Personnel Responding to Hazardous Materials Incidents, and the Joint Commission on Accreditation of Healthcare Organizations). Federal Emergency Management Agency, Federal Response Plan, Bulletin 9230.1-PL ,1992. Federal Emergency Management Agency, FEMA 229, change 11, 1997. Office of Personnel Management, WMD preparedness Executive Workshop Manual, p. 149, 2003.


Part 4

Applied Research



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Brucellosis-A Biowarfare Threat and Public Health Concern Philip H. ELZER Louisiana State University AgCenter and School of Veterinary Medicine Department of Veterinary Science 111 Dalrymple Building, Baton Rouge, LA; 70803; USA Abstract. Brucellosis is a zoonotic disease with potential for use as a biowarfare agent. The genus Brucella contains six recognized species including B. abortus, B. melitensis, B. suis, B. canis, B. ovis, and B. neotomae. All of the brucellae are Gram negative, facultative intracellular pathogens, which survive and replicate in host macrophages. The hallmarks of animal brucellosis, in both domestic and wild animals, are abortion, infertility and reproductive failure. If used in an agroterrorist attack, these organisms could decimate a generation of livestock and companion animals. Fever, chills, malaise, arthritis, dementia, and possibly even death characterize the disease in man. Human brucellosis, also known as undulant fever or Malta fever, is caused by only four species of brucellae. B. melitensis is the most infectious to man in that 1-10 colony forming units are thought to cause disease followed by B. suis (1000-10,000), B. abortus (100,000), and finally B. canis (> 1,000,000 in an immuno-compromised individual). There are several animal vaccines that are safe and effective; however, they are all pathogenic to man. Currently there are no vaccines approved for use in humans. Brucellae are characterized as BSL-3 organisms due to their ability to infect humans through aerosol exposure, which makes them an ideal bacterial agent for use by terrorists. If the general public were exposed to this biowarfare agent, medical resources would be stretched 10 fold to take care of the large number of people that would be debilitated by this organism. With the recent tragic events in the United States and throughout the world, three species of Brucella are considered "agents of mass destruction." The need for a human vaccine is paramount.

There are six recognized species in the genus Brucella characterized by their host specificity and ability to cause chronic infections in many animals and man. Brucellae are know to cause abortion and infertility in wild and domestic ungulates [1,2,3]- In addition to the agricultural economic loss associated with brucellosis, several of the brucellae are zoonotic agents. After the tragic events associated with September 11, 2001, in the United States, three species of Brucella are also considered "agents of mass destruction." Therefore the development of a safe and efficacious vaccine for all ungulates and humans is a paramount goal for brucellosis researchers. The properties of the ideal biological weapon are that the agent should be highly contagious and consistently produce a known disease or syndrome. It is best if it can be disseminated throughout the environment, i.e. aerosolized; and it needs to be stable under production, storage, and delivery to target. However the organism should have a short survival time in the target area so it is not a threat at later time points to delivery personnel. It is preferable that the target populations have little or no natural resistance. Brucellae are the

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consummate biological warfare agents in that they meet all of these criteria. They are highly infectious (see Table 11), can be easily aerosolized, are stable during production; and due to its sensitivity to direct sunlight, it is destroyed in the environment over time. Since there are no human vaccines against brucellosis, most, if not all populations, have little or no natural immunity to this organism. Brucella species were weaponized in the United States following WWII. These species were field tested in cluster bombs in 1955. However all of the munitions using this agent were destroyed in 1969. If used in an agroterrorist attack, these organisms could decimate a generation of livestock and companion animals. With the concentration of livestock, lack of genetic diversity, increased farm sizes, importation of animals, and increased international travel, agriculture around the world is very vulnerable to a terrorism attack. The effects of agroterrorism would be immediate in that there would be mass hysteria manifested by panic buying of the stocked products. There would be demoralization due to the loss of food, plants, companion animals, and economic devastation. This economic destruction could become worldwide if a country's main agricultural export product or products were boycotted by numerous nations [4]. All of the brucellae are Gram negative, facultative intracellular pathogens, which survive and replicate in host macrophages. In man, brucellosis, also known as undulant fever or Malta fever, is caused by only four species of brucellae (B. abortus, B. melitensis, B. suis, and B. canis). Human infection is caused by ingestion of infected raw milk products, exposure to infected animals, and aerosolization of the organism. Brucellosis in man is characterized by a cyclical fever that starts two to three weeks post-exposure. Night sweats, headaches, backaches, and general malaise are symptoms associated with acute infection. Chronic brucellosis can lead to a debilitating condition, including arthritis, dementia and even death. Patients with chronic brucellosis have frequent relapses, and 2/3 of these individuals develop psychoneurosis. Human brucellosis can be treated with the administration of tetracycline or doxycycline in combination therapy with rifampin or gentamicin [5]. Due to the potential use of biowarfare agents, public health officials need to be constantly aware of possible intentional exposures to infectious agents. There are needs for rapid and specific detection devices for environmental releases and medical samples. Improved surveillance and diagnostics with effective communication amongst numerous regulatory and emergency agencies is necessary to contain and control an exposure or outbreak. An example of this is illustrated by a case report of a 38 yr old woman from New Hampshire, USA, who was thought to have been exposed to Brucella spp. This report highlights several aspects of the needed public health response to a possible biowarfare agent [6]. The CDC classifies B. abortus, B. melitensis and B. suis as "agents of mass destruction" and as category B organisms. Brucella canis, a less virulent strain, can cause human disease but only when contracted by an immuno-suppressed individual. B. melitensis is the most infectious to man in that 1-10 colony forming units (cfu) are thought to cause disease followed by B. suis (1000-10,000 cfu), B. abortus (100,000 cfu), and finally B. canis (> 1,000,000 cfu) in immuno-compromised individuals (Table 11). Brucellae are characterized as BSL-3 organisms due to their ability to infect humans through aerosol exposure, which makes them an ideal bacterial agent of mass destruction [7]. If the general public were exposed to this biowarfare agent, medical resources would be stretched 10 fold to take care of the large number of people that would be debilitated by this organism. Currently there is no approved vaccine for human use, and a vast majority of the animal vaccines are virulent to man. Thus, there is a need to find a safe and efficacious vaccine that can be used in humans [7].

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Table 11. Human Susceptibility to Brucella spp

goats, sheep

Human Virulence HIGH

Number of organisms 1-10

B. suis

swine

High-Moderate

1,000-10,000

B. abortus

cattle

Moderate

100,000

B. canis

dogs

Low/immuno-

> 1,000,000

Brucella spp. B. melitensis

Natural Host

suppressed

References [1]

[2] [3] [4] [5] [6] [7]

Timoney, J.F., J.H. Gillespie, F.W. Scott, and J.E. Barlough. 1988. The Genus Brucella. Hagan and Brunner's Microbiology and Infectious Diseases of Domestic Animals. 8th edition. Cornell Univ. Press, Comstock Publishing Assoc., Ithaca, New York. Burrows, W. 1968. Brucella. In Textbook of Microbiology. 19th edition. W.B. Saunders Co.,Philadelphia, Pennsylvania. Nicoletti, P. 1980. The epidemiology of bovine brucellosis. Adv. Vet. Sci. Compar. Med.24:70. Huxsoll, D.L., W.C. Patrick III, and C.D. Parrott. 1987. Veterinary services in biological disasters. J. Am. Vet. Med. Assoc. 190:714-722. Young, E.J. 1989. Clinical manifestations of human brucellosis, p.97-126.In:E.J. Young and M.J. Corbel (Ed.), Brucellosis: Clinical and laboratory aspects. CRC Press, Boca Raton, FL. Suspected Brucellosis Case Prompts Investigation of Possible Bioterrorism-Related Activity—New Hampshire and Massachusetts, 1999. MMWR 49 (23); 509-512. Acha, P.N., and B. Szyfres. 1980. Zoonoses and communicable diseases common to man and animals, p. 28-45. Pan American Health Organization, Washington, D.C.

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Medical Countermeasures Research at Dstl Porton Down, UK Petra C. F. OYSTON CBS Porton Down, Salisbury, Wiltshire, SP4 OJQ, United Kingdom Abstract. For almost a century Porton Down has been the site of defence research to provide protective measures for British Service personnel against chemical and biological warfare (BW). A wide variety of BW agents potentially pose a threat and Countermeasures are being devised and evaluated at Dstl Porton Down to combat this threat. Two approaches to provide effective medical Countermeasures are being undertaken. The first approach is to use prophylaxis or post-exposure therapies, such as antibiotics and anti-toxins. The second approach is to use vaccines, which are given before exposure to the agent and have the potential to provide very high levels of protection from disease. Many of the vaccines available against BW pathogens are not suitable for use. We are therefore undertaking research to produce effective, licensed vaccines for protection against BW agents, such as plague and tularemia.

1. A Brief History of Porton Down On the 22nd April 1915, the Algerian Division of the French Army was attacked with 150 tons of chlorine gas released by the German Army over a front of about 4 miles. The effect was devastating. More attacks followed, with the British Army first experiencing an attack on 1st May. This led to calls for retaliation in kind and for the development of a defensive capability to protect the troops. As a result, the research establishment was set up at Porton Down, to research an offensive chemical warfare (CW) capability and to provide defensive measures for the Armed Forces. This research continued for several decades, and biological warfare (BW) agents were added to the research programme. However, the moral and political climate was changing rapidly in the second part of the last century. Thus, by the late 1960s the UK renounced the development of BW weapons and became a signatory to the 1972 Convention on the Prohibition of the Development, Production and Stockpiling of Biological and Toxin Weapons, and the Chemical Weapons Convention. All research now undertaken at Porton Down is defensive in nature, with the aim of protecting the Armed Forces in the event of chemical or biological attack. The history of Porton Down can be found elsewhere [1, 2] for those seeking more detail.

2. Medical Countermesures against BW agents Most biological defence is subsumed in chemical defence, in that the respirator will prevent inhalation of aerosolised agents. However, a critical aspect of such a strategy is knowing when to don the respirator in the event of an attack, or when it is safe to remove it. It would be

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expected that significant numbers of personnel would be affected in the event of a BW attack, and for this reason there has been a programme of work at Porton Down evaluating various approaches to protect the Armed Forces should such a scenario arise. 3. Antivirals, Antibiotics and Antitoxins Biopharmaceutical companies spend millions of dollars a year developing new antimicrobial compounds, but the number of new antibiotics and antivirals appearing are few. At CBS Porton Down, we screen commercially available and new lead compounds against some of the pathogens of concern in order to ensure we have the most effective treatment possible, whether as a prophylactic or as a post-exposure therapy. Antibiotics are the best known example of antimicrobials, but are active only against bacteria. In principle, antibiotics could provide protection against all bacterial BW agents, but many bacterial BW agents are inherently resistant to antibiotics. For example the pathogenic Burkholderia are resistant to most available antibiotics, and possess multi-drug eflux pumps which render many compounds useless [3]. For some BW agents, antibiotics control the disease but the disease reappears when the antibiotic is withdrawn. This has been widely reported for melioidosis, where the period between therapy and recrudescence can be as long as over twenty years [4]. The use of antibiotics is also complicated by the fact that an aggressor may develop antibiotic-resistant strains of BW pathogens. There have been reports, for example, of a clinical isolate of plague which was multiply antibiotic resistant [5], showing how easily pathogens acquire resistance genes. Furthermore, Stepanov et al reported the production of a multiply antibiotic resistant strain of anthrax [6]. Such resistance, whether natural or engineered, would complicate therapy in the event of infection. Finally, the logistics of giving repeated doses of antibiotics to thousands of troops over an extended period of time, coupled with the side-effects this may induce in the soldiers, makes this an undesirable approach to prophylaxis in a BW theatre. There are few, if any, suitable commercial antivirals. The problem in devising antivirals lies with the way in which the virus uses the host cell to replicate, meaning that it is difficult to interfere with viral growth without also detrimentally affecting the host. In addition, most antivirals developed to date are virus-specific. An antitoxin is anything capable of inhibiting the activity of a toxin. This could be either a compound capable of inhibiting the activity of the toxin, or an antibody which binds to the toxin and thus blocks its action. Currently there are no inhibitory compounds available to treat intoxication, and all antitoxins at present are antibody preparations. The toxic doses of toxins such as botulinum toxin or ricin are quite high, so it is likely that an attack would trigger detectors on the battlefield warning of the hazard and possible exposure of personnel. In such a scenario antitoxins could be used for treatment of exposed individuals to prevent the onset of toxic symptoms. Antisera are generated by immunising animals, such a goats or sheep, with an inactivated form of the toxin (such as a toxoid) followed by purification of the antitoxin from repeated production bleeds. The plasma obtained is purified to IgG. Adverse affects such as analphylaxis are common when patients are given whole IgG products derived from a foreign host and at Porton Down we are looking at ways of producing effective antisera from fragments of the IgG, namely F(ab')2, Fab' and Fab. The rational behind these studies is that the reactogenic Fc portion of the molecule has been removed and thus the smaller molecules should result in fewer side effects. The smaller size of the fragments has the added advantage that they may reach their site of action more quickly than IgG and be more effective for post

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exposure therapy [7]. This approach is currently being investigated for antitoxins against botulinum toxin and ricin. A further point to note is that in general it is much faster to obtain a licence for use of antitoxins in humans than to licence a vaccine, permitting a usable product to be generated relatively quickly. 4. Vaccines Vaccines have the potential to provide high levels of protection against BW agents. They must be given prior to exposure, but once the course is complete then the individual requires only boosters at lengthy intervals. However, the pathogens of concern in a BW scenario are not usually those of concern to every day public health. As such, there are either BW agents against which we have no vaccine, or the vaccines are less than ideal. For example, the painful reaction many soldiers suffered following the initial dose of the anthrax vaccine in the Gulf War dissuaded many of them from returning to complete the course. Also, the vaccine must be able to induce an immune response capable of protecting against pneumonic infection. For example, the current plague vaccine was shown not to protect against pneumonic plague [8]. To reduce the risk of side-effects and to ensure effective immunity, the UK vaccination policy is to use only licensed vaccines to protect the Armed Forces. Research is underway at CBS Porton Down to develop licensed vaccines against the BW agents of concern, some of which is described in detail below.

4.1. Plague Two killed whole-cell vaccines are currently available for use. The first (Commonwealth Serum Laboratories Ltd., Australia) consists of heat-killed cells of fully virulent Y. pestis in saline. The second (Miles Inc., Canada) contains formaldehyde-killed Y. pestis cells. The regime consists of three primary doses administered intra-muscularly, followed by annual boosters. The protection induced by these killed whole cell vaccines has been shown to be variable and short-lived. Since the protection induced is highly variable and high levels of adverse reactions occur, the killed vaccines are unsuitable for general use, so are restricted to individuals who work with the pathogen or are potentially exposed to virulent strains. The killed vaccine fails to induce secretory IgA [9] and may not protect against pneumonic transmission [8]. A live vaccine is also available, derived from strain EV76. Strain EV76 is poorly defined, and resulted in excessive adverse reactions and highly variable responses between individuals [10]. In addition, EV76 was lethal for non-human primates [11] and resulted in hospitalisation of a proportion of human recipients [10]. Thus this strain is not suitable for general use. A second-generation vaccine of recombinant proteins developed at CBS Porton Down is currently undergoing clinical trials. The antigens in the formulation are the capsular Fl antigen and the V antigen, a component of the Type III secretion system [12]. These had been identified in 1970 as the essential antigens which must be produced by an attenuated Y. pestis strain for induction of an effective immune response [10]. The first challenge in developing this second generation vaccine was obtaining a source of antigens which could be produced on a large scale. The caf operon encoding Fl antigen is carried on one of the three Y. pestis virulence plasmids [13]. The monomeric unit of the Fl antigen has a molecular mass of 15.5

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kDa, but there is aggregation of the monomers into large complexes with masses in excess of 3 Mda [14]. This aggregation occurs spontaneously in solution. Cloning the cafoperon allowed high level expression of recombinant Fl antigen, a much safer source than isolating the antigen from plague cultures. The V antigen has a key structural and regulatory role in the type III secretion system of Y. pestis [15]. The protein has a molecular mass of 37 kDa, and similarly to Fl antigen it spontaneously aggregates [12]. The folding of V antigen is important to immunogenicity, as studies have shown that B-cell epitopes in the protein are conformational [16, 17]. V antigen is more difficult to produce than Fl antigen due to inherent instability of the protein. It is produced as a GST fusion, which is purified and subsequently cleaved to give pure V antigen. The Fl and V antigens have been shown to be able to induce a protective immune response individually, but a combination of the proteins had an additive effect. The optimum ratio for immunisation was shown to be 2:1 (F1:V) [18]. The interdependency of immunogenicity and conformation led us to deduce that combined free sub-unit was the approach to follow, rather than the production of a genetic fusion of the two antigens, an approach undertaken by other workers. At present the antigens are being taken into clinical trials in a formulation with alhydrogel as an adjuvant, given intramuscularly. Although this vaccine was shown to induce protection in mice against aerosol challenge, work is now underway to develop a vaccine which is mucosally delivered. In addition to inducing immune responses in the respiratory tract, mucosal delivery has the advantage of being needle-free. Much work has been undertaken to deliver the antigens using biodegradable polymeric microencapsulation. A preparation suitable for nasal administration has been produced which is fully protective against aerosol challenge in the mouse model after just two doses [19]. 4.2. Tularemia Tularemia in humans can occur in several forms depending to a large extent on the route of entry of the bacterium into the body. Tularemia can be a severely debilitating disease, especially when caused by F. tularensis subspecies tularensis. The most acute form of disease is associated with the inhalation of bacteria, although pneumonic disease can also occur as a complication of other forms of tularemia. The Live Vaccine Strain (LVS) vaccine remains the only effective vaccine against tularemia developed to date. However, this vaccine is not currently available, though work to licence it is underway in the USA. That this attenuated mutant of F. tularensis can induce protective immunity suggests that this approach to vaccine development is feasible. In a range of other pathogens, the introduction of defined mutations into genes required for growth of the pathogen in vivo has yielded safe and effective vaccines. The construction of a defined attenuated mutant of F. tularensis could provide a safe, effective and licensable tularemia vaccine. The aromatic amino acid and purine biosynthetic pathways have already been identified from genome sequence information as targets for the construction of a defined attenuated mutant [20]. However, the utility of this approach is limited because there are as yet no methods for the construction of allelic replacement mutants of F. tularensis. Work is currently underway at CBS Porton Down to develop methods to create isogenic allelic replacement mutants. Mutants of F. novocida compromised in their ability to replicate inside macrophages have been produced at the University of Victoria [21]. We are now examining these mutants for attenuation in the mouse infection model, and for their ability to induce a protective immune response. Interestingly, in one of these mutants the transposon interrupts a

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gene required for purine biosynthesis, and as stated above, purine mutants are of interest because in other bacterial species they have been used as attenuated vaccine strains. A second approach we are following is the evaluation of subunit vaccine antigens. One such potential protective subunit is lipopolysaccharide. Immunisation with lipopolysaccharide was able to protect mice against challenge with the LVS strain, but not against the more virulent Schu4 [22]. Therefore, this may form one part of a vaccine, but would require additional antigens for full protection. Other antigens would probably be proteins expressed by the pathogen. A crude outer membrane preparation of F. tularensis was able to protect mice, but so far individual proteins have been unable to induce a protective immune response [23]. Future progress towards identifying suitable antigens should be accelerated by the availability of the F. tularensis strain Schu4 genome sequence data. Analysis of the immune response to the LVS vaccine has shown a heterogeneity of immunogenic epitopes recognised in humans and this indicates it is likely that a sub-unit vaccine will need to be composed of a number of protective antigens to provide protection against virulent strains.

5. Summary In order to protect the UK Armed Forces, medical countermeasures such as antibiotics and vaccines against BW agents are being evaluated at CBS Porton Down. A second generation plague vaccine is currently undergoing clinical trials, and further work to develop a needlefree, mucosally delivered vaccine is on-going. For some of the pathogens, for example F. tularensis, nothing is known about the basis of virulence of the pathogen or suitable antigens for vaccine development. Genome sequence data will underpin all research efforts on these pathogens, but a priority remains the development of good genetic tools for manipulation of some of these organisms.

6. Acknowledgements Crown Copyright 2003 Dstl References [1] [2] [3]

[4] [5] [6] [7]

Carter GB. Porton Down: 75 years of Chemical and Biological Research. London: HMSO; 1992. Carter GB. Chemical and Biological Defence at Porton Down 1916-2000. London: The Stationery Office; 2000. Moore RA, DeShazer D, Reckseidler S, Weissman A, Woods DE. Efflux-mediated aminoglycoside and macrolide resistance in Burkholderia pseudomallei. Antimicrobial Agents and Chemotherapy 1999; 43: 465-470. Sanford JP. Melioidosis: forgotten but not gone. Trans. Am. Clin.Climat. Assoc. 1977; 89: 201-205. Guiyoule A, Gerbaud G, Buchrieser C, Galimand M, Rahalison L, Chanteau S, Courvalin P, Carniel E. Transferable plasmid-mediated resistance to streptomycin in a clinical isolate of Yersinia pestis. Emerging Infectious Diseases 2001; 7: 43-48. Stepanov AV, Marinin LI, Pomerantsev AP, Staritsin NA. Development of novel vaccines against anthrax in man. Journal of Biotechnology 1996; 44: 155-160. Mayers CN, Hoiley JL, Brooks T. Antitoxin Therapy for botulinum intoxication. Reviews in Medical Microbiology 2001; 12: 1-9.

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[8] [9]

[10] [11] [12] [13] [14]

[15] [16] [17] [18] [19] [20]

[21] [22] [23]

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Russell P, Eley SM, Hibbs SE, Manchee RJ, Stagg AJ, Titball RW. A comparison of plague vaccine, USP and EV76 vaccine induced protection against Yersiniapestis in a murine model. Vaccine 1995; 13: 1551-1556. Oyston PCF, Williamson ED, Leary SEC, Eley SM, Griffin KF, Titball RW. Immunization with live recombinant Salmonella typhimurium aroA producing Fl antigen protects against plague. Infection and Immunity 1995; 63: 563-568. Meyer KF. Effectiveness of live of killed plague vaccines in man. Bull. Wld. Hlth. Org. 1970; 42: 653666. Meyer KF, Cavanaugh DC, Bartelloni PJ, Marshall JD. Plague immunization. I. Past and present trends. J. Inf. Dis. 1974; 129: S13-S18. Titball RW, Williamson ED. Vaccination against bubonic and pneumonic plague. Vaccine 2001; 19: 4175-4184. Protsenko OA, Anisimov AP, Mosarov OT, Donnov NP, Popov YA, Kokushkin AM. Detection and characterization of Yersinia pestis plasmids determining pesticic I, fraction I and mouse toxin synthesis. Genetika 1983; 19: 1081-1090. Miller J, Williamson ED, Lakey JH, Pearce MJ, Jones SM, Titball RW. Macromolecular organisation of recombinant Yersinia pestis Fl antigen and the effect of structure on immunogenicity. Ferns Immunology and Medical Microbiology 1998; 21: 213-221. Price SB, Leung KY, Barve SS, Straley SC. Molecular analysis oflcrGVH, the V antigen operon of Yersinia pestis. J. Bacterial. 1989; 171: 5646-5653. Hill J, Leary SEC, Griffin KF, Williamson ED, Titball RW. Regions of Yersinia pestis V antigen that contribute to protection against plague identified by passive and active immunization. Infection and Immunity 1997; 65: 4476-4482. Pullen JK, Andersen GL, Welkos SL, Friedlander AM. Analysis of the Yersinia pestis V protein for the presence of linear antibody epitopes. Infection and Immunity 1998; 66: 521-527. Williamson ED, Vesey PM, Gillhespy KJ, Eley SM, Green M, Titball RW. An IgGl titre to the Fl and V antigens correlates with protection against plague in the mouse model. Clinical and Experimental Immunology 1999; 116: 107-114. Eyles JE, Sharp GJE, Williamson ED, Spiers ID, Alpar HO. Intra nasal administration of poly-lactic acid microsphere co- encapsulated Yersinia pestis subunits confers protection from pneumonic plague in the mouse. Vaccine 1998; 16: 698-707. Prior RG, Klasson L, Larsson P, Williams K, Lindler L, Sjostedt A, Svensson T, Tamas I, Wren BW, Oyston PCF, Andersson SGE, Titball RW. Preliminary analysis and annotation of the partial genome sequence of Francisella tularensis strain Schu 4. Journal of Applied Microbiology 2001; 91: 614-620. Gray CG, Cowley SC, Cheung KKM, Nano FE. The identification of five genetic loci of Francisella novicida associated with intracellular growth. FEMS Microbiology Letters 2002; 215:53-56. Fulop M, Mastroeni P, Green M, Titball RW. Role of antibody to lipopolysaccharide in protection against low- and high-virulence strains of Francisella tularensis. Vaccine 2001; 19: 4465-4472. Fulop M, Manchee R, Titball R. Role of Lipopolysaccharide and a Major Outer-Membrane Protein from Francisella-Tularensis in the Induction of Immunity against Tularemia. Vaccine 1995; 13: 1220-1225.

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Current Problems Regarding Detection and Identification of Biological Threats Michal BARTOSZCZE Military Institute of Hygiene and Epidemiology, Lubelska 2, 24-100 Pulawy, Poland 1. Introduction Bioterroristic attacks with B.anthracis spores in USA visualized to world opinion the scale of biological factor threats. On 22 persons infected by B.anthracis spores 5 died. Antibiotic therapy was applied in 32000 peoples (non-infected). The attack was accompanied with panic and disorganization: ransoming the medicines in drugstores, purchasing the protective equipment and also change in human behavior (limitation in people movement and isolation in houses). The economic losses as a result of biological attack are very high (buildings, persons and environment disinfection). Protection against unconventional biological attack is very difficult and demands unusual fancy for foreseeing the possible scenario of attack. In preparedness for attack consider for the indirect target of attack many substances (cosmetics, drugs even the chewing gums). In preparedness for protection against biological attack essential will be intellectual efforts (prejudice the enemy intentions). Proper prepared and efficiently acting defensive infrastructure will influence on the effectiveness of the protection against biological factors. Development of the molecular biology methods opened the great chances for using them for all men goods, but also caused the fear for using them for military offensive purposes. Thanks to the genetic engineering methods obtaining the biological factors (highly virulent and highly resistance on antibiotics) are become possible. The efficiency of production of the pathogens and theirs toxins was also enlarged. Attainable is transfer of virulence factors from one pathogen to another. Obtained in this method the " wolf in sheep's skin " can procure considerable difficulties in microbiological diagnostics. Accessible is also the modern technologies enlarging the pathogen resistance on environmental factors how as: temperature, light, drying (biopolymers). We should also mentioned about possibilities to "programming" the length of pathogen lives, which after "fulfilling the assignment" decay, giving the chance of safe return for winners after extermination of enemy. Among many decisive factors, which can have the influence on the possibility of decreases the results of use biological agents should be mentioned obligatory: rapid detection and identification of biological factor used, the proper preventive treatment and the medical management.

M Bartoszcze / Detection and Identification of Biological Threats

The aims of identification • To identify the factor • To estimate the area of contamination • To evaluate the possible countermeasure efforts (antibiotics, disinfectants) • To assess the effectiveness of the decontamination efforts (decontamination of the persons, equipment, buildings, environment etc.) The objects of identification • Bacteria and bacteria's spores • Viruses • Toxines • Genetically modified factors • Etc. The list of factors used in the NATO SIBCA exercise • B. anthracis • Coxiella burnetii • Burkholderia mallei • Francisella tularensis • Brucella melitensis • V. cholerae, • Y. pestis • Vaccinia • VEE • Yellow fever virus The nature of the samples • Aerosol • Soil • Water • Food • Swabs from surfaces • Unconventional (envelope, chocolate) • Human and animal, insect etc. Requirement • The rapidity of the tests • The preciseness of the results • The generality (the possibility of uses for identification purposes the different samples: water, soil etc.) • Possibility of identification of different factors • The simplicity of the tests • The possibility of conducting the tests in field conditions • Equipment adaptation to different field conditions e.g. shock resistance

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2. Present technologies 2. 1. Detection methods There are technologies (1), which are assigned for aerosol detection, coming from low distance. The LRBSDS (Long Distance Biological Standoff Detection System), assigned for aerosol cloud detection within the radius of 30 km is an example. Equipped with laser transmitter for infrared, receiving telescope and detector. However, this system is not fully automatic. The problems with signals detection were reported, which were often disturbed, what was the significant detection non-suitability. The system was improved, and the system JBSDS (The Joint Biological Standoff Detection System) was created - the completely automatic system, able to monitor the aerosol cloud movement. It possesses abilities to differ the biological and non-biological clouds, gives possibilities of early warning and report. Following projects regarded detection elements, but identification of biological agents, too. The IBADS (The Interim Biological Agent Detection System) is one of them; it is semi automatic system, which possesses the aerosol concentrator and dynamic particle measuring device. The immunochromatographic tests are applied for micro-organisms identification. The technology of this system is cheap and allows to execute the initial microbe identification only. JPS (The Joint Portal Shields) is the first highly automatized system, improved because of sensor net increasing the detection sensitivity. All operations of this system are controlled by central computer unit. After concentration and physical characterisation, aerosol is next taken for automatic analysis with immunochromatographic methods, with the ability of 8 biological agents identification within 25 minutes. The JBPDS (The Joint Biological Point Detection System) is the next detection and identification system, build with two modules. Comparing with the previous one, it is characterised by significantly higher sensitivity and specificity. It creates the possibility of bioparticle presence detection within 60 seconds, with the ability of 10 organisms identification within 20 minutes. JBAIDS (The Joint Biological Agent Identification and Diagnostic System) - it is the portable device, prepared for simultaneous pathogen identification, as well from environmental or from clinical samples. The ability of fluorescence induction with the laser light beam, analysed thanks to sensitive photo-detectors, was used for biological agents detection, among others. This method allows to determine the living biological agents from the dead ones, and to determine the size and shape of particles. The technologically advanced systems are capable to analyse data fast, together with the alarm signal generation, if the critical level are exceeded, and transfer the obtained data to the commanding centres. The modern FLAPS (Fluorescence Aerodynamic Particle Seizers) system, executing fast analysis of the aerosol type, the shape and concentration, is the sample of this type technology. This type devices were used for examination of parcels suspected to contain the B. anthracis spores in some post offices in the USA (2). The common feature of mentioned above systems is, that they are mainly assigned for biological aerosols detection. However, the bioterrorist attack may be executed with not only aerosol way, but with the use of food and water and non-conventional methods (3, 4). Concerning the above, the mentioned above systems secure only the part of defensive necessities for bio-terrorist attack. Later, the technology allowing for biological agents detection in different environments shall be described.

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2.2. Luminometry It is the method allowing to detect the ATP (adenosino- triphosphate) content in living cells. The method based on the principle of emitted light detection, created at enzymatic decomposition of ATP. Emitted energy is proportional to the ATP content in the sample and is determined with the use of sensitive photo - detector - luminometer (5). The luminometric method is suitable for i.e. fast detection of bacteria presence in fluids, powders, lyophilizates. The PROFILE system, which allows to distinguish eucariotic and priocariotic cells, is one of few technologies assigned for ATP measurement. Because of adequate additional equipment, it allows to execute the examination of the contaminated surfaces, food, water, air, etc. It makes the fast detection of bacterial spores presence in samples possible, too, what may be used for screening examination of samples, suspected of having B. anthracis spores (6). As newest researches show, because of gammalisine application the luminometric method is capable to identify B. anthracis (7). The own research shown the possibility of Salmonella spp., rods identification with the luminometric method (8). Thinking about the versatility of bioluminometry in the aspect of bioterrorist threats, the WIHiE (Military Institute for Hygiene and Epidemiology) prepared and introduced for application the Field System of Bacterial Contaminations Detection (PSWZB).

2.3. Biological agents identification methods Conventional, classic microbiological methods, though they allow to detect and identify the biological agents, have many disadvantages from the military point of view. They are, i.e.: long time necessary for results obtaining, the need of having laboratory subsidiaries, trained personnel and significant material consumption.

2.4. Bio-sensor technologies In bio-sensor techniques, at the searching for i.e. antigens, light-pipe sensors covered with antibody are frequently used. The complex created after the antibody binding with the searched antigen, is detected with the use of antibody marked with fluorescent dye. Thanks to laser light beam , the strong induction of the marker in the reaction place takes place, producing the signal registered by the detector. Thanks to that, the determination on "dirty" samples are accessible to be done. The system Analyte 2000, based on this conception, may identify with the use of four probes, four samples at the same time. The test sensitivity amounts from 3 to 30 bacteria /ml, and the time of analysis equals 20 minutes (9). System RAPTOR (10) presents the development of the method mentioned above, completely automatised, portable, where the sensitivity of determinations is increased and the time of identification is reduced. Exchangeable multi-use blocks, where the tested material flows, were applied. The system allows to detect bacteria as well as their toxins and viruses, what is the significant advantage. The device may work in the permanent system, in the connection with air sample collector. Methods described above did not allow, mostly, to execute genetic tests. RAPID system - field automised system, which allows to identify pathogens with the "Real time" PCR method (11) became the brake-though. Thanks to adequate fluorescent dyes and marking

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probes and use of FRET phenomenon (Fluorescence Resonance Energy Transfer), the amplification process is observed constantly on the monitor screen. The apparatus is easy to run, requires the precision at the test sample preparing, only. We should mention about its connection with the early warning system "Leaders" (Lightweight Epidemiology Advanced Detection and Emergency Response). The RAPID system application for FMD virus identification on the cattle farm was important success.

2.5. Field diagnostic tests Contrary to more or less complicated and usually expensive technologies described before, there are some simple immunochromatographic tests, practically used, allowing to obtain the result within 10-15 minutes (3). Mono - and polyclonal antibodies, marked with colloidal gold are used in this method. The positive reaction is observed optically as a coloured thin line. The test sensitiveness is quite high and amounts from 10 3 to 10 4 CFU/ml. In the case of bioterrorist attack, the used agent concentration may be high (from 10 8 to 10 11). To increase the reading sensitivity, the bar readers are used, what eliminates the reading subjectivity. The Guardian reader is an example of this solution. The immunochromatographic method sensitiveness may be increased, using the UCP (Upconverted Phosphor) instead of colloidal gold. Thanks the induction of these particles with the light, similar to infrared rays, the visible light is emitted , registered by detector (UCPRS - Upconverting Phosphore Reporting System). Comparing to the method with the colloidal gold use, the UCPRS method is 10 - times more sensitive, with the efficient elimination of background "shining". Thanks to different substrate application, it is possible to detect with the use of this method several biological agents simultaneously (12). It should be pointed, that all described technologies allow to execute the more or less precise , but initial diagnostics. The initial identification confirmation is executed by reference laboratories with the adequate level of biological safety, equipped with unique devices and employing high class specialists.

2.6. Molecular biology methods in biological agents identification The basic method applied in genetics diagnostic is the PCR - polimerase chain reaction which allows to amplify selectively the chosen DNA fragments. Thanks to specific starters (primers), it is possible to detect bacterial genes localised on plasmids or chromosome (13). In the case of detection of agents containing RNA, the RT - PCR method is used. Classic PCR method allows to obtain good results, at clean colony and clinical material examination, especially. At the environmental samples examination, because of many PCR reaction inhibitors occurrence, much better identification results are obtained with the Nested PCR method, where two pairs of external and internal starters are used. The amplification reaction occurs with two stages. Products, created in the first phase (pre - existing amplification products) become the matrix for internal pair of primers, what, as a result, significantly improves the reaction sensitivity. Because of the genetic material exchange between bacteria in natural conditions, several, and even more than ten different primers should be used. The PCR - ELISA test, which is development of method mentioned above, is the connection of Nested PCR and ELISA technique. It consists on introduction of dUTP marked with digoxygenine into dNTP mix ("master mix"), what makes the creation of marked


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amplicones in the amplification process. In the next step, the hybridisation of created products with biotinilic probe of internal primers is executed. Obtained hybrid is placed on polystyrene plates, coated with streptavidine, where it is caught because of strong streptavidine - biotine binding. The hybrid detection is executed with the use of antibodies for digoxygenine, marked with enzyme. Thanks to enzyme and suitable substrate, the colour reaction occurs, marked on ELISA reader. It was shown that this test is from 10 times to 100 times more sensitive than classic PCR method. Multiplex PCR - allows for simultaneous amplification of several genome regions with the use of different pairs of primers. It has its advantages, because it allows detection of several agents in the same time, causing the decrease of examination time and decrease of reagents use. During the own examinations (14) Multiplex PCR was used for B. anthracis, Francisella tularensis and Vibrio cholerae detection. Other genetic methods used for pathogens identification were described in one of works (13). Nowadays, intensive research works take place over elaboration of simple tests "hand handle tickets", adjusted for genetic examinations execution (stripe method) in field conditions. Their sensitivity is similar to classic PCR method. 2.7. Mass spectrometry Mass spectrometry is one of more promising identification methods. The aerosol particles undergo the process of sonification for proteins liberation. Obtained products are purified, concentrated and separated with chromatography method, and then undergo with UV ionisation and are analysed in mass spectrometer. The method allows to identify bacteria, including the inter-strain differences. Further progress of this method was observed after the use of infrared rays (UR) for sample ionisation, instead of UV light, thanks to what the more specific signal and higher sensitivity were obtained. Moreover, what is worth of underlying, the method requires no special sample elaboration (15). PGCIMS (Pyrolisis - gas chromatography - ion mobility spectrometry). Allows to execute the chemical - biological identification (16). After concentration, the sample is treated with the high temperature, what causes the markers liberation, then analysed with the apparatus. That way, for instance, it is easy to detect the picoline acid of B. anthracis with this method. Produced apparatuses are not bigger than shoe-box, nowadays. 2.8. Flow cytometry This method found its application for detection pathogens as well as bacterial toxins. This method value is directly depending on reagents quality (antibodies, antigens). Except antibodies and antigens application, the possibility of use of dyes themselves for diagnostic purposes is worth of attention, which may selectively bind itself with one - or double-stranded nucleinic acids. The vast data concerning the topic of flow cytometry in microbiological diagnostics are contained in works of Stopa (17). The research on UCPRP use for biological agents identification with flow cytometry method seems to be promising (18). The suitability of the method for nucleic acids fragments was proved, what may be used for identification purposes (19).

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2.9. DNA "CHIP" technology It gives the possibility to execute the significant amount of hybridise experiments in the same time (15). The reaction takes place on extremely small plate, where oligonucleotides were placed. The tested DNA is marked with fluorescent marker and observed with conphocal fluorescent microscope. Point emitting fluorescent signal indicate the occurred hybridisation. Different types of mentioned technology create wide range of research and application possibilities (15). Last time, the significant progress of new biological agents detection and identification technologies took place (19). Part of them, after laboratory and field tests, shall be introduced to microbiological practice soon, giving their contribution for more efficient fight against biological threats.

3. General problems and challenges Rapid detection of biological threats gives us the possibility to usage (in time) the non-specific protective methods: usage the HEPA filter masks, individual protective clothes and the collective protection. Ideal will be the situation in which biological factors become detected very quickly, so the introduction of preventing undertakings is highly possible. Nowadays exists many different detection technologies of biological threats, but they need time to distinguish between the real biological factor from the artificial - simulants. Should be also mentioned, that purposeful use of simulants can create, at least at the beginning, the considerable complications (panic, chaos, disorganization) analogous to situation, when the real biological factor were used. Regarding to this only the quick identification of biological factor can prevent the dangerous consequences of spurious the attack. Nowadays exist many technological tendencies, which appear very promising. For example the bio-sensor technique (the immunosensors, optic sensors, taste chips.) also the bioanalysis technologies (flow cytometry, bioluminescence, Volatile Organic compound analysis, DNA analysis for forensic identification etc.). But in the majority they are still in test stage. Among quick identification methods immunochromatographic techniques should be mentioned. However the results of this methods also require the confirmation by the genetic tests. In turn, the genetic tests can create the incorrect results especially, when the environmental samples were tested (the phenomena of naturally exchange of genetic material between bacteria's). Should be brought to the attention that in special cases, even in field conditions, there are needs of usage the classical microbiology methods e.g. culturing the suspicious material for obtaining the precise results. Sometimes the results obtained by the classical microbiology methods are the final confirmatory tests for the results obtained by different methods. According to this the microbiology techniques should be improved and adapt to the field conditions. The limitation of wider implementation to practice the different diagnostic methods the mass spectrometry is e.g. the high price of the device. Apart from the costs of apparatus considerable limitation is also the weight and size of equipment - flow cytometer. The modern identification methods e.g.: the chip and microarray techniques are still not implemented for field diagnosis needs.


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The diagnostic possibilities of the NATO members' countries have the crucial meaning in fighting with world bioterrorism. The problem in protection against bioterrorism in regional and world scale is lack of synergistic laboratories. Necessary it is also the standardization and unification of identification methods for comparing results. Indispensable is the necessity of organization by medical service the international training concentrated on identifications of biological factors. In last years we observed considerable progress in microbiological diagnostic, especially on the genetic level, but however such techniques are not in common use (high cost of modern technology). This cause, that fast reaction on local level will not be possible in case of attack. The limitations are also: weak laboratories infrastructure, lack of experienced staff, impediment to laboratory standards, lack of unification diagnostic procedures. We should also mentioned, that many technologies are designated for laboratory conditions. The technologies, which possess the highest value, are designated and were tested in field conditions. Also unusually essential is continues training of the diagnostic personnel, continues stand by the system, possession of unalterable reserve the reagents designated for identification purposes in case of bioterroristic attack. The most important elements in field of protection are possession of the diagnostic potential for detection genetically modified biological factors. Using the genetically modified biological factors is challenging for Health Service Units, which have to undertake the challenge concerning the public health.

References [1] [2] [3] [4] [5] [6]

[7] [8] [9] [10] [11]

Walt D R, Franz D R. Biological warfare detection. Anal Biochem 2000; 72:738A-46A. Leoma G A. Detecting Biological Agents in Mail Using Fluorescence Particle Sizing. Detection of Bioterrorism Agents. Biodetection Technologies. Furthering Science through Information. Knowledge Press 2003 ;9-21. Bartoszcze M, Niemcewicz M, Malinski M. Recognition of Biological Threats. AAVDM Newsletter 2001; 6:113-4. Bartoszcze M, Niemcewicz M., Chomiczewski K. Biological threats (aerosol, water, unconventional). Joint Service Scientific Conference on Chemical and Biological Defense Research 2002 Nov 19-21; Hunt Valley, Maryland; 21. Bartoszcze M, Bielawska A. The past, present and future of luminometric methods in Biological Detection. Rapid methods of Analysis of Biological Materials in the Environment. Ed. P. Stopa and M.Bartoszcze. Kluwer Academic Publisher Dordrecht/Boston/London 2000;73-7. Bartoszcze M, Arciuch H, Chomiczewski K, Matras J. Some Problems Concerning Application of the Luminometric Methods in the Detection of Bacillus anthracis Spores. Proc.of the 1996 ERDEC Conference on Chemical and Biological Defense Research 1997 Nov 19-22; Aberdeen Proving Ground; 711-2. Nelson D, Loomis L, Fischatti V A. Using Bacterial Enzymes for Rapid Identification of Bacteria. Laboratory of Bacterial Pathogenesis. The Rockefeller University, NY and New Horizons Diagnostics Corporation, Columbia, MD; Information leaflet; 2002. Lidacki A, Bartoszcze M, Arciuch H, Skoczek A, Mierzejewski J. The evaluation of IMS method for biological detection. Proc. of the ERDEC Scientific Conference on Chemical and Biological Defence Research 1995 Nov. 14-17; Aberdeen Proving Ground;775-7. Tempelman L A, King K, Anderson G P, Liglers F S. Quantitating Staphylococcal Enterotoxin B in Diverse Media Using a Portable Fiber Optic Biosensor. Anal Biochem 1996;233:50-7. Lim D. Real time/near real time biosensor. Detection of Bioterrorism Agents. Biodetection Technologies. Furthering Science through Information. Knowledge Press; 2003;1 17-36. Ritter T. Fighting Germs on the Front Lines: an Integrated Laboratory Approach to Field and Lab Analysis and Surveillance. Detection of Bioterrorism Agents. Biodetection Technologies. Furthering Science through Information. Knowledge Press; 2003;95-103.

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Cooper D E. Upconverting Phosphor Technology Overview. Biological Agent Detection and Identification. DARPA, 1999April 27-30; Santa Fe, New Mexico; 76-100. Matras J, Bartoszcze M: Bacillus anthracis. Post.Mikrobiol 2002;41:3-19 Niemcewicz M, Osiak B, Gawel J, Bartoszcze M. Zastosowanie PFGE i PCR w roznicowaniu niechorobotworczych (bezplazmidowych) szczep6w B. Anthracis, B.spp.813. II Konferencja naukowa. Ochrona przed Zagrozeniami Biologicznymi; 2002 listopad 19; Pulawy;27. Donlon M. Biosensors - The tool for fast detection. NATO ARW. 2003 Jan 15-18 Warsaw;31. Spangler G E. Miniaturizing Gas Chromatography in Combination with Ion Mobility Spectrometry. DARPA, 1999 April 27-30; Santa Fe, New Mexico; 302 Stopa P J. The Application of Flow Cytometry For the Detection and Identification of Microbiological Agents. Ph.D. Dissertation. Military Institute of Hygiene and Epidemiology Warsaw; 1999. Wright B. Compact Flow Cytometer. Biological Agent Detection and Identification. DARPA Santa Fe, New Mexico 1999 April 27-3 ;94-100 CBNSP-Annual Report Technology Development. Chemical &Biological National Security Program. http//www.nn.doe.gov/cbnp/tech-dev.sht


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Rediscovering Remote Sensing: Improving Infectious Disease Surveillance Debra M. NIEMEYER, MS, MA, Ph.D., MT (ASCP)1 Lt Col, USAF, BSC Program Executive Office for Chemical and Biological Defense 5109 Leesburg Pike, Skyline #6, Suite 401B, Falls Church VA 22041 Abstract Satellites have been used over the last 30 years to examine and map terrestrial features, and to study properties of the earth's surface. Recently, researchers have explored using remotely sensed data to evaluate associations between vector- and waterborne diseases, habitat changes, human health and transmission risks. With the wealth of archival sensor data, and on-going efforts to launch improved sensor systems, we need to devote our efforts and resources to exploring remote sensing applications to better assess environmental hazards and prevent disease. A new application of an existing technology, remote sensing will improve force protection by enhancing our ability to prevent, monitor and record untoward exposure of our Armed Forces to "chemical, biological and similar hazards" (Public Law 105-85 (10 USC 1074f), Section 765) [1]. Through assessment of archival, baseline and documented changes of environmental factors, we can improve epidemiological surveillance for a variety of hazards, man-made and naturally occurring, prior to and during deployments. Through linkage of satellite imaging and other disparate data with electronic medical surveillance systems, we will be able to use smaller, "expeditionary" personnel packages and equipment footprints to provide near- to real-time assessment in garrison and in theater. Discussion will focus on the application of remotely sensed data for monitoring distribution of communicable diseases.

Disclaimer. The conclusions and opinions expressed in this document are those of the author. They do not reflect the official position of the United States Government, Department of Defense, Joint Program Executive Office for Chemical & Biological Defense, United States Army or Air Force.

1. "Take the High Ground" In 1972, the launch of Lansat-1 initiated the collection and analysis of remotely sensed data for mapping the earth's surface [2]. Since the dawn of time, man has sought the "high ground" for improved observation. Especially important in warfare mountaintops, balloons, and then airplanes provided a better vantage, enhancing surveillance. During the FrancoPrussian War (1870-1871), the first use of balloons for military observation was documented [3]. Aerial reconnaissance and photography was used during the American Civil War to follow enemy troop movements, direct artillery barrages, map gun emplacements and chart harbor defenses and coast lines [4,5,6]. In 1909, Wilber Wright first used the aircraft as an aerial surveillance platform, taking motion pictures over Italy [6]. During World War I, the British

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conducted aerial photography using balloons and aircraft to document large regions for operations planning [7]. Following that war, unmanned craft such as kites and balloons were used for a variety of peaceful surveillance applications ranging from forestry conservation to topographical mapping, and charting of sea coasts [8]. The application of aerial photography for environmental monitoring and meteorological research followed and continues to be used for many functions to include the management of forests and waters, in pollution and vegetation damage control, and in urban planning [8]. Scientists obtain snapshots thousands of feet up to assess environmental changes. As early as 1970, scientists recognized the value of and applied "remote" satellite techniques to improve epidemiological surveillance [9]. The first published remote sensing application for disease surveillance was part of a dracunculiasis eradication campaign in the Republic of Benin [2,10]. Waterborne disease surveillance is also feasible as evidenced by the tracking of global climatic changes associated with cholera outbreaks [11,12].

2. Future Applications For some time, experts have urged measures be taken to more effectively deal with emerging infectious diseases. Recommendations followed for development of a global infectious disease surveillance system [13-15]. Numerous electronic medical surveillance systems are under development and being used by federal, state and Department of Defense (DoD) agencies (Table 12). The systems work well as electronic medical records or for enhancing syndromic surveillance, and several provide data mining tools for the epidemiologist. Lacking, however, is the ability to integrate disparate environmental assessment data, such as Global Information Systems (GIS) to include satellite data [15], and Global Positioning Systems (GPS) to mark and track environmental anomalies and provide prospective analysis for outbreak prediction. As an example, one system being developed by a U SAP-corporate consortium and tested by DoD, the Lightweight Epidemiological Advanced Detection and Emergency Response System (LEADERS), will incorporate disparate data for outbreak prediction [23]. Another system being evaluated by the Air Force Force Protection Battlelab at Lackland Air Force Base, the Deployed Environmental Surveillance System (DESS) will pull data from a variety of sources, to include tactical chemical and biological sensors to enhance the commander's situational awareness [26]. A recent concepts deployment to Honduras resulted in the successful demonstration of rapid screening for Dengue virus and strain specific identification for the vector, Aedes, using realtime PCR [27,28]. Studies are underway to evaluate system linkage with LEADERS and other electronic patient encounter systems, rapid testing technologies, and a Combat Command and Control System (CSC2) capable of GPS and secured (SIPRNET) data receipt [29]. The next step will include incorporation of remote sensing data feeds of select overseas locations for epidemiological analysis [30]. The goal is to concepts test the analysis of remote sensing data to predict occurrence of and track vector-borne diseases and diseases associated with refugee migration and poor sanitation, and to integrate this information onto a common medical surveillance platform. An excellent model of a stand-alone system that employs climate and satellite data for disease surveillance is the collaborative project by DoD Global Emerging Infections Surveillance and Response System (GEIS) and the National Aeronautics and Space Administration (NASA) to monitor Rift Valley Fever (RFV) epidemics in Eastern Africa [31]. Climate and satellite data, regional maps of areas at risk will be available on the Web to

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facilitate a better understanding of the environmental factors associated with disease outbreak and spread, thereby improving opportunities for disease control and prevention. Table 12. Examples of DoD Electronic Medical Surveillance Systems' System

Sponsor2

Employed3

DMSS Defense Medical Surveillance System

DoD / USACHPPM

Retrospective DoD patient database maintained by AMSA.

ESSENCE Electronic Surveillance for the Early Notification of Community Based Epidemics

DoD GEIS developed in response to PDD NSTC-7 on emerging infections (18)

In use by DoD; all MTF data visible to all users

GEMS Global Expeditionary Medical System

AF / HQ ACC/SG

Used in CENTAF, USAFE, being evaluated for PACAF and DoD use, i.e., DEPMEDS and Navy Fleet (SAMS linkage); integration with TMIP under evaluation

LEADERS Lightweight Epidemiological Advanced and Emergency Response System

AF/ HQ USAF/SGX

Utility assessment done at WHMC Fall 01, and being evaluated at WRAMC, Navy Medical Center, Bethesda as DoD initiative. Started as 1year COSSI initiative - AFIndustry partnering to leverage development in the commercial IM/IT sector.

SAMS Shipboard NonTactical ADP Program (SNAP) Automated Medical System

Navy / BUMED

On-board ships

Features4 Primary data repository containing up-to-date and historical data on diseases and medical events (e.g., hospitalizations, ambulatory visits, reportable diseases, HIV tests, acute respiratory diseases, and health risk appraisals) and longitudinal data on personnel and deployment (16). AMSA routinely publishes summaries of notifiable diseases, trends of illnesses of special surveillance interest and field reports describing outbreaks and case occurrences in the Medical Surveillance Monthly Report (MSMR) [16,17]. A sensitive, specific, standardized, timely and flexible health indicator surveillance system (syndromic surveillance). Transparent acquisition, analysis, and dissemination via secure web site of aggregated daily Ambulatory Data System (ADS). Post 9/11 ESSENCE expanded to include ADS data from 413 DoD MTFs in addition to NCR primary care clinics and ERs. ADS data from 121 Army, 110 Navy, 80 Air Force, and 2 Coast Guard installations are processed daily [19,20]. Three part system: 1) PEM - Patient Encounter Module (electronic medical record), 2) TEM - Theater Epidemiological Module (multiple MTF status visible at theater level, i.e., number of cases, bed status, etc.), 3) TOM - Theater Occupational Module - for occupational health assessments (under development) [21,22]. Application Service Provider (ASP)-based; electronic patient encounter, epidemiological data mining tools with automatic alerts and reporting, incident and events management, links for "Labs, Rads, Pharm" data. Evaluation underway to link, disparate databases for nonconventional data feeds (i.e., Veterinary/Public Health, meteorological weather, community OTC pharmaceutical sales); evaluation for TMIP, CHCS/2, GEMS interface on-going. Post 9-11 collaborated with CDC to bring 250+ NY hospitals on-line [23,24]. An automated medical administrative management system designed to address the requirements of shipboard Medical Departments. Included features for the management of health care, administration functions, monitoring functions, medical supplies and health protection programs [25].

Originally published in the Society of Armed Forces Medical Laboratory Scientists (SAFMLS) Newsletter, Society Scope Winter 2003; 6(1):1. Reprint coordinated through Editor; modified for the NATO Conference Series Book.

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DM. Niemeyer /Rediscovering Remote Sensing Notes: 1) 2)

3) 4)

Listing is not intended to be all-inclusive USACHPPM--U.S. Army Center for Health Promotion and Preventive Medicine; GEIS--DoD Global Emerging Infections Surveillance and Response System; HQ ACC/SG--Office of the Command Surgeon, Air Combat Command, Langley AFB VA; HQ USAF/SGX-Directorate of Expeditionary Operations, Science & Technology, Office of the Air Force Surgeon General, Boiling AFB DC; BUMED-Navy Bureau of Medicine and Surgery, Washington DC. AMSA-Army Medical Surveillance Agency; MTF-Medical Treatment Facility; DEPMEDS-Deployable Medical System; WHMC-Wilford Hall Medical Center, Lackland AFB, TX; WRAMC-Walter Reed Army Medical Center, Washington DC; National Navy Medical Center, Bethesda, MD; COSSI--Commercial Operations & Support Savings Initiative. NCR-National Capital Region; CDC--Center for Disease Control & Prevention

3. Conclusion An ever-expanding global mission pushes deployments to and establishment of fixed facilities in regions with endemic infectious diseases and substandard sanitary conditions. Coupled with natural disaster, refugees, eroding infrastructure, and possibility of attack by overt or covert release of biological agents, the need for an integrated multifaceted medical surveillance system is pressing. Evaluating how to use remote sensing to enhance disease surveillance is not only prudent, but it is necessary. Tools to see and monitor "over the horizon" are important to accurately assess the environment. These tools provide early hazard warning to enhance the commander's situational awareness of the battlespace. Laboratory specialists contribute important data for medical surveillance thereby improving situational awareness and enhancing battlespace management. Electronic laboratory reporting can markedly improve surveillance, but it is not without it's challenges [32]. Therefore, key is a team approach with laboratory specialists working alongside preventive medicine, infectious disease and public health specialists, civil and environmental engineers, industrial hygienists, informatics and systems experts to ensure provision of accurate and timely biohazard data. Critical laboratory data includes biological warfare agent and pathogen identification, antimicrobial resistance, therapeutic drug monitoring and nonspecific infectious disease marker results. The on-going challenge remains the search for and understanding of emerging and re-assessment of current technologies for novel applications to enhance force protection in the new expeditionary environment. References [1] [2] [3] [4] [5] [6] [7] [8] [9]

Public Law 105-85 (10 USC 1074f), SECTION 765. Improved Medical Tracking System for Members Deployed Overseas in Contingency or Combat Operations; retrieved at: http://www4.law.cornell.edU/uscode/l 0/1074f.html. Beck, LR, BM Lobitz, BL Wood. Remote sensing and human health: new sensors and new opportunities. Emerg Infect Dis 2000 May-Jun;6(3):217-27; retrieved at: http://www.cdc.gov/ncidod/eid/vol6no3/beck.htm. Balloon; retrieved at: http://encarta.msn.com/FinaVConcise.asp?ti=060E3000. Civil War "Firsts;" retrieved at: http://www.civilwarhome.com/civilwarfirsts.htm. Lowe, TSC, Personal Observation, The Balloons with the Army of the Potomac; retrieved at: http://www.civilwarhome.com/balloons.htm. Center for Remote Sensing and Spatial Analysis, Rutgers University. History of Aerial Photography, retrieved at: http://deathstar.rutgers.edu/courses/airphoto/airphoto 1 /sld023 .htm. History of Ballooning; retrieved at: http://airtime.hvpermart.net/history.htm. Aerial photography in Canada: A brief history; retrieved at: http://airphotos.nrcan.gc.ca/history.html. Cline, BL. New eyes for epidemiologists: aerial photography and other remote sensing techniques. Am J Epidemiol 1970 Aug;92(2):85-9; abstract retrieved at:


[10] [11]

[12]

[13] [14] [15] [16] [ 17] [18]

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[24] [25] [26] [27] [28] [29] [30] [31]

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http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&listuids=5464318&dopt= Abstract. Clark KC, JR Osleeb, JM Sheery, JP Meert, RW Larsson. The use of remote sensing and geographic infomation systems in UNICEF's dracunculiasis (Guinea Worm) eradication effort. Prev Vet Med 1990;229-235. Colwell, RR. Global climate and infectious disease: the cholera paradigm. Science 1996 Dec 20;274 (5295):2025-31; abstract retrieved at: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list uids=8953025 &dopt=Abstract. Lobitz, B, Beck L, Huq A, Wood B, Fuchs G, Faruque AS, Colwell R. Climate and infectious disease: use of remote sensing for detection of Vibrio cholerae by indirect measurement. Proc Natl Acad Sci USA, 2000 Feb 15;97(4):1438-43; retrieved at: http://www.pubmedcentral.nih.gov/articlerender.fcgi?tool-pubmed&punmedid=l-677480. Morse, SS, B Rosenburg, J Woodall. Global Monitoring of Emerging Diseases: Design for a Demonstration Program; retrieved from: http://www.fas.org/promed/papers/proposal.html. Shalala, D. Collaboration in the Fight Against Infectious Diseases, Emerg Infect Dis 1998 Jul-Sep;4(3); 354-7; retrieved at: http://www.cdc.gov/ncidod/eid/vol4no3/shalala.htm. Nuttall, I., DW Rumisha, TRK Pilatwe, et, al., GIS Management Tools for the Control of Tropical Diseases: Applications in Botswana, Senegal, and Morocco; The International Development Research Centre Web site: http://www.idrc.ca/books/focus/766/nutta.html. Defense Medical Surveillance System (DMSS), Army Medical Surveillance Agency (AMSA); retrieved at: http://www.amsa.army.mil/AMSA/amsa_home.htm. Medical Surveillance Monthly Report (MSMR), AMSA. National Science and Technology Council Presidential Decision Directive 7 (NSTC-7, 1996) documented the lack of U.S. readiness to identify and respond to emerging infectious diseases and ordered DoD and other agencies to improve surveillance, response, and prevention capabilities; retrieved at: http://www.fas.org/irp/offdocs/pdd ntsc7.htm. Electronic Surveillance for the Early Notification of Community Based Epidemics (ESSENSE) Back ground Paper, Department of the Army, Office of The Surgeon General. DoD Global Emerging Infections Surveillance and Response System, retrieved at: http://www.geis.ha.osd.mil/. Morrow R.C., KO Schafer, RL Williams. Quality of deployment surveillance data in Southwest Asia. Mil, Mil Med 2001 Jun:166(6):475-9; abstract: http://www.ncbi.nlm.nih/gov/entrez/query.fcgi?cmd+Retrieve&db=PubMed&list uids=11413722&dopt =Abstract. Humphrey, E, presentation entitled, "Global Expeditionary Medical System." Headquarters Air Combat Command Office of the Command Surgeon, 5 Mar 2002. Niemeyer, D, E Hanson, R, R. Rowley, R Munson, K Schafer. Real-time Medical Surveillance for Early Warning and Mitigation of Environmental Hazards. Proceedings of the International Conference on Protection Against Biological Threats, Sponsored by DARPA and the General Karol Kaczkowski Military Institute of Hygiene and Epidemiology, Warsaw, Poland, June 2001. Bunk, S. Early Warning. The Scientist 16(9): 14, April 29, 2002. Virtual Navy Hospital. Military Sealift Command Medical Manual; Appendix A, Glossary. Shipboard Non-Tactical ADP Program (SNAP) Automated Medical System (SAMS); retrived at: http://www.vnh.org/Admin/MSCMedManual/AppendixA.htmlffdefinitions. Niemeyer, D. Kenny Battlelab Initiative, Deployed Environmental Surveillance System (DESS), Air Force, Force Protection Battlelab, May 2000. Swaby, J, D Lowe. Deployed Environmental Surveillance System (DESS) Initiative Briefing Joint Services Integration Group, Mar 2001. Personal Correspondence, Col J Swaby, Air Force Institute of Operational Health, Brooks City-Base TX, Sep 2002. Combat Support Command and Control System (CSC2) Joint Expeditionary Experiment 2000 (JEFX 2000) After Initiative Report. Niemeyer, D, J Swaby. Proposed Kenny Battlelab Initiative, Remote Sensing for Infectious Disease Surveillance from a Common Expeditionary Platform (draft). Climate and Disease Connections—Rift Valley Fever Monitor. DoD GEIS; retrieved at: http://www.geis.ha.osd.mil/RVFWeb/index.htm.

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[32]


Overhage JM, J Suico, CJ McDonald. Electronic laboratory reporting: barriers, solutions and findings. J Public Health Manag Pract 2001 Nov;7(6):60-6; abstract retrieved at: http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd±=Retrieve&db=PubMed&list uids=11713754&dopt=Abstract


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The Joint Biological Agent Identification and Diagnostic System (JBAIDS) Debra M. NIEMEYER, MS, MA, PhD, MT (ASCP)1,2,3 Joint Program Executive Office for Chemical and Biological Defense 5109 Leesburg Pike, Skyline#6, Suite 401B, Falls Church VA 2204 Abstract. The Joint Biological Agent Identification and Diagnostic System (JBAIDS) program will field a reusable, portable, modified commercial system for military use. The system will be capable of simultaneous, reliable identification of multiple Biological Warfare Agents and pathogens of military significance. JBAIDS will enhance force protection by providing medical personnel and commanders an expedient means to reliably and quickly identify biological threats in the operational environment, whether naturally occurring or man-made. Information derived from JBAIDS will improve situational awareness and facilitate the application of effective preventive measures, prophylaxis, and appropriate treatment. Medical personnel will use the JBAIDS to quickly identify exposure to or infection by biological agents and screen for endemic diseases. System employment will be at multiple levels of health service support to include deployed medical units, fixed medical facilities, ground vehicles, aircraft, and aboard ship for contingency and humanitarian mission, and homeland security medical support. The acquisition, production and fielding of JBAIDS will occur in tree increments or blocks. Block I will be a nucleic acid amplification system that will identify a limited number or biological agents. Block II includes the capabilities of Block I, plus the capability to identify a limited number of biological toxins. Block III will identify Block I and Block II agents, plus additional bacterial, viruses, fungi, and toxins referenced from standard threat lists, region-tailored based on the Combatant Commander guidance. The Block III system will be lightweight, handheld device employed by the medic for point of care testing. With automated sample preparation, testing and results interpretation, first responders will use it for biohazard assessments, recon and surveillance activities. Block III will interface with existing medical surveillance networks and feed pertinent information into Chemical, Biological, Radiological, and Nuclear Warning and reporting systems. Each block will incrementally improve decision-making through enhancement of the Commander's situational awareness of biological hazards in the battle space. A review of the program, to include the near-term acquisition strategy and technical requirements will be discussed.

Disclaimer. The conclusions and opinions expressed in this document are those of the author. They do not reflect the official position of the U.S. Government, Department of Defense, Joint 1 JBAIDS Team: Bakeer B, Belrose B, Bentley C, Craig P, Korte K, Melling D, Wilson S, Joint Program Executive Office for Chemical and Biological Defense (JPEO-CBD), Falls Church VA; Bayless b, JPEO-CBD, 311th HSW, Brooks City-Base TX; Allen k, U.S. Army Medical Material Command, Legal Office, Fort Detrick MD, Potter W, Selfridge L, U.S. Army Space and Missile Defense command (USASMDC), JPEO-CBD, Chemical Bioloical Medical Systems (CBMS), Frederick MD; 2 Other/Support: Danley D, Ranhofer R, JPEOCBD, CBMS, Frederick, MD, Fanelli W, House K, JPEO-CBD, Falls Church VA. 3 Submitted to the Society of Armed Forces Medical Laboratory Scientists (SAFMLS) Newsletter, Society Scope, for publication. Reprint coordinated through Editor, modified for the NATO Conference Series Book. Note: Cleared for Open Publication.

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DM. Niemeyer / The Joint Biological Agent Identification and Diagnostic System

Program Executive Office for Chemical and Biological Defense, United States Army or United States Air Force. 1. Introduction The Joint Biological Agent Identification and Diagnostic System (JBAIDS) exemplifies the new United States (U.S.) Department of Defense (DoD) approach to the acquisition of products and materials under the streamlined acquisition process instituted by DoD and the U.S. Congress, August 2002. This program constitutes DoD's first effort to develop and field a common medical test platform that will identify biological agents - Biological Warfare (BW) agents and pathogens of operational concern. The system will be configured to support reliable, fast and specific identification of biological agents from a variety of clinical specimens and environmental sample sources. JBAIDS will enhance force protection by providing commanders and medical personnel the capability to determine appropriate treatment, effective preventive medical measures and medical prophylaxis in response to the presence of biological agents [1]. "Lessons learned" from the Persian Gulf War pointed to the need for BW agent detectors and subsequent material solutions alleviated capability gaps. However, another deficiency was identified in response to "Gulf War Syndrome" and other ailments suffered by military personnel - medical personnel needed a portable diagnostic tool to quickly identify disease-causing biologic agents. Thus, the concept for JBAIDS was proposed [2]. Its need is reinforced by the threat of biological attack faced by our forces deployed around the world. Furthermore, on-going operations in war-torn locations regularly present with infectious disease challenges that demand far-forward agent identification capabilities to retain troop readiness, and quickly access specific patient treatment options to include medical evacuation [3]As a result of the September 11th attack on the World Trade Center in New York City, and the subsequent release of Anthrax spores in the U.S. postal system, the U.S. Military moved forward to purchase and field medical BW agent identification equipment for installation and carrier battle group defense. However, still lacking is a common "workhorse system", a complete package with support equipment, supplies, and an extensive array of validated agent assays, sample protocols, and standardized training. Additionally, a consolidated DoD plan is needed to obtain system clearance from the U.S. Food and Drug Administration (FDA) for diagnostic use. The JBAIDS program encompasses that complete system package and FDA plan [1,2].

2. Incremental Capabilities Development JBAIDS is a competitive development and production program designed to meet military end user requirements. The JBAIDS Office is housed within Chemical-Biological Medical Systems (CBMS) of the Joint Program Executive Office for Chemical and Biological Defense (JPEOCBD). Different from other medical products such as vaccines, protective skin lotions, and anticonvulsants, a multi-block configuration, spiral development and fielding approach is proposed to obtain a licensed multi-agent identification system. The JBAIDS Block I program consists of the development and testing of BW agent identification hardware, along with Production Block I options for manufacture, integration,


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and fielding of 1KB AIDS systems late 2004 to early 2005. System clearance by FDA will be initiated in Block I and carried throughout for each Block upgrade. The system will comprise platform test equipment, assay test kits with controls, protocols for sample preparation and system operation, ancillary equipment and consumable supplies, maintenance manuals, and training materials (Table 13). Block I will provide a capability to identify ten biological agents (Table 14). The JBAIDS Office will procure a modified Commercial-Off-The-Shelve (COTS) or modify a Non-Development-Item (NDI) Table 13. Components of JBAIDS

JBAIDS System Protocols • •

Sample Preparation System Operation

Platform Test Equipment

Expendable Supplies

• • • •

• •

Assay Analysis Hardware Laptop Computer Associated Software Storage/Shipping Case

•

Assay Test Kits Shelf-Stable Reagent Kits Other Laboratory Consumables

Table 14. Select Block I and II Agents1 AGENT OR DISEASE

ORGANISM

Block 1 Anthrax Brucellosis Ebola-Marburg Plague Q-Fever Salmonellosis Smallpox Tularemia Typhus Fever

Bacillus anthracis Brucellae Filoviridae Yersinia pestis Coxiella bumetii Salmonellae Orthopox viruses Francisella tularensis Rickettsia typhi

Block II Clostridium botulinum Botulium Ricin Ricinus communis Staphylococcus Enterotoxins (i.e., SEB) Staphyloccus aureus Notes: 1) Representative agents for which assays will evaluated for development 2) Does not provide a complete list 3) Agents are not listed in any priority or importance

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system design to meet the requirement. The COTS/NDI system will be used by medical laboratory specialists configured to support forward deployed operations for force health protection. The Block I system will undergo environmental, reliability, performance and suitability testing in late 2003 and early 2004. FDA testing for 510(k) submissions will be performed in parallel. The Block II increment is an evolutionary enhancement to field a toxin identification capability by expanding the modular design of the Block I hardware, software, and addition of test reagents to handle toxin identification. Additional tests will be added to the Block I assay complement (Table 14), expanding test panel options for the laboratory operator. Block III will fully integrate microorganism and toxin identification technologies into a single, lightweight, handheld or smaller unit that can correctly identify at least 50 different biological agents (bacteria, viruses, and toxins) in less than 15 minutes. Block III requirements will be fine-tuned when mature technologies surface in the future that have potential to meet JBAIDS out year requirements, to include automated sample processing and linkage to theaterlevel electronic medical information systems that will tie into a lager early warning and reporting systems. Emerging candidate technologies range from nucleic acid and antigen microarray platforms, to nanotechnology for single cell analysis - the "lab on a chip" concept [4,5]. The Government's acquisition plan allowed for commercial companies to bid any technology in response to the Pre-solicitation announcing the star of a Market Survey for suitable off-the-shelf solutions [6]. Only relatively mature technology was evaluated, having to meet the identified end user screening requirements, for example, the system must already exist either in production or functioning prototypes. Additionally, proposed technology had to pass a two-week long performance-based "Fly-Off Laboratory Test and be suitable for accelerated Developmental and Operational testing and evaluation with rapid fielding of requirementscompliant systems by December 2004. The down-selected contractor is to posses the capacity to manufacture in accordance with Good Manufacturing Practices (cGMP) and be compliant with new biological surety requirements [7]. Following contract award, summer-fall 2003, the contractor needs to be ready to submit the platform, with sample protocols, and key assays (i.e., Anthrax and Smallpox) to the FDA for clearance as a diagnostic device. Assays for the remaining Block I biological agents will be incrementally submitted as the reagent kits are produced. 3. Streamlined Acquisition JBAIDS is a unique program that combines a streamlined acquisition process with an evolutionary block, spiral development strategy by leveraging available commercial biotechnology and manufacturing capabilities, and utilizing the research strength of government laboratories. This new process provides a template by which to evaluate and down-select against established end user requirements, mature, off-the-shelf technologies for inclusion into current and future Joint Service Chemical and Biological Defense programs. Representatives from across the DoD other U.S. federal agencies (i.e., Departments of Health and Human Services, Agriculture, Energy, and the Environmental Protection Agency) and the North American Treaty Organization participate on Integrated Product Teams. These different agencies have banded together to select promising candidate systems to be further developed to provide a much-needed capability, setting a precedent for resource-sharing. This streamlined approach ultimately benefits the user community by dramatically reducing acquisition time and


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coasts. It serves as a model depicting for Program Managers and Material Developers how to take advantage of off-the-shelf technologies to rapidly field much needed multi-use products. JBAIDS will be a flexible, multi-use technology employed on the conventional battlefield and in far-forward locations around the globe to combat terrorism, improve force health protection and patient care, and enhance Homeland Defense. 4. Summary JBAIDS, the first program in which FDA clearance will be sought for rapid biological agent analysis, will serve as the benchmark for. future Government and Industry partnering. Furthermore, an innovative fielding plan employs parallel processing, and will reduce delivery time by delivering a Block I capability for environmental sample testing and surveillance while the system moves through FDA review. Unlike medical devices currently employed for biological defense, a complete system package will be delivered, ready for immediate use. Additionally, the JBAIDS Office is currently working with JPEO-CBD Critical Reagents Program, the Armed Forces Institute of Pathology, and other Centers of Excellence, such as the Air Force Institute For Operational Medicine, the U.S. Army Medical Research Institute of Infectious Diseases, and the U.S. Army Soldier Biological Chemical Command, to set up an overarching Quality Assurance and Proficiency Program to assess operator performance in testing samples containing biological agents. Operator certification standards will be established along with re-certification requirements. Furthermore, a New Equipment Training course will be designed utilizing the best of existing military and civilian training programs. This process will also identify course updates in military schools necessary for successful system fielding. The JBAIDS program long-term acquisition strategy ensures continuous improvement by leveraging off of the "latest and greatest" technological solution Industry has to offer. The same acquisition strategy will be used for Block II, toxin identification, with a market survey projected late 2003 following Block I contract award. A DoD first, this strategy will ensure that the best, most modern and readily available capability is quickly and efficiency evaluated against approved end user requirements to rapidly field a material solution to the warfighter.

References [1]

Joint Biological Agent Identification and Diagnostic System (JBAIDS) Operational Requirements Document, 6 Sep 2002.

[2] [3]

Annual Report to Congress and Performance Plan, Washington DC, DoD Program, 2003. Niemeyer D. Improved Public Health Surveillance: Advanced Technologies and Techniques (invited manuscript submitted to JCLA for Summer 2003 publication). Niemeyer D. Genomics and Bioinformatics: The Next Technological Leap. Society Scope, Summer; 5(2) and Fall 02;5(3):2 (Editor's Page). Rudert F. genomic and Proteomics Tools for the Clinic. Curr Opin Mol Ther 2000 Dec;2(6):633-42; abstract retrieved at:

[4] [5]

http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=l 1249740&dopt=Abstract

[6]

JBAIDS Pre Solicitation Notice, Federal Business Opportunities Solicitation Number DASG60-02-R-0008 (closed 28 Mar 2002). FBO Archived document, on-line, Internet, available at: http://www.eps.eov/servlet/Solicitation/R/USA/SMDC/DASG60/DASG60-02-R-0008.

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[7]


Possession, use and transfer of select agents and toxins, in 42 Code of federal Regulations (CFR) Part 73, Interim Final Rule, on-line, Internet, available at: http://www.cdc.gov/od/sap.


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Biosensors - The tool for fast detection Mildred A. DONLON Program Manager, Biological Warfare Defense DARPA/SPO 3701 North Fairfax Drive,Arlington, VA 22203-1714, USA Abstract. The threat of attack on military and civilian targets with chemical and biological weapons is a growing national concern. The Defense Advanced Research Projects Agency (DARPA) is developing technologies for detecting biological materials in the natural environment. While several technologies show promise as broadband detectors, there is no "silver bullet" that detects all chemical and biological materials at the requisite levels of sensitivity and specificity. DARPA is developing enabling components for use in biological detectors.

The threat of attack on military and civilian targets employing chemical and biological weapons is a growing national concern. Technologies for detection of these materials in the natural environment are being developed by DARPA. While several technologies show great promise as broadband detectors, there is no "silver bullet" that detects all chemical and biological materials at the requisite levels of sensitivity and specificity. DARPA is developing biosensor technologies to identify and develop new component technologies which will enable the development of fast, highly sensitive, and highly specific biosensor systems that will reduce false alarm rates and increase the ability to detect and identify multiple biological warfare agents. Most current biosensor systems use antibody-based components with fluorescent reporters to detect the BW agent and the Polymerase Chain Reaction (PCR) to amplify the DNA genome. This multistep approach is both time consuming and complex, and limits the development of both rapid and unattended detection and identification systems. Biosensor technologies are currently under development to support this goal: the enhancement/replacement of antibodies, mass spectrometry technologies, and a phylogenic microchip. Enhancement/Replacement of Antibodies is being approached by developing small molecular weight compounds with high affinities and specificities that will enhance current antibody identification protocols, with the ultimate objective of using these kinds of moieties to replace the antibody as the principal detection/identification molecule in biosensors. The mass spectrometer is known as the most powerful laboratory analytical tool for analysis of a broad spectrum of chemical and biological materials. A Phylogenic Microchip, containing an expanded hierarchical set of more than 100 oligonucleotide probes, is being developed which will enable the parallel detection and identification of a variety of species of organisms allowing rapid determination in unknown samples.

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M.A. Donlon /Biosensors - The Tool for Fast Detection

Figure 33. FACS analysis showing specific binding of fluorescently-labeled mAbs to B. anthracis spores

This project has prepared anii-Bacillus anthracis spore and anti-exosporium monoclonal antibodies (mAbs) and showed that many of the mAbs were species specific for B. Anthracis spores (Figure 33). The mAbs were used to produce smaller polypeptides for example Fab fragments, single-chain antibodies (scFvs), variable and complementary determining regions. These fragments were tested as ligands for spore binding. Fab fragments bound cognate spores nearly as well as intact mAbs. Many of these mAbs appear to bind a single exosporium-associated glycoprotein, which is part of the exosporium hair-like nap. Commercial phage display peptide libraries were also screened (5 peptides/phage) to identify short peptides (5-6 amino acids) that bind B. anthracis spores. The purified peptides are being modified to increase valence and improve specificity. Smaller fragments are presently being tested. Infra-Red Mass Spectrometer Mass spectrometry is a technique for the determination of the masses of molecules and specific fragmentation products formed following vaporization and ionization. Detailed analysis of the mass distribution of the molecule and its fragments leads to molecular identification. These molecular measurements can be carried out at the attomole (10-18 mole) level of material using specialized laboratory-based instruments. The combination of specific molecular identification and extreme sensitivity makes mass spectrometry one of the most powerful analytical laboratory tools. While such capability has existed in the laboratory for many years, the development of a small, portable mass spectrometer for potential field detection of chemical and biological substances remains under development. Usually such instruments have employed UV lasers at 337nm. However, a number of laboratories have reported that IR lasers offer various advantages for desorption of proteins and especially for desorption of proteins from thick samples. MALDI mass spectra were obtained on a prototype Mantis time-of-flight instrument (Science and Engineering Services, Inc., Burtonsville, MD, USA). The instrument has a fourinch end-cap reflection and was operated in the positive mode, at 20kV accelerating voltage with a 0.3 us delay time. A PTI (London, Ontario, Canada) model PL2300 nitrogen laser was used as a UV laser source and a prototype of an SESI (Burtonsville, MD) model TMIR-1000


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all-solid-state optical parametric oscillator (OPO) based tunable laser source was used at 2.94 um in IR experiments. Tablel5. Mass units associated with various bacterial spores and treatments

Organism

IR MALDI (1)

UV MALDI (1)

B. cereus

4833

4833

7930 9534 (4,5) 12453 B. subtilis

B. globigii

6847 (4,5) 6949 (4,5) 7779 (4) 8064 (4) 8651 (4) 9136 (4,5)

6949 7779 8064 8651

6910 7082 7346 8898

7082 7346

UV MALDI and 5% TFA (2) 4833 6712 6834 7082 7930 (3) 9534 (3) 12453 6847 6949 (3) 7779 9130 (3) 6910 7082 7346 8898 (3)

Spectra obtained from B. cereus, B. subtilis and B. globigii spores using IR- and UVMALDI were obtained. Respectively Table 15 summarizes the biomarker ions that were observed from these microorganisms. The number of biomarker peaks desorbed in the mass range 4000-14000 Da is greater when the IR laser is used. In the analysis of B. cereus spores, the peak at m/z 4833 dominates in both IR and UV spectra. However, IR-MALDI desorbs several additional biomarker ions above 6000 Da.

Phylogenic Microchip The most characteristic molecules for identification of an organism are the genetic materials DNA and RNA. Researchers working at Argonne National Laboratory (ANL), from the Englehardt Institute in Moscow, have developed a tool for rapid identification of microorganisms using a three dimensional gel pad chip. The target moiety in their investigation is ribosomal RNA (rRNA). Since rRNA is present in many thousand copies per cell, the selection of this target obviates the need for signal amplification using PCR-based methods. Therefore, analysis of RNA dramatically speeds the analysis as compared to other nucleic acid identification approaches. In the evolutionary history of life, the RNA molecule and the ribosome structure were among the first objects developed. In particular, the 16S fraction of rRNA is intriguing to study as it appears to be one of the genetic elements which encodes the roadmap of evolutionary progression and divergence from simple to complex organisms. The 16S rRNA consists of 1 500nucleotides in a characteristic structure. As life has evolved, this

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molecule has evolved with it. Every living creature from bacteria to humans has rRNA materials within its cells. In the RNA structure there are highly conserved regions along with regions that are show considerable variability. By judicious selection of RNA complementary probes, regions of the 16S rRNA can be used to detect and phylogenetically distinguish organisms at the genus and species level-using the RNA chip. The scientists working at ANL have been able to develop a two dimensional array of complementary nucleic acid probes immobilized within a 10 X 60 micron gel pad. The gel pads act as a very small test tubes for carrying out the annealing reactions between the complementary probes and the rRNA oligonucleotides. Detection is carried out by breaking open the cells of interest, cleaving the rRNA, allowing the resulting oligonucleotides to flow over the chip, and measuring the fluorescence of the individual pads using a fluorescent microscope. From a knowledge of the complementary sequence, the sequence of a particular section of RNA can be determined and identification of the organism can be obtained. This type of chip is capable of distinguishing organisms that differ in only a single base pair at the target site. The first application of this technology has been the development of a "Bacillus Chip." The genus Bacillus is quite important for biological detection applications because it includes the organism responsible for anthrax. However, the surface structure of spores of the genus Bacillus is such that highly specific antibodies for anthrax are not as yet available. The "Bacillus Chip" is a technology that allows the specific identification of anthrax from closely related organisms. One caveat of the rRNA based chip is that it is of little use in detecting viruses which do not contain an amplified target such as rRNA and proteinaceous toxins which may only contain small variable amounts of contaminating nucleic acids. These caveats may be overcome by the amplifying the signal through PCR based methods or enhancing the detection of low copy signals using other technologies such as the mass spectrometer for readout.


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A flexible approach to biomanufacturing K.P. O'CONNELL, P. E. ANDERSON, D.C. LUKENS, M. H. KIM, A.S. KHAN, R. G. THOMPSON, J.T. PARK, J. J. VALDES U.S. Army Edgewood Chemical Biological Center, APG, USA N. BEEK, T. CHASE, .W. BENTLEY Chesapeake PERL, Inc., 387 Technology Drive, College Park, USA Jordan Kostov and Govind Rao University of Maryland Baltimore County, USA Abstract. A flexible, creative, and rapidly responsive biomanufacturing infrastructure is an essential part of an effective overall strategy for bioterrorism preparedness and biological defense. A variety of approaches and technologies are evolving to provide the capacity to bring innovations in biological threat detection, prophylaxis, and therapeutics from the laboratory bench to advanced development and ultimately to the end user and/or the marketplace. Biotechnology products, including affinity reagents, real time PCR probes and primers, molecular elements for microarray design and manufacture, therapeutic peptides, and vaccines, each have unique requirements for their production at useful scales. Another critical feature of a flexible bio-manufacturing facility is the ability to archive a variety of biological materials in a secure fashion. We will describe in this paper the multifaceted approach to biological manufacturing being advanced by the U.S. Army Edgewood Chemical Biological Center and its partners in government, academia and industry. State-of-the-art biological manufacturing methods (efficient cell culture reactors, cost analysis studies) as well as traditional methods (fermentation) and an advanced cryorepository are being used to solve problems in biological agent detection, agent simulation, environmental decontamination, and the production of biologicals for human clinical trials. Results from research on real-time optical monitoring of in vivo production of recombinant proteins are also described.

Introduction All immunologically based and DNA/RNA hybridization-based sensors that detect biological warfare agents require agent-specific molecules to form a specific and sensitive interface between the sensor apparatus and the threat agent. For immunosensors, those reagents are commonly polyclonal or monoclonal antibodies. Antibodies are proteins produced by the immune system that selectively recognize and bind target molecules or organisms with high affinity and selectivity. As such, they are the essential component in immunosensors that detect biological warfare agents and are the source of their sensitivity and selectivity. The biological defense research establishments of most countries incorporate into their sensor platforms antibodies produced either in whole animals (polyclonal) or in mammalian cell cultures (monoclonal). There is considerable lot-to-lot variability in the current production of antibodies. This is especially true for polyclonal antibodies, which require the injection of a disarmed B W agent into animals. The individual response of each animal to an agent can vary dramatically. The process of developing antibodies in animals or in mammalian cell culture is also time-consuming, which limits the capacity for "just-in-time" or surge production in time

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of conflict. We discuss here our efforts towards providing flexible, rapid, and scalable manufacturing solutions for the production of biological defense reagents. We have established (or are establishing) these capabilities with the additional goal of providing broader biotechnology manufacturing services to researchers in government, academic, and biotechnology industry laboratories. 1. Reagent Development 1. 1. Recombinant Antibodies For Simulant Detection In addition to antibodies that bind BW agents, there is a need for antibodies that bind and detect organisms and substances that simulate BW agents. A panel of BW agent simulants that are non-toxic and non-pathogenic is widely used for the development and testing of biosensors and environmental samplers, in a work setting without the need for high levels of biological containment. Two such simulants are bacteriophage MS2 (a non-pathogenic virus of the bacterium Escherichia coli, which is used to simulate viruses) and ovalbumin (a benign protein which is used to simulate protein toxins, such as ricin). To meet the need for highquality, inexpensive antibody reagents that bind these BW simulants, we have used a powerful technique called phage display [1,2] to isolate antibody genes from immunized mice. The resulting antibody molecules are called Fabs, indicating that they are comprised of heavy and light chain antibody sequences, which form the antigen-binding variable region, but do not contain the IgG constant region. We have cloned and characterized three new reagents, two that bind ovalbumin and one that binds MS2. Briefly, recombinant antibodies are cloned from a library of heavy and light chain genes cloned into a DNA expression vector appropriate for the organism in which the antibodies will be expressed. In our case, the vector also created a fusion between each antibody in the library and one of the coat proteins of the filamentous bacteriophage Ml3 (Figure 34). Expression of the antibody genes in E. coli cells in the presence of helper phage creates a population of Ml3 particles each displaying a different antibody. The resulting particles each contain the gene that encodes the antibody displayed on its surface. The display of the recombinant antibodies on the surface of the phage allowed us to obtain clones of antigen-specific antibodies through binding recombinant MS2 coat protein immobilized in the wells of microtiter plates (a form of affinity purification called "biopanning"). To minimize the possibility of contaminating bacterial cultures with intact MS2 and to obtain the purest antigen possible for affinity screening, we expressed and purified a recombinant version of the MS2 coat protein. Figure 34. Schematic of antibody displaying filamentous phage particle. A phage particle in the display library contains DNA encoding an antibody from the immunized mouse, and displays that same antibody on its surface. The entire repertoire of antibodies cloned from the mouse is thereby converted to a recombinant form that can be screened by applying the population to a surface coated in the antigen of interest.

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After purification, the recombinant coat protein was used to screen the antibody library for clones that bind MS2. Ovalbumin for both immunization and biopanning was obtained commercially (Pierce Co, Rockford, IL). Recombinant anti-MS2 and anti-ovalbumin antibodies were used in enzyme-linked immunosorbent assays (ELISAs) to detect their corresponding antigens. Purified MS2 was detectable down to a level of 250 ng (protein equivalent) in this experiment (Figure 35). The recombinant coat protein used for biopanning and screening, however, was detected to a level of approximately 20 ng [3]. This apparent greater sensitivity for the recombinant coat protein over the intact virus may reflect the isolation of the antibody using the recombinant protein as the biopanning target. It may also indicate that the epitope bound by the anti-MS2 Fab is partly obscured when the coat protein is in the three-dimensional context of the intact viral coat. Use of intact virions as the target, as well as performing the affinity capture of anti-body-displaying phage in solution will allow the isolation of Fabs with even greater affinity for intact MS2. Anti-MS2 did not bind phage Ml3, BSA, or ovalbumin (data not shown). Using partially purified recombinant antibodies OVA-3 and OVA-4, ovalbumin was detectable down to a level of 1 microgram (data not shown). 1.2. Peptides as Affinity Reagents To complement the development of recombinant antibodies, we have employed other combinatorial/genetic approaches to derive novel reagents for affinity-based biosensors. Antibodies, being relatively large molecules of moderate stability, require storage conditions not always available to persons in the field, outside highly controlled environments. The traditional hybridoma approach for developing antigen-specific antibodies is also costly, labor intensive and may access only a fraction of the variants of antibodies thought to be encoded within the immunological genome [4]. The understanding that only a relatively short stretch of the antibody polypeptide actually comprises the antigen-binding site makes searching for short peptides with antigen-binding capacity a reasonable undertaking. [5-7].

Figure 35. ELISA detection of (A) MS2 virions and (B) recombinant MS2 coat protein using purified anti-MS2 Fab antibody. Values represent the mean of duplicate measurements, adjusted by subtracting the absorbance measured in the no antigen control. The antibody appears to be more sensitive for the detection of the recombinant coat protein.

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We have employed a combinatorial random peptide display library expressed in E. coli to identify short, linear peptide sequences that bind to ricin and staphylococcal enterotoxin B. Kinetic analysis of peptide binding to the toxins shows lower equilibrium binding constants for the peptides than monoclonal antibodies. This is attributed to both slower association rate constants and faster dissociation rates for the peptides. The ricin peptides bound to ricin with a KD of luM versus the antibody's KD of 14 nM. The SEB peptide bound to SEB with an affinity of 31 nM compared to the antibody's affinity of 0.35 nM. Further characterization, testing and refining of these reagents is currently underway. 2. Antibody production 2.1. General Remarks As mentioned above, antibodies form the molecular interface between immunosensors and the target molecules or organisms they detect. Many of the reagents used in these sensor systems are monoclonal antibodies (mAbs) that are specific for target organisms of concern. The use of mAb specific for biological warfare (BW) agents in fielded assays requires the maintenance of a high-quality and economical supply of these reagents. A common method for producing monoclonal antibodies from an existing hybridoma cell line is to inject a laboratory animal (usually a mouse) with a population of the cell line. The hybridomas grow in the peritoneum of the mouse and produce antibody in a fluid that accumulates there, called ascites. However, occasionally hybridoma cells produce abdominal tumors rather than ascites fluid, necessitating the growth of the cell line in vitro to obtain antibodies [8]. In addition, there is a growing ethical concern about the humaneness of producing mAb using this method. An increased emphasis on development of better in vitro methods has generated commercially available methods for production of mAb without the use of live animal hosts [8]. We have worked with and evaluated three in-vitro cell culture methods for the production of monoclonal antibodies (mAb): gas-permeable bags, Integra CELLine membrane flasks, and a hollow fiber bioreactor. 2.2. Production of Monoclonal Antibodies in Three Types of Bioreactors In developing and optimizing methods for producing 0.1 to 10 grams of monoclonal antibodies, we examined three in vitro technologies. In vitro methods were chosen to avoid the regulatory issues encountered when establishing animal colonies, and to develop more controlled, defined manufacturing processes. The methods/devices examined were: • A Cell-Pharm® System 1500™ Hollow Fiber Bioreactor (Unisyn, Hopkinton MA, USA) in this study. In a hollow fiber bioreactor system, nutrient medium is separated from cells and secreted mAb by semi-permeable capillaries. Because most of the cells were attached in the capillary surface, the cell density in the bioreactor could not be determined during the culture. The antibody produced in the Unisyn CP1500 bioreactor (a hollow-fiber cartridge with a l0kd molecular weight cutoff) was approximately 450 mg in a 3-month continuous incubation period. • Integra CELLine flasks (Integra Biosciences Inc., Liamsville MD, USA). These units provided easy access to both the nutrient medium compartment and the cell


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compartment to allow frequent monitoring of cultures [9, 10]. Maintaining producing cultures required periodic splitting of cells and exchange of the nutrient medium. Cells and spent medium were removed easily for counting cells and harvesting antibodies, and the number and viability of cells in the cell compartment of the flask were able to be tightly controlled. The nutrient medium was poured easily from the reservoir, allowing for quick medium exchange while permitting viable cells to remain in the cell compartment. Total cell density reached approximately 7xl07 cells/ml within 4 weeks of inoculation and remained at that level thereafter. The viability of cells in the cell compartment decreased steadily and then remained steady at approximately 20%. The high cell concentrations in the small volume cell compartment of Integra CELLine allowed us to achieve high concentrations of mAb, routinely as high as 0.6 mg/ml as measured by analytical protein-A HPLC. Single one-liter flasks produced as much as 110 mg of mAb in a 3-month period. Higher antibody concentrations are probably possible with further optimization of the frequency of antibody harvests and nutrient medium exchange, and reactor cell population. Gas-permeable bags (TC Technologies, Minneapolis MM, USA) are simply that; polymer bags into which cells and medium are placed and incubated in a 5% CO2 atmosphere at 37 °C. Initially the viability of cells is high, but diminishes with time; the production of mAb is usually inversely correlated with cell viability. We found that hybridoma cells cultured in 2-liter gas-permeable bags produced the most antibody per unit of medium consumed, in the least amount of time. In a representative bag culture, total cell concentration increased to approximately 6.4x106 cells/ml within 2 weeks of inoculation. Viable cell concentration increased within the first week of the culture and diminished slowly to approximately 5.8xl05 cells/ml. The viability of cells in the bag peaked at 1 week and decreased steadily thereafter. The concentration of antibodies in gas-permeable bags increased most rapidly during the second week of the culture after inoculation, reaching and maintaining a maximum concentration of 0.15 mg/ml after 15 days. A typical single 2-liter gas permeable bag produced approximately 240 mg of mAb from our test cell line in a 1-month period. 2.3. Results and Summary Remarks Antibodies were harvested from media drawn from each of these three types of reactors using single-step preparative chromatography [11-13]. Purified mAbs produced all three types of reactors were found to be active against the target antigen (in this case, cells of Francisella tularensis) (data not shown). Naturally, the viability of cells and antibody yield vary considerably among cell lines. The performance of each method was evaluated by considering productivity, cost, ease of handling and risk of contamination (Tables 16 and 17). Based on this work, we have chosen gas-permeable bags as our preferred cell culture method for routine production of mAbs on both research (< 1 gram) and production (> 1 gram) scales. The frequent need to open the flask bioreactors, remove spent culture medium and feed with fresh medium created opportunities for contamination. Unlike the flask bioreactors, gas-permeable bags require essentially a single access (depositing the initial load of cells and medium). Subsequent samples to monitor viability and mAb production are taken with a syringe through a septum, greatly reducing the opportunity for contamination.

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Table 16. Operational characteristics of three in vitro hybridoma culture methods

Variable

Integra CELLine

Hollow Fiber Bioreactor

Gas-Permeable Bag

Cell Density

High

High

Low

Principle of Operation

Perfusion

Perfusion

Stationary

Capital Equipment

No

Yes

No

CO2 Incubator Required?

Yes

No

Yes

Contamination Risk

Significant

Significant

Low

Table 17. Comparison of the three different cell culture methods examined in this study Gas Permeable Bag (2-Liter)

Variable

Integra CELLine: CL1000

Hollow Fiber Bioreactor: CP1500

Productivity (mg MAb/unit/mo)

40

130

240

Antibody concentration (mg/ml)

0.6

0.5

0.12

Media harvested (1/g MAb)

1.7

2.0

8.3

Total media consumed (1/g MAb)

50

150

8.3

Cost for culturing (materials only)

Moderate

High

Low

Recovery of purification process

85%

92%

78%

Purity (by gel filtration chromatography)

98.4%

99.4%

97.1%

Activity (by ELISA)

Active

Active

Active

3. Other Biotechnology Products 3.1. Organophosphate-Cleaving Enzymes Neurotoxic chemical warfare (CW) agents in the hands of extremist terrorist organizations and rogue nations in recent years have significantly increased the prospect of intentional exposure


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of civilian populations and military personnel. Currently available decontaminants are corrosive to equipment, and pose environmental concerns. The use of catalytic enzymes in place of highly reactive decontaminants offers obvious advantages, i.e. they are non-toxic, noncorrosive, and environmentally benign. Two CW-degrading enzymes, OPH (organophosphorus hydrolase) from Pseudomonas diminuta and Flavobacterium sp. for highly toxic V-type [14-16] and OPAA (organophosphorus acid anhydrolase) from Alteromonas JD6.5 for G-agents such as Soman and Sarin [17,18] have been cloned and expressed in Escherichia coli strains. In addition, a modified OPAA, Q-OPAA designed for a long-term protection of exposed skin to chemical Gagents has been expressed in E. coli cells. In single-batch incubation of cells in a 1-L shaker flask, typical OPH and Q-OPAA yields ranged 10-20 and 30-40 mg/L, respectively. We have also developed larger-scale fermentations to explore the mass production of these enzymes. We are currently focused on scaling up the production of these enzymes in bacterial fermentations. Several 20 liter-scale fermentation runs in Luria broth were conducted to optimize the production of OPH, prior to attempting a 1000-liter fermentation. Culture growth characteristics (growth rates, pH, dissolved oxygen consumption profile, and final cell density) among the four runs were reproducible; however, OPH activity varied considerably. Only the 20-L fermentation run #4 that had been added with 1 mM CoC12 showed a drastic increase in OPH enzyme activity (from 238 to 2012 U/ml in crude cell lysate). In the 1000 liter run, adding 1 mM CoCl2 prior to cell harvest resulted in obtaining 20.7 mg OPH per liter. 3.2. Production of Biologicals Under current Good Manufacturing Practices (cGMP) Recently, a 1400 square feet clean room facility has been designed and currently under construction at our facility. The clean room facility provides a controlled single-pass HEPA (High Efficiency Particulate Air) filtered suites to meet environmental air quality according to the current Good Manufacturing Practices (cGMP) set forth by the U. S. Food and Drug Administration (21 CFR). Our manufacturing facility has been segregated into flexible GMP suites for microbial fermentation, mammalian cell culture, recovery, and downstream purification. The design of GMP suites also allows two simultaneous production campaigns: cell culture and fermentation. Separated from the production space will be a quality control suite and associated support facilities for archival storage of strains and cell lines, and controlled storage for the holding and release of raw materials during the quality control (QC) process. The QC suite is planned to include two areas: a QC analytical laboratory for routine analysis of raw materials, samples from utilities, and finished products, and a QC microbiology laboratory for biological testing (to ensure sterility and absence of contaminants, endotoxins, etc.). Once commissioned and fully validated according to the cGMP requirements for product safety and quality, we will be able to support a broad spectrum of research and product testing (for example, phase I and II clinical trials) and customers (federal and state research agencies, small- to medium biotechnology firms).

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4. Novel Manufacturing Methods 4.1. The Glycosylation Problem As mentioned above, several groups have described bacteriophage-based systems for the selection and cloning of specific antibody gene sequences. We described previously the advantages of recombinant antibodies [3]. They are genetically completely defined, in that their sequences are fully known; malleable, in that their sequences can be manipulated to alter their properties; and producible in a variety of organisms into which the antibody genes can be inserted. These systems include bacteria (e.g., E. coli). However, a drawback to the production of antibodies in bacteria is their lack of ability to properly modify the expressed antibodies with sugar molecules, a process called glycosylation. Glycosylation of antibodies is desirable when the antibodies are to be used as human therapeutics, as these sugar moieties reduce the antigenicity of the antibodies, and therefore reduce the body's tendency to recognize them as foreign. Glycosylation also affects how the immune system interacts with recombinant antibodies to facilitate binding to an antigen. To obtain glycosylation of recombinant proteins, worker can express them in eukaryotic cells, such as yeast or cultured mammalian cells, or in whole transgenic higher organisms including mammals and plants. The time required to develop expression systems in transgenic animals or plants can be costly, making these approaches economically feasible only for very large-scale expression. The use of mammals in the production of antibodies and other proteins is becoming increasingly controversial for ethical reasons [19].

4.2. Insect Cells and Larvae as Bioreactors for Recombinant Protein Products An attractive alternative to transgenic animal or plant expression for antibodies or indeed, any other proteins, is to express the desired genes in transformed insect larvae. Insects are sufficiently molecularly sophisticated to glycosylate antibodies [20, 21]. Certain species, such as Trichoplusia ni, are also inexpensive to raise and can be grown in very large numbers in automated facilities. Most importantly, insect viruses (most harmless to humans) can be used to infect insects via ingestion and can be used as delivery vehicles for the genes of interest [22]. Transfected cells of infected larvae can produce the desired protein product in large quantities; however, gene expression in transformed insects is not foolproof, and not every larva expresses the desired gene at the same level. To reduce the amount of "background" insect protein in a preparation relative to the recombinant product, it is desirable to identify larvae that are the highest producers, and to extract the recombinant product from those larvae preferentially. To this end, we established a protein expression system for use in larvae that would allow a manufacturer to readily identify larvae producing large amounts of a recombinant protein. The method involves the fusion of the gene encoding the desired product to a reporter gene encoding a colored protein, in this case DsRed, an auto-fluorescing protein derived from Discosoma coral.

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4.3. Cloning Strategy and Results The DsRed gene was cloned into the vector in a manner permitting the fusion of the DsRed gene to any gene cloned between it and the promoter region. Accumulation of the product would therefore be tied directly to the accumulation of DsRed, which can be monitored by illuminating the larvae with light in the visible portion of the EM spectrum (excitation 545nm, emission = 620nm). In this way larvae producing a desired recombinant protein can be easily identified and selectively harvested. Such larvae could be selected either by hand, or by an automated larva "picker" guided by machine vision. A similar technology was described by Cha et al. [23] in which green fluorescent protein (GFP) was used as the reporter. We next demonstrated the expression of a useful protein using the DsRed fusion vector. This required the engineering of a set of antibody genes and their cloning into the DsRed fusion vector so that one of the antibody's light chain gene and the DsRed reporter protein would be expressed as a fusion protein. This fusion allowed the determination of expression levels of the antibody in cells or larvae before beginning the process of antibody extraction and purification. The fusion protein was engineered in a way that allows the selective removal of the DsRed fluorescent marker after purification. To accomplish these goals, an existing gene set encoding the heavy and light chains of a recombinant anti-botulinum toxin Fab was modified for expression in insect cells and cloned into both the original vector (without DsRed) and the DsRed-modified vector. The vector chosen for the initial proof-of-principle experiments was the insect baculovirus expression vector pAcAB3, and our modified version of pAcAB3 containing the DsRed gene. Preliminary experiments examining the expression of DsRed protein alone gave striking results: larvae expressing quantities of DsRed so great that the larvae turn bright pink (data not shown). DsRed expression was also noted to be strong in cultured Sf9 cells (an insect cell line; N. van Beek, unpublished results). When the antibody-encoding genes were cloned into the DsRed fusion vector and introduced into larvae, some larvae were estimated to produce as much as 1-3 mg antibody per larva (data not shown). Samples of the antibody are currently being purified and will be tested with control lots of the Botfab to compare the affinities and specific activities. The results obtained here are preliminary to the incorporation of the antibody geneDsRed construct into baculoviruses and the introduction of the baculoviruses into T. ni larvae. This work will be a proof of concept experiment to demonstrate that the insect gene expression system. A complete demonstration of the system will include the expression of a desired recombinant protein in larvae fused to DsRed, the identification of high producers visually using DsRed as the marker, the purification of the recombinant protein, and determination that the recombinant protein has the desired activity. Features designed to test all these aspects of the system have been engineered into the vectors. 4.4. Initial Studies in Machine Vision A long-term goal of this work is the rapid identification of larvae that are producing large quantities of recombinant product relative to the overall transfected population. The dynamics of protein production and its balance with the endogeneous protease activities create a very narrow window of opportunity to achieve high protein yield. This window can be defined by frequent sampling of larval tissues; however this could kill the larvae. Ultimately, we intend to accomplish this goal using robotics guided by machine vision that is capable of identifying

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high-producing larvae using a fluorometer to detect the reporter protein-product fusion. The resulting concentrations were visualized and measured non-invasively through its green fluorescence under UV excitation. Thus, physical sampling of the product can be avoided, and the decrease of the protein concentration eliminated. Additional advantage of the protein fusion is the substantial increase in production of proteolytically sensitive proteins. In preliminary studies, a portable fluorometer was constructed (ref SPIE) and used to detect DsRed protein in expressed in larvae for various durations after infection. Larvae were grown, harvested, and frozen prior to observation. DsRed was expressed to different extent in the tissues. As the infection begins in the gut, usually the belly is the brightest. The sides of the larvae are of similar brightness, and the back is the dimmest. In the sensor designed for this study, larvae were positioned beneath the device in a laying position, simulating real conditions. A frozen larva usually has an S-shape, thus the measurements were taken from the sides of the larvae. Despite the fact that only two side measurements were taken, the data for each larvae batch showed good correlation with the amount of DsRed, determined from the fluorescence intensity of the total soluble protein (TSP) (data not shown). In most of the cases the correlation between the amount of DsRed and the device readings was lower than the correlation between DsRed and the average of the fourside fluorescence. This suggests that a better estimate of the DsRed amount could be attained if more than one sensor is used, i.e. positioned at different angles above the larvae so different portions of its circumference are observed. It is interesting to note that while measured fluorescence was a strong predictor of total DsRed in larvae, it did not correlate with the percentage of the DsRed in TSP at 92 and 120 hours after infection. This is likely a result of bioproduction dynamics. From average of 12 mg per larvae at 67 hours post infection (SD=6.7 mg), TSP dropped to 4.2 mg at 92 hours (SD=2.6 mg) and later recovered to 11.1 mg at 120 hours (SD=5.9). However, the percentage of DsRed remained essentially unchanged. This might suggest that there is some upper level of the DsRed concentration in the cell and the further increase of the DsRed amount is achieved only in parallel with the production of other proteins. The poor correlation between total larval fluorescence and the percent DsRed in TSP could also be the result of the defrosting of frozen larvae, during which some melanization was observed. The fluorescence from strongly melanized larvae was anomalously high, possibly because of the high light scattering. However, protein gel analysis suggested that the DsRed quantity is similar to the amount in the non-melanized larvae. This suggests that after freezing, the larvae should be measured before thawing, or the melanin formation could introduce a significant error in the production estimate. Our work to date indicates that the concentration of the fluorescent recombinant protein can be successfully measured in frozen larvae with a high degree of correlation with the protein concentration determined by traditional methods. The method is applicable both for tracking the in-vivo larvae production of protein (by freezing samples for later measurements) and for sorting. The results suggest that a single reading from the side of the larvae may not be enough to perform reliable measurements. It is preferable to have multisided measurements from at least three sides. Given the low cost of the fluorometer sensors, the method is readily applicable for industrial purposes.

5. Secure Repositories Edgewood Chemical Biological Center (ECBC) operates the Critical Reagent Repository Program (CRP). The Critical Reagent Repository program works closely with laboratories


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across DoD, industry, academia, other government agencies, and the defense research establishments in allied countries to ensure that the best biodefense reagents, methods, and information are available to our customers. To support the antibody development program, a number of different antibodies are produced from hybridomas sent here by program participants. Antibodies are produced and distributed to DoD and allied laboratories for evaluation and study. This task often requires that several cell lines be grown simultaneously for production of antibodies on a research scale (< 1 gram) in the CRP production laboratories at ECBC. 6. Concluding Remarks This work is part of an overall strategy to develop an improved suite of biodetection reagents by establishing the methods for cloning, isolation and large-scale production of recombinant polypeptides. The antibodies being developed in this program complement another ongoing effort in our group to isolate and characterize small peptide aptamers that bind and detect BW agents. Our work in reagent development is, in turn, a subset of our larger effort to provide flexible and scalable biotechnology solutions to customers in government, academia, and the biotechnology industry. Recombinant protein production in bacteria, cell culture, or lower eukaryotes is in step with a growing consensus that current methods of producing recombinant products in mammals (especially the use of ascites culture for monoclonal antibodies) in quantity are inhumane. Traditionally, monoclonal antibodies are produced by injecting hybridoma cells into animal hosts, and subsequently collecting abdominal fluids (ascites) that contain the essentially pure antibody. This method produces great discomfort in animals and is strongly discouraged for large-scale antibody production (National Research Council, 1999). Phage display library construction and subsequent cloning of antibody genes, minimizes animal use by using only those animals initially immunized in the process. Gene expression in insect cells should provide a useful means for the production of materials for human therapeutics. The manufacture of products for use in humans requires a strict adherence to cGMP (current Good Manufacturing Practices) to ensure the safety, quality and purity of product. An economic analysis will need to be performed once data on product yield is obtained. Preliminary data indicate that, in the case of Botfab, approximately 300-1000 larvae may be required to produce one gram of antibody. Other proteins will need to be expressed, and the cost of raising, infecting, growing/harvesting larvae, and subsequent purification of product will have to be determined. Since several technological obstacles have already been overcome for the automation of a larval production line, it seems likely that only minor molecular modifications will be required to obtain efficient recombinant protein manufacturing. Market analysis, including the going rates for recombinant proteins made by other methods, and the overall demand for recombinant proteins, will ultimately determine the economic viability of this and other protein production systems.

References [1]

Parmley, SF, Smith GP. 1988. Antibody-selectable filamentous fd phage vectors: affinity purification of target genes. Gene 73:305-318.

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[2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [ 14] [15] [16] [17] [18] [19] [20]

[21] [22] [23] [24]


Hogrefe H, Shopes R. 1994. Construction of Phagemid Display Libraries with PCR-amplified Lmmunoglobulin Sequences. PCR Methods Appl. S109-S122. O'Connell KP, Anderson PE, Khan AS, Valdes JJ, Stinchcombe TJ, Shopes R, Khalil M, Eldefrawi ME. Recombinant antibodies for the detection of bacteriophage MS2 and ovalbumin. Proceedings of the 21st Army Science Conference, Baltimore MD, December 2000. Winter G,MiIsteinC. 1991. Man-made antibodies. Nature 349:293-99. Devlin JJ, Panganiban LC, Devlin PE. 1990. Peptide libraries: a source of specific protein binding molecules. Science. 249:404-6. Davies J, Riechmann L. 1995. Antibody VH domains as small recognition units. Biotechnology 13:4759. Horwell DC. 1995. The'peptoid' approach to the design of non-peptide, small molecule agonists and antagonists of neuropeptides. Trends Biotechnol. 13:132-4. Jackson LR, Trudel LJ, Lipman NS. 1999. Small-scale monoclonal antibody production in vitro: methods and resources. Lab Animal (autumn issue), pp 20-30. Trebak M, Chong JM, Herlyn D, Speicher DW. 1999. Efficient laboratory-scale production of monoclonal antibodies using membrane based high-density cell culture technology. J. Immunol. Methods 230:59-70. Wolf ML, DeSutter T. 1999. High-density cell cultures in a new passive membrane-based bioreactor. Pages 293-301 in Biotechnology International II (Connor and Fox, eds.) San Francisco: Universal Medical Press, Inc. Gagnon, P. 1996. Protein A affinity chromatography. Chapter 9 in: Purification Tools for Monoclonal Antibodies Tucson, AZ: Validated Biosystems, Inc. Fulton SP, Meys M, Varady L, Jansen R, Afeyan NB. 1991. Antibody quantification in seconds using affinity perfusion chromatography. BioChromatography 11:226-231. Blank GS, Vetterlein, D. 1990. Quantification of monoclonal antibodies in complex mixtures by protein G high-performance liquid affinity chromatography. Anal. Biochem. 190: 317-320. Lai K, Stolowich NJ, Wild JR. 1995. Characterization of P-S bond hydrolysis in organophosphorothioate pesticides by organophosphorus hydrolase. Arch. Biochem. Biophys. 318: 5964. Kolakowski JE, DeFrank JJ, Harvey SP, Szafraniec LL, Beaudry WT, Lai K, Wild JR. 1997. Enzymatic hydrolysis of chemical warfare agent, VX and its neurotoxic analogues by organophosphorus hydrolase. Biocatalysis and Biotransformation 15: 297-312. Rastogi VK, DeFrank J J, Cheng TC, Wild JR. 1997. Enzymatic hydrolysis of Russian-VX by organophosphorus hydrolase. Biochem. Biophys. Res. Commun. 241: 294-296. Cheng TC, Harvey SP, Chen GL. 1996. Cloning and expression of a gene encoding a bacterial enzyme for decontamination of organophosphorus nerve agents and nucleotide sequence of the enzyme. Appl. Environ. Microbiol. 62: 1636-1641. Cheng TC, Liu L, Wang B, Wu J, DeFrank, JJ, Anderson DM, Rastogi VK, Hamilton AB. 1997. Nucleotide sequence of a gene encoding an organophosphorus nerve agent-degrading enzyme from Alteromonas haloplanktis. J. Ind. Microbiol. & Biotech. 18: 49-55. Committee on Methods of Producing Monoclonal Antibodies, Institute for laboratory Animal Research, National Research Council. 1999. Pages 45-47 in Monoclonal Antibody Production. Washington, D.C.: National Academy Press. Hsu TA, Takahashi N, Tsukamoto Y, Kato K, Shimada I, Masuda K, Whiteley EM, Fan JQ, Lee YC, Betenbaugh MJ. 1997. Differential N-glycan patterns of secreted and intracellular IgG produced in Trichoplusia ni cells. J Biol Chem. 272:9062-70. Davis TR, Wood HA. 1995. Intrinsic glycosylation potentials of insect cell cultures and insect larvae. In Vitro Cell Dev Biol Anim. 31:659-63. Pham MQ, Naggie S, Wier M, Cha HJ, Bentley WE. 1999. Human interleukin-2 production in insect (Trichoplusia ni) larvae: effects and partial control of proteolysis. Biotechnol Bioeng. 62:175-82. Cha HJ, Dalai NG, Pham MQ, Bentley WE. 1999. Purification of human interleukin-2 fusion protein produced in insect larvae is facilitated by fusion with green fluorescent protein and metal affinity ligand. Biotechnol Prog. 15:283-6. Kostov, Y., Tolosa, L., O'Connell, K., Anderson, P., Liu, Y., van Beek, N., Rao, G. 2003. Monitoring of DsRed protein concentration in frozen insect larvae. SPIE Proceedings, Vol. #4967, Genetically engineered and optical probes for biomedical applications.


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Development of Rapid Fingerprinting and Detection Assays for Biological Agents of Mass Destruction Vito G. DelVecchio Director of Research, Institute of Molecular Biology and Medicine, University of Scranton, Scranton, PA, USA Abstract. Recent advances in genomics and proteomics have generated new strategies for the development of nucleic acid- and immunochemical-based detection assays as well as rapid fingerprinting systems for biological warfare agents (BWA). The increasing list of sequenced and annotated microorganisms has enhanced the data obtained from comparative genomics and proteomics. In silico studies have facilitated the identification of putative signature sequences for various BWA and provided repetitive sequences that may be exploited in future rapid fingerprinting methods. Suppressive subtractive hybridization investigations of closely related strains and species of BWA have also pinpointed signature sequences for use in probe assays. Proteomics studies using 2 D-gels and MALDI-TOF of spores, secretomes under simulated host cell conditions, as well as vegetative cells have been used in determining differences that can be applied to probe assays. Protein chip SELDI-TOF can be used to observe differences of BWA outer membrane and exosporium proteins. All of these strategies are now being applied to technical platforms that will allow rapid detection and fingerprinting.

Recent world events have heightened the need for rapid, user-friendly, and field-worthy assays for biological agents of mass destruction (BWMD). The genomic era has opened the way for new strategies for detection and diagnosis of BWMD. The increasing list of sequenced and annotated microorganisms will certainly impact on the specificity of such assays and help devise new fingerprinting systems. Post or functional genomic will also generate new methodologies that will not only be applied to BWMD but to the identification of genes found in a diverse pathogens and a vast range of disease conditions. Our laboratory has employed several genomic strategies to identify biomarkers or target sequences that can be used in probing assays for BWMD. These include the use of bioinfomatics, rep-PCR polymorphisms, and suppressive subtractive hybridization. Proteomic techniques such as comparisons of secreted proteins, exosporia, and global analysis of closely related species have been used in biomarker discovery. Certain criteria must be adhered to in the development of molecular probing assays. A specific nucleic acid or antigen target should be unique for the BWA in question, the target must be conserved or found in all isolates of the BWA, and the target should have no polymorphisms or areas of plasticity in the nucleotide composition. Molecular fingerprinting is used to discriminate between closely related strains or isolates of a microorganism. Fingerprinting can determine if an infectious outbreak is the result of

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several or one strain of a pathogen or are the different isolate epidemiologically related. It can also determine the source of a BWMD by comparison of the fingerprint of the strain in question with a database of fingerprints of strains from many geographic locations. Thus it may be able to determine if a certain BWMD originated from a particular terrorist group or hostile state. Traditionally scientists have utilized both molecular and non-molecular methods to type microorganisms. These have included RFLP, ribotyping, PFGE, phage typing, and plasmid profiling. These methods have advantages and disadvantages such as being labor-intensive, taking long periods of time to accomplish, requiring Southern blotting, or needing to liberate DNA from cell embedded in agarose blocks, or can take days or even weeks to complete. The ideal system should be easy to perform, rapid, cost effective, discriminatory and reproducible, serve as a bar code or signature for strain identification, and enable determination of the source of an outbreak. One method that has involved a minimum of time and work expenditure has been repetitive sequenced-based PCR (Rep-PCR) [3]. Takes advantage of the fact that both prokaryote an eukaryote genomes contain dispersed repetitive sequences separating longer single-copy DNA sequences. The repetitive elements vary in position and number on the chromosome of different strains of a pathogen. PCR primers, directed outward from the rep elements, amplify the regions of long single copy DNA sequences situated between the rep elements. The areas between the repetitive sequences are the only part of the genome that is amplified. Since these vary in size, they generate PCR products of different sizes. Electrophoresis of these fragments yields a fingerprints that act as barcodes. Advantages of this method are: DNA is easily and quickly liberated from cells, PCR is so sensitive that only small amounts of template DNA are needed, does not require expensive equipment, and these procedures are relatively rapid—being completed in less than 24 hours.

Figure 36. Rep-PCR fingerprint of different Bacilli. The bands enclosed in green were digitally enhanced to differentiate minor differences. Bands unique to one species can be excised from the gel, cloned, sequenced, and used as a putative candidate for probe development.

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The proteome is defined as the sum total of all proteins produced by an organism under a defined set of conditions or at a particular time in the life of a cell. Proteomics deals with the study of multiprotein systems in which the focus is on the interplay of multiple distinct proteins and their roles as part of a larger network [6]. Protein identification using proteomic technology is achieved by partial sequence analysis with the aid of database matching tools. Traditional proteomics investigations use two-dimensional SDS-PAGE, in which the proteins are first separated by isoelectric focusing (IEF) in the first dimension and by SDS-PAGE in the second dimension. This is a powerful technique for resolving complex mixtures of proteins and permitting the simultaneous analysis of hundreds or thousands of proteins. IEF separates proteins on the basis of their isoelectric point (pI). The pI of a protein is the pH at which the protein has a zero net charge. When a protein mixture is applied to an IEF gel, each protein migrates until it reaches the pH that matches its pI. At this point the protein cannot migrate. Proteins with different pIs are therefore align at different points throughout the gel. IEF offers the greatest resolving power in separating proteins according to net charge. Proteins on IEF strips are then separated in the second dimension by polyacrlyamide gel electrophoresis in the presence of the detergent SDS. SDS binds to most proteins by hydrophobic interactions in amounts proportional to the molecular mass of the protein, about one molecule of SDS for every two amino acid residues. The bound SDS contributes a large negative charge, rendering the intrinsic charge of the protein insignificant. Furthermore, when SDS is bound, most proteins assume an almond shape, resulting in a similar ratio of charge to mass. Protein separation therefore is based almost exclusively on the basis of mass, with smaller polypeptides migrating most rapidly. After electrophoresis, the proteins are visualized by staining with SYPRO® Ruby, a ruthenium-based fluorescent stain shown to have several advantages over other commonly used protein stains with respect to sensitivity and linear dynamic range [7]. The stained gels are imaged and the spots are picked for protein identification. The protein spots are subjected to protease (e.g. trypsin) digestion to yield smaller peptide fragments, the number and size of which are characteristic for an individual protein. The digested protein is then spotted on a plate and identified by mass spectrometry (MS). In general, there are 3 essential components in a mass spectrometer, i.e., an ion source, a mass analyzer, and a detector [1]. One commonly used instrument for most MS work is Matrix-Assisted Laser Desorption lonization Time of Flight (MALDI-TOF) Mass Spectrometer. In this instrument, the ion source, equipped with a laser produces ions from the sample. The sample to be analyzed is mixed with an aromatic matrix that absorbs light at a specific wavelength. Typical matrix compounds include 2,5 dihydroxybenzoic acid, 3,5dimethoxy-4-hydroxycinnamic acid and a-cyano-4-hydroxycinnamic acid. The mixture of sample and matrix is spotted onto a metal plate and allowed to evaporate in air. After drying, the target plate is inserted into the vacuum chamber of the MS. A laser directed at the sample is fired, resulting in the ionization and desorption events. A high voltage applied to the target plate provides potential to push the ions into the flight tube. The ions formed in the MALDI source are then extracted and directed into the TOF mass analyzer. The mass analyzer resolves ions based on their mass/charge (m/z) ratio that is proportional to their velocity. This is dependent upon the time required for the ions to travel the length of the flight tube, i.e., the time between application of voltage to the plate and the registration of signal by the detector. The smaller the m/z value, the shorter is the flight time

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and the faster the ions reach the detector. The detector then converts the kinetic energy of the arriving particles into electrical signals. The mass spectrum generated from MALDI-TOF-MS is called a mass fingerprint that is characteristic of a particular protein. The identity of an unknown protein is determined by comparing its peptide mass fingerprint with the theoretical spectrum generated by digestion of each of the proteins in a database by using a search engine like Mascot from Matrix Science Ltd. (http://www.matrixscience.com). Protein identification from enzymatically-derived peptides depends on the frequency of specific cleavage sites within a protein. The cleavage sites yield a set of potential peptide masses that are unique to that sequence entry when compared to all other entries in the database. If a significant number of the experimentally determined peptide molecular weights match the m/z values in the theoretical mass spectrum, a match is obtained and the protein is identified. Some of the databases commonly used for protein identification include NCBInr, SWISS-PROT, TrEMBL and OWL. Thus, a rapid and automated protein characterization is achieved. Alternatively and more advantageous in terms of speed and accuracy of identification, is matching of MS spectra with translations of the nucleotide sequence from an annotated genome of the same organism, allowing the investigator to match expressed proteins to their corresponding ORFs. For instance, the availability of a completely sequenced and annotated B. melitensis genome has paved the way for a highly comprehensive and rapid analysis of its proteome.

Figure 37. Method of protein identification using the Mascot search engine

The results of proteome analysis indicate which genes are expressed under a given set of conditions, how protein products are modified and how they might interact. Unlike the genome, the proteome is not static. It changes with the state of development, under conditions of environmental stress and during disease states of a tissue. There are many more proteins in a

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proteome (mainly due to posttranslational modifications) than genes in a genome. Fig.37 summarizes the steps that we used in the global analysis of B. melitensis proteome. The utilization of fully automated and robotized instruments from Genomic Solutions, Inc. (Ann Arbor, MI, USA) for staining and imaging of gels, protein spot picking, protein digestion, and spotting onto MALDI target plates. For details regarding the capabilities of these instruments and the ongoing collection of proteomic data please see the IMBM Proteomics website at http://www.proteome.scranton.edu. SYPRO® Ruby protein gel stain was used for a more sensitive fluorescence detection of proteins since it has a linear dynamic range that exceeds that of silver by a factor of 5 to 10. Further, SYPRO® Ruby staining is fully compatible with Edman sequencing and mass spectrometry. To date, a total of 937 B. melitensis 16M protein spots have been identified using twodimensional gel electrophoresis and peptide mass fingerprinting. These proteins corresponded to 269 discrete open reading frames in the B. melitensis genome and were classified under appropriate metabolic categories [2]. A majority of these proteins were hydrophilic and only a few were hydrophobic, with transmembrane domain-containing proteins being underrepresented. Proteome analysis has shown that the two circular chromosomes of B. melitensis are functionally active [12]. The expressed ORFs identified to date by proteomics analysis revealed that they are evenly distributed over the physical map of both chromosomes. These data suggest that the two chromosomes are indispensable for the survival of this organism. Proteins involved in membrane transport as well as in carbohydrate and protein metabolism composed the majority of proteins identified in B. melitensis. A significant number of proteins with unknown functions were included under the "hypothetical proteins" category. This category was assigned by Integrated Genomics based on putative genes predicted from the annotated sequence of the B. melitensis genome (10). Comparative proteomics is a powerful tool in identifying similarities and differences in the biochemical pathways that ultimately determine the metabolic state of an organism. By looking at the expression levels of hundreds or even thousands of proteins simultaneously, one gets a global picture of what pathways are blocked, upregulated, downregulated or unaffected. Ultimately, perturbations of affected pathways have predictive values in determining the final phenotype of an organism. Such information is extremely important in probe development. Our laboratory has also conducted a global comparative analysis of Brucella abortus and B. melitensis proteomes. Initial results have indicated that their proteomes have significant differences in both the number of protein spots and their qualitative and quantitative expression pattterns on 2-D gels. Focusing on these proteomic differences may contribute to our understanding of the metabolic differences that impart host preference and virulence in the different species of Brucella. Bacillus anthracis, the causative agent of the often-fatal disease anthrax, has attracted worldwide attention due to its recent use as an agent of bioterrorism. Fully virulent strains of B. anthracis carry two large plasmids, pXOl (181.7 kb) and pXO2 (96.2 kb) that encode for toxin production and capsule formation, respectively. The genes pagA, cya and lefon pXO 1 code for the synthesis of protective antigen (PA), lethal factor (LF), and edema factor (EF), respectively [4; 8]. Systemic infection following the inhalation of B. anthracis spores usually results in shock and death within a few days following the onset of symptoms. Treatment with antimicrobials is effective only if initiated in the early stages of infection during the incubation period, which indicates the importance of rapid, sensitive and accurate diagnosis of anthrax infection [4].

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Knowledge of proteins that are induced in the host during infection (i.e., stress factors) and contribute to pathogenicity would aide in the design of safe, efficient vaccines against B. anthracis, lead to the discovery of a new generation of effective anti-anthrax drugs [8] and will greatly impact on probe development. B. anthracis is encapsulated in vivo, but produces a capsule in vitro only in the presence of bicarbonate and CO2 by growing B. anthracis on CAP plates in an atmosphere of 5% CO2 95% air[10]. It has been described that bicarbonate influences the transcription of the toxin genes, in addition to capB. Since bicarbonate is present in all body fluids in vivo (CO2 + H+ + OH" H+ + HCO3 at physiological pH), it may act as a signal for the expression of all B. anthracis virulence factors. In this scenario, bicarbonate would signal the vegetative cells that they are no longer in a soil environment but in a warm-blooded organism [10; 5; 11]. Bacteria, particularly those of the Bacillus genus, are known to secrete proteins into their growth medium in large quantity. The secretome of a microorganism is the sum total of proteins secreted by the organism under a defined set of conditions. Thus it feasible to identify proteins secreted by B. anthracis, which elucidate virulence and stress factors that have not yet been characterized. A synthetic, minimal medium is available that induces B. anthracis to produce a toxins for use in anthrax vaccine development [9]. By growing B. anthracis in a protein-free medium the yield of toxin production increased two to five-fold above previously reported values. It has been shown that the transcription of the toxin genes, pagA, lefand cya are "coordinately regulated," (i.e. "induced" by bicarbonate and enhanced at 37°C), [8]. The effect of temperature on toxin induction supports the hypothesis that these conditions correspond to an in vivo environment surrogate [10]. Protein identification is possible due to advances in two-dimensional gel electrophoresis (2D-E), improved mass spectrometry (MS) techniques and the availability of complete genomic sequences of various bacilli (http://www.tigr.org/tdb/mdb/mdbcomplete.html: http://www.ncbi.nlm.nih.gov/) The identification and characterization of proteins expressed in an environment that simulates the in vivo condition for B. anthracis yielded valuable information associated with the virulence and pathogenicity of this organism, especially concerning the role of the chromosome. The proteins that were identified under each set of growth conditions were compared. Forty-one spots (open circle) were matched between the average gels of the two conditions tested (Fig. 38). Thirty-five spots were induction-independent (i.e. expressed at the same level under both sets of conditions), (Fig. 38, category A). The proteins that were CO2-HCO3inducible (Fig. 38, category B) or CO2-HCO3 -repressible (Fig.38, category C) were also examined. There were at least five proteins that appeared to be CO2-HCO3~-inducible. The category D represents spots which need further resolution (i.e. pI vs MW).

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Figure 38. Super-imposed average gels non-induced (red) versus induced (green). Category A (CO2-indifferent), Category B (CO2-inducible), Category C (CO2repressible), Category D (spot zooming needed).

In this study 27 proteins isolated under induction were assigned probable identifications; eighteen without-induction proteins were identified. One protein was correlated with an immunoreactive protein in a different organism; this could lead to a development of new methods of nucleic-acid based and immunochemical biomarkers for B. anthracis. This investigation could also lead to the identification of virulence factors not yet elucidated. A comparative proteomic study of the exosporium of B. anthracis, B. cereus, and B. thuringeinsis is presently in progress. This could also generate future probes.

References [1]

Corthals, G.L., S.P. Gygi, R. Aebersold and S.D. Patterson. 1999. Identification of proteins by mass spectrometry. p. 197-231. In Rabilloud, T. (ed), Proteome research: 2-D gel electrophoresis and detection methods, Springer-Verlag, NY.

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DelVecchio, V.G., V. Kapatral, R. J. Redkar, G. Patra, C. Mujer, T. Los, N. Ivanova, I. Anderson, A. Bhattacharyya, A. Lykidis, G. Reznik, L. Jablonski, N. Larsen, M. D'Souza, A. Bernal, M. Mazur, E. Goltsman, E. Selkov, P. H. Elzer, S. Hagius, D. O'Callaghan, J. J. Letesson, R. Haselkorn, N. Kyrpides, and R. Overbeek. 2002. The genome sequence of the facultative intracellular pathogen Brucella melitensis. Proc. Natl. Acad. Sci. USA. 99: 443-448. DelVecchio, V.G., J.M. Petroziello, M.J. Gress, F.K. McCleskey, G.P. Melcher, H.K. Crouch, and J.R. Lupski. 1995. Molecular Genotyping of methicillin-resistant Staphylococcus aureus via fluorophore enhanced repetitive-sequence PCR. J. Clin Microbiol. 33:2141. Dixon, T.C., M. Meselson, J.Guillemin, and P.C. Hanna. 1999. Anthrax N.Engl.J.Med. 341:815-826. Fouet, A., M. Mock. 1996. Differential influence of the two Bacillus anthracis plasmids on regulation of virulence gene expression Infect.Immun. 64: 4928-4932. Liebler, D.C. 2002. Introduction to Proteomics: Tools for the New Biology. Humana Press Inc., Totowa, NJ, 198 pages. Lopez, M.F., K. Berggren, E. Chernokalskaya, A. Lazarev, M. Robinson and W.F. Patton. 2000. A comparison of silver stain and SYPRO Ruby Protein Gel Stain with respect to protein detection in twodimensional gels and identification by peptide mass profiling. Electrophoresis. 21: 3673-3683. Mock, M., A. Fouet. 2001. Anthrax Annu.Rev.Microbiol. 55: 647-671. Ristroph, J.D., B.E. Ivins. 1983. Elaboration of Bacillus anthracis antigens in a new, defined culture medium Infect.Immun. 39: 483-486. Sirard, J.C., M. Mock, and A. Fouet. 1994. The three Bacillus anthracis toxin genes are coordinately regulated by bicarbonate and temperature J.Bacteriol. 176: 5188-5192. Stretton, S., A.E. Goodman. 1998. Carbon dioxide as a regulator of gene expression in microorganisms Antonie Van Leeuwenhoek 73: 79-85. Wagner, M. A., M. Eschenbrenner, T.A. Horn, J.A. Kraycer, C.V. Mujer, S. Hagius, P. Elzer and V. G. DelVecchio. 2002. Global analysis of the Brucella melitensis proteome: identification of proteins expressed in laboratory-grown culture. Proteomics. 2: 1047-1060.


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Decontamination of Drinking Water and Liquid Media by Cold Plasma in the Special Periods Alexander A. PIVOVAROV Ukrainian State Chemical-Technolgy University 8, Gagarin Ave., Dnepropetrovsk, Ukraine 49005, [email protected] Abstract. The results of researching to decontamination of drinking water and waste water under action glow dischargeplasma are considered. Perspective of application of such way of decontamination of liquid media colonised by pathogenic and potentially pathogenic micro organisms and viruses in real conditions and in the specific of time is shown. As a result decontamination drinking water can meet the existing standards and be suitable for the subsequent use.

1. Introduction Ukraine is a problem country as to sanitary quality of drinking water and waste water discharged in to the rivers and reservoirs. The existing enterprises for preparation of drinking water do not provide its due decontamination at the final stage of preparation, which is the reason of often mass epidemics of the population. On the other hand, being on the ways of penetration of illegal immigrants from the countries of Near East and Southeast Asia in to Western Europe, Ukraine is under constant and growing threat of possible terrorist act of biological or any other character. No less acute is problem of maintenance by quality drinking water of the population during natural calamities, such as droughts, floods, high waters and floodings of the basic sources of water supply. Plentiful atmospheric precipitation and floods caused by them in Europe, Ukraine and in the south of Russia in the summer of 2002 testify to the necessity of taking emergency measures connected, first of all, with maintenance by highquality water for the inhabitants of cities, countryside and hardly accessible areas. Besides in the recent years growing tendency of universal deterioration of ecological conditions not only in Ukraine, but also in the countries around it and as a result the reducing of general immune status of the population is registered. The number of diseases caused by pathogenic microorganisms and viruses continuously grows. The increase of water consumption connected with the growth of cities, low degree of purification and growth of volume of industrial and household waste water promotes more intensive microbial pollution of the reservoirs used as sources of centralised industrial-drinking water supply. As there is no reliable technology of clearing of water purification against of virus and bacterial pollution, drinking water continues to remain one of the main factors of transfer of many infectious diseases [1]. There is a significant amount of works showing that pollution by enteroviruses of objects of the environment, including drinking water, causes a number of complex problems.

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First of all, it is caused by a long survival of enteroviruses outside human body and their ability to cause serious pathologies like polio and related diseases, encephalitis, hepatitis, gastroeteritis, aphtosa syndrome, acute respiratory enteroviruses diseases, miocarditis in newborn babies, acute haemorrhoids conjunctivitis etc. [2,3]. The level of contamination largely depends on the quality of drinking water. Presence of serovariety of enteroviruses (more than 68), absence of cross immunity to them are the reasons that during lifetime a person can be infected repeatedly by enteroviruses. A convincing proof of the possibility by enteroviruses through water are the outbreaks of various enteroviruses infections, including polio, which took place in the different countries in various years with the sickness rate from several tens up to several thousands [4,5]. Methods of drinking water decontamination used during its preparation are ineffective in relation to enteroviruses [6]. According to the statistical data in a city with the population of one million through the use of water which does not meet sanitary requirements, these arises daily 600 clinical and subclinical forms of diseases with enteroviruses aetiology [7]. Content of 0.3-0.5 mg/1 of free chlorine in water does not provide its complete purification from enteroviruses [8,9]. In research [7] a decrease of a titre of poliovirus by 104 times was observed at the content of chlorine 5-20 mg/1. During outbreaks of cholera, which took place on the territory of Ukraine and other NIS, the basic preventive measure was the increase of concentration of chlorine in drinking water 4-5 times in comparison with usual water. Thus, natural or artificial infringement in the system water preparation at any moment can result in epidemiologie complications [10]. Now there are no official normative documents determining the admissible content of enteroviruses in drinking water. According to the recommendations of WHO it should not to exceed one virus particle per 100-1000 1. Some authors [7] believe the virus agents in it should not be present at all. In least decades in Ukraine from water supply there were isolated 2109 strains of various representatives of vibriogenus, wich makes up 1.2 % of the number of the investigated tests. Even more widely (up to 20 % of positive results) they are distributed in reservoirs, water from which goes to purification facilities. Unfortunately, the isolation of vibrios from water is hampered because of the absence of reliable ways to concentrate them [11]. Among all decontamination methods used chlorination despite of is now in widest use. So, by this method more than 500 km3 of natural waters in the world are decontaminate using about 2 million tons of chlorine annually [12]. It is a fact that for some geographical areas the application of chemical methods of water decontamination is connected with significant difficulties. In conditions of low temperatures decontamination action of chlorine is not effected and at high temperatures its unproductive losses take place. The transportation of reagents to remote or scarcely populated areas costs dearly besides for reagent processing capital purification facilities are necessary. Under such conditions purification methods based on use of electrical energy are most perspective. The advantages of methods of electroprocessing of liquids in comparison with traditional ways of purification are obvious. Most evident are simplicity of the technological scheme, absence of necessity in premises for a storing reagents and equipment for their preparation, simplicity of process automation and opportunity to improve working conditions, a wide spectrum of the improved characteristics of liquids and smaller specific volumes, weight and dimensions of facilities [13-15]. The application of new methods of water decontamination up to sanitary norms and above makes possible a steep decrease in enteroviral infections incidence. Most perspective are the methods based on application of nonequilibrium low-temperature plasma (otherwise known as glow discharge plasma (GDP)) [16,17]. They have by a number of advantages: smaller size of the equipment, opportunity of automation both of the process and quality control of processed

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environment, low engagement of human resources, opportunity of the use of new solutions, though poorly investigated, but having useful potential and properties. The basis for the process is the contact plasma discharge on the surface of a liquid phase formed between an electrode in a gas phase and a surface of a liquid, in which the second electrode is immersed. GDP serves a source of chemically active atoms and molecules in a liquid causing set of chemical reactions of redox character, rendering disinfection action on pathogenic and potentially pathogenic microorganisms and viruses contained in drinking water or other liquid media [18,19]. 2. Experimental Sanitary - microbiological and hygienic evaluation of processing of water by GDP is has been done in Research Institute of Hygiene of Academy of Medicine, Research Institute Practical Toxicology and Disinfection, Moscow, Russia and Scientific-Research Institute of Hygiene, Kiev, Ukraine. The processes of decontamination of drinking water in the terms pathogenic and potentially pathogenic microorganisms were experimentally studied: Esherichia Coli, Staphylococcus aureus, Staphylococcus epidermidis, Proteus vulgaris, Candida albicans, Clostridium, Salmonella typhimurium, Salmonella munhen, Salmonella infantis, Salmonella derbi, Pseudomonas pneumoniae, Vibrio cholerae, Cornebacterium diphtheria gravis and also Poliomyelitius virus, Hepatitis virus B In the experiments to study decontaminating of GDP effect water was infected by cultures of the mentioned above bacteria in concentration, 24-hour to real conditions of possible pollution of drinking water (from 103 up to 106 microorganisms per litre). To specify a degree of GDP effect on the spore forms of bacteria having the greatest stability to any sort of adverse influences of physical, chemical and biological nature for this purpose strains of Clostridium were taken. It is known, that their vegetative form are less resistant, therefore in the experiment their culture was previously warmed up within 20 minutes at 70 °C, which resulted in destruction of vegetative forms and preservation of spore ones. Infecting of water was carried out by spore forms of Clostridium and vegetative ones separately. For bacteriological research of drinking water before and after its processing by glow discharge plasma dense and liquid nutritious medium were used. In experiments with waste water dechlorinated drinking water was used with addition 0.1 % and 1.0 % of a waste liquid from sewage network (before purification and decontamination) and also waste liquid, to which pathogenic, enterobacteria (Salmonella of 4 strains): Salmonella typhimurium, Salmonella munchen, Salmonella infantis, Salmonella derby were added. Initial concentration of Salmonella (control) and the results of GDP effect processed media were determined by inoculation of medium of dense bismuth sulphite 0.1-1.0 ml medium by inactivation of the processed waste water on liquid nutritious medium (magnesium broth of double concentration), which made it possible to determine the amount of bacteria water under study. All other bacteria indicated in the study of drinking water were determined in waste water without preliminary infection. Besides taken into account were such resistant kind of microorganisms as Enterococcus. Their isolation was done in the SlanecBertley medium. After 24-hour incubation of crops in a thermostat at 37 °C carried out calculation of GMN (general microbial number of colonies), identification and calculation of the rest of micro flora was done. To define biochemical activity of investigated cultures isolated from water differential Kigler media were used. In case of necessity (for Salmonella first of all) serological diagnosis of "suspicious" cultures was carried out. For modelling virus pollution in

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A. A. Pivovarov / Decontamination of Drinking Water and Liquid Media

the experiment a phage of E.coly and virus of polio were used. Coliphages are widely used now in experimental research as virus models. Besides, on coliphage control of drinking water purification is carried out in sanitary - epidemiological stations of various countries to determine viral contamination [36]. Attenuated strain of polio virus 1 is close in its resistance to physical and chemical factors to wild strains of various enteroviruses and is applied in experimental conditions to estimate efficiency of purification and decontamination in terms of viruses in the most countries of the world. The technique of experimental research consists in the following. In certain volume (from 0.5 up to 3.0 1) of dechlorinated drinking water and active filtered waste water coliphage and polio virus were introduced up to final concentrations from ones up to hundreds PFU in one litre and from tens up to thousands PFU in a ml. Microorganisms suspensors obtained in researched waters were placed in special reactor with volume 30 ml and subjected to GDP effect for 1, 3, 5 and 10 minutes. In case of the minimal content of microorganisms (ones and hundreds in litre) as well as under action of plasma for a long time (5 and 10 minutes) the volume of each research test equalled to 30-1000 ml. The isolation of coliphages in tests of water was carried out by two methods: by direct inoculation and method of additional growth according to the technique given in work [20]. While studying tests with volume of 30 ml all liquid was inoculated on firm nutritious media. After cultivation in a thermostat a recalculation of PFU (pateh forming units) of coliphages for 1 ml or for 1 1 of researched liquid was done. A polio virus for isolated on the culture inoculated of Hep-2. For each test cultivation 4 test tube with culture of cells were used. The registration of results was done by cytopathic effect in 24 hours registration on the third, fifth, seventh and tenth day. On the tenth day the titre of the polio virus in TCD5o/ml was calculated according to [21]. In the absence of cytopathic effect in the test an additional passage for culture of tissue was done. The research assess the quality of water also included a study organoleptic and phisicochemical properties of water, content of organic substances and basic anthropogenic pollutants, metals and non-metal elements. In several series of research the influence of processing of drinking and river water with a various level of pollution by glow discharge plasma investigated. Preparation of model waters was done by the organic pollutants: chlorine and introducing phosphororganic pesticides, chloroform, benzapirene, carcinogenic substances, phenols as well as salts of metals: A12(SO4)318H2O, Fe2(SO4)3 9H2O, CuCl2, Cd(NO3)2 4H2O, CoCl26H2O, Pb(NO3)2, NiCl 2 6H 2 O, KMnO4, K2Cr2O7, Ba(NO3)2, Zn(NO3)2 6H2O, (NH4)6Mo7O244H2O. 3. Results and Discussion GDP effect on inactivation of coliphages and viruses in drinking water. At the first stage of experimental research dynamics of inactivation of various concentration coliphages in drinking water was studied for various time duration of GDP influence. The results of research are submitted in Tab. 18. The given data testify that the degree of inactivation of coliphages at a high level of pollution of drinking water (tens of thousands PFU/ml) after GDP treatment for 1 minute equalled 95.71 %, which is caused by the presence in the suspension of highly resistant and less resistant populations of microorganisms. Further increase of time of GDP influence resulted in the growth of a degree of inactivation. At maximal time of processing (10 min.) the amount of coliphages was reduced in water supply to 99.92 %.

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Table 18. Inactivation of coliphages in water supply in dependence on time duration of GDP effect (average date) Time of treatment, min.

PFU/ml.

Control 1.0 3.0 5.0 10.0

1.2-104 5.0-102 1.7-102 64 8.8

Inactivation, %

95.71 98.53 99.41 99.92

Time of treatment, mm. Control 1.0 3.0 5.0 10.0

PFU/ml.

1,2-102 46 0.4 0 0

Inactivation, %

61.67 99.66 100.0 100.0

Time of treatment, mm. Control 1.0 3.0 5.0 10.0

PFU/ml.

Inactivation, %

64 37 4.1 1.0 0

42.46 93.60 93.44 100.0

At initial pollution of drinking water at the level of hundreds and tens PFU/ml after one minute processing the amount coliphages was reduced on the average by 61.67 % and 42.46 %, correspondingly and only the increase of time of GDP effect on water for 3 minutes resulted in one more essential inactivation of coliphages (99.66 and 93.6 %). The analysis of the received data has shown that irrespective of the initial pollution of drinking water, after a 3-minute GDP influence was observed appraise equal decrease of the content coliphages in drinking water. Further the time necessary for complete inactivation of coliphages depended on initial concentration of microorganisms in the water. Of significant interest is the study of GDP effect to inactivate microorganisms contained in drinking water at the level of ones, tens and hundreds PFU in 1 litre, which is most frequent in natural conditions in existing methods of purification of water or under nonobservance of modes of its treatment. In this connection a series of studies to define the degree of coliphages inactivation was done to assess GDP effect under conditions of their presence in drinking water in the concentration given above. The results of research are given in Tab. 19. Table 19. Inactivation of coliphages in drinking water depending on duration of GDP affect (average date) Time of treatment, min. Control 1.0 3.0

PFU/1

Inactivation, %

9.4- 102 4.6-102 0

51.10 100.0

Time of treatment, min. Control 1.0 3.0

PFU/1

Inactivation, %

30 0 0

100.0 100.0

The data sited show that in drinking water pollution at the level of hundreds PFU/1 at one-minute GDP influence the degree of inactivation makes 51.10 % and after a 3-minute treatment of such water the degree of inactivation achieves 100 %. In a lower initial pollution (at the level of tens PFU/1) 1 min. of treatment is enough for complete coliphages inactivation. Along with the above research the GDP effect on inactivation of polio virus was studied. The results of this research are submitted in Tab. 20. Seen from the tab. 18 the polio virus contained in drinking water at the level of thousands of virions in 1 ml is inactivated by 45.1 % after oneminutes GDP treatment and by 99.9 % after the effect of glow discharge plasma after 3 minutes. Complete inactivation of the virus was observed after a 5-minute GDP treatment. At the content of the virus in drinking water at the level of hundreds PFU/ml after one-minute treatment it is inactivation by 38.1 % and is not found in tests of water after 3 minutes of GDP treatment. At the lowest concentration of the virus in drinking water (at the level of a few) it is not found in tests of water after one-minutes GDP influence.

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Table 20. Dynamics of inactivation of the polio virus in drinking water under GDP influence Time of treatment, min. Control 1.0 3.0 5.0

Titreofthe virus in TCD5o/ml

Inactivation, %

3.2±0,2 1.79±0,23 traces 0

45.1 99.9 100.0

Time of treatment, min. Control 1.0 3.0 5.0

Titre of the virus in TCDso/ml

Inactivation, %

2.19+0,23 1.34±0,2 0 0

38.1 100.0 100.0

Time of treatment, min. Control 1.0 3.0 5.0

Titre of the virus in TCD50/ml

Inactivation, %

0.84+0,2 0 0 0

100.0 100.0 100.0

It is necessary to note that at 5-minute of GDP treatment water renders toxic effect on cells of tissue culture and Esherichia Coli (strein HB2). This phenomenon was repeatedly observed in the research and was confirmed in passages. Thus, the given data of experimental research testify to high inactivation GDP effect as to indicators (coliphages) and viruses in drinking water. GDP effect on coliphages and viruses inactivation in waste water. The study into coliphages inactivation in waste water was carried out with use of imitate and filtrate of native waste water. The results of research are presented in Tab. 21 Table 21. Inactivation of coliphages in waste water in dependence on duration of GDP influence (average date) Time of treatment, min.

Control 2.0 3.0 5.0 10.0

Imitate of waste water

Filtrate native waste water

PFU/ml.

Inactivation, %

PFU/ml.

Inactivation, %

5.6-104 1.6-104 2.3-102 9.7 0

71.50 99.98 99.98 100.0

5.6-104 1.2.103 1.9-102 0.67 0

97.54 99.61 99.99 100.0

The presented data testify that after 1.0 min. treatment of waste water coliphages are inactivated in the imitate of waste water by 71.5 % and in the filtrate by 97.54 %. After an increase of time of GDP influence up to 3 min. the degree of inactivation practically remained the same equalled 99.58 and 99.61 % correspondingly. The greatest degree of inactivation of coliphages (more than 3-4 orders) in these waters was registered after 5 minutes of GDP treatment. After 5.0 min. of processing it was established that in nutritious media on the inoculated water tests grow of Esherichia Coli (strain HB2) was not registered. In the study of inactivation process of the polio virus in waste water it was established that after 1 minute of GDP treatment the degree of inactivation reaches 52.5 and 41.3 % depending on initial concentration of the virus in waste water (Tab. 22). The increase of time of GDP treatment up to 3 min. resulted in inactivation of the virus by 99.64 % (at higher initial concentration of the virus in waste water) and by 99.9 % at lower concentration of the virus. After 5 min. of GDP treatment the virus was found in tests in insignificant quantity (in 1 of 4 infected test tube) and was not found in tests of water in the second case. Thus received data testify to high inactivating effect of GDP as to the polio virus and coliphages contained in waste water. Practically 5-minute GDP treatment of such water results in practically complete inactivation of viruses contained in it.

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Table 22. Dynamics of inactivation of the polio virus in the filtrate of native waste water under GDP influence Time of treatment, min. Control 1.0 3.0 5.0

Titre of the virus in TCD5o/ml 3.28±0,2 1.55±0,23 0.84±0,2 traces

Inactivation, %

Time of treatment, min.

-

Control 1.0 3.0 5.0

52.8 99.64 99.99

Inactivation, %

Titre of the virus in TCD5o/ml 2.64±0,2 1.55±0,3 traces 0

41.3 99.9 100.0

Efficiency of decontamination of water containing spores of B.cereus was studied in term of initial concentration of the spores in water at various parameters of plant work. Antimicrobial effect of the GDP was found to shown in the itself relation of Staphylococcus aureus and Micobacterias B5 after 30 min., P.auruginosa - 15 min., yeast-like fungi Candida albicans - 5 min. Most resistant to GDP influence among vegetative forms of microorganisms are S.aureus and Micobacterias B5. Less resistant are yeast-like fungi Candida albicans and P.auruginosa. On the basis of the data obtained for the further research S.aureus and Micobacterias B5 were selected. The results of decontamination of water contaminated by S.aureus and Micobacterias B5 treated by GDP are presented in Tab. 23 Study of efficiency of decontamination of water containing test - virus (vaccine strain of the polio virus of type 1) used in the development of modes of disinfection of products of medical purpose carried out under GDP influence on water for 1, 3, 5, 10, 15 and 30 min. The control for the tests was effected through tests of waters containing the virus before its GDP treatment. The results of research are given in tab. 24. Table 23. Decontamination of water contaminated with S.aureus and Micobacterias under GDP influence Time of treatment, mm.

Microorganism.

Quantity of viable cells in 1 ml of water. Before treatment After treatment 3.86-105

Staphylococcus aureus

5.0 10.0 15.0 30.0

2.64-105 1.82-102 1.60-101 0

Micobacterias 15.0 30.0 45.0 The note: "+" - presence of growth; "-" - absence of growth.

+

+ -

Table 24. Decontamination of water containing of polio virus under GDP influence Time of treatment, min.

Quantity of the virus, Ig TCD5o/ml Before treatment

After treatment

5.26+0,07

1.0 3.0 5.0

3.26+0,07 0.43+0,08 0

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Organoleptic properties and phisico-chemical composition of water treated with glow discharge plasma. Drinking water. Estimation of the parallel phenomena in water as a result of GDP effect during decontamination of drinking water was done along the following basic guidelines: study of possible negative influence of the given treatment method on the quality of water obtained, study of the efficiency of additional purification of water from various kinds of chemical pollution. The analysis of results of sanitary - chemical tests has shown that under GDP effect (at the maximal GDP exposition found by the results of microbiological study to be 5 min.) organoleptic parameters of the treated water did not change greatly. The odour of water practically remained at the same level, colour intensity of water after treatment was reduced on the average by 35 %, at the initial level appropriate to the hygienic rules (20°). Turbidity of water after treatment was considerably reduced. Change of physical properties of water at the same time was registered. After treatment the pH shift to alkaline medium (from 7.5 to 9.4 units) was observed which exceeds the hygienic standards for drinking water; temperature of the water rose on the average by 10-15°C. Insignificant decrease of salt content in the treated water was found to meet optimum standards for drinking waters of hydrocarbonate class (250-500 mg/1). The treatment of running of water by GDP resulted in the change of the amount of nitrogen containing compounds; the content of ammonium nitrogen in the treated water decreased on the average by 22 %, and the concentration of nitrites grew approximately by 30 %, but did not exceed the hygienic norm; the content of nitrates practically did not change. As a result of study into the quality of water by the indices of salt compositions before and after GDP treatment it was fond that in the treated drinking water, in comparison with control, some decrease in general salt content is registered. It is explained by the basic reduction of concentration of bicarbonates as well as insignificant decrease of salt hardness (calcium and magnesium). The received data testify that indirect parameters namely content of organic substances in water under study (permanganate oxidability, the organic carbon) changes differently. Thus permanganate oxidability in GDP treated water grew and the organic compounds diminished which apparently, is caused by processes of oxidation of organic substances and their transition into easily oxidised forms. The concentration of organic carbon was reduced on the average by 60 % in comparison to the initial level. The analysis of quality of running water by other parameters of organic pollution of water, in particular by halogen containing compounds (GCC), formed during chlorination of water testifies to high efficiency of water purification from GCC as a result of its GDP treatment. The concentration of GCC in this case did not exceed the hygienic standards. The similar regularity was established for other widespread anthropogenic organic pollutants of water (surface - active substances, phenols etc.) at their initial content in the level of the ultimate concentrations. River water. The experimental study was done on the bases of model water solutions described by higher levels of chemical pollution. For this purpose river water was used with previously introduced various kinds of chemical pollutants (phenols, carcinogenic substance, polyform compounds, and salts of heavy metals). The analysis of results of organoleptic tests has shown that during GDP water treatment the odour of water practically did not vary at high initial colour intensity (38°), the treated of water had colour intensity far below the hygienic standards for sources of industrial and drinking water supply (10°). Alongside with it, as well as in the previous series of tests, the shift of the hydrogen parameter index from 7.5 down to 9.8 was registered. The oxidation of inorganic nitrogen containing substances was estimated by the change of the content of ammonia, nitrites and


223

nitrates. In water after glow discharge treatment the content of ammonia was reduced by 1.3 times, nitrites remained at the same level as before treatment and nitrite content rose a little (0.7 times). By the change of the content of organic substances in river water it is possible to note some increase of the content of light organic compounds (by the permanganate oxidation index) after its GDP treatment (7.4 mg O2/l). The content of organic carbon after treatment water was reduced by 3 times (from 19.5 down to 6.5 mg/1). The analysis of research results has shown that at on GDP influence on river water containing phenols there is a decrease of their concentration practically by 2 times. The efficiency of purification is high enough, however the content of phenol in the initial and treated water exceeds maximum norms (admissible concentration limit (ACL) - 0.001 Mr ). The similar regularity is established also allowable for benzapirene. The obtained data testify that the decrease of concentration of benzapirene (82 %) is observed; after water treatment its content in the treated water does not exceed treatment maximum allowable concentration. The highest efficiency of purification is registered for halogen-containicy compounds in river water. The research has shown that during treatment of river water glow discharge plasma there occurs a considerable decrease of concentration of halogen-containicy compounds the contents GCC in the water is at the level of the hygienic norm (30 mkg/1). The result of the analysis of the data obtained makes it possible to make the conclusion that the use of the given method alongside with decontamination provides effective purification river water of a number of organic compounds and, first of all, from galoidform compounds, phenols and carcinogenic substances. The influence of GDP treatment on water and water solutions containing ions of various metals, was also investigated for two cases. In the first case selected for research was drinking water with a low content of compounds of metals polluting such water; in the second case the research object was river water containing compounds of metals of various concentrations characteristic of river water from reservoirs adjacent to industrial cities. As a result of research it is established that the concentration alkaline and alkaline-earth metals in drinking water before treatment varies insignificantly. The content of magnesium is reduced by 26 % and concentrations of calcium, potassium and sodium practically do not change. As regards to other researched elements a significant decrease of concentration of ions of metals after GDP treatment of drinking and river water is registered. Most effective is the purification of water of zinc, copper, manganese, iron, aluminium and other polyvalent metals. Especially it is necessary to note, that the GDP treatment of water with the high content of metal compounds is as effective as of the water with their low content. General result of the GDP treatment of drinking and river water is the decrease of metal compounds to the levels required by hygienic norms. In 1995-1997 JSC "Dneprovsky Machine-Building Factory", Dnepropetrovsk, Ukraine, under research supervision of the author of this article produced pilot plasmachemical plant (Fig. 39) with productivity up to 1.0 -1.6 m3/hour of treated liquid media.

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Figure:39. Pilot plasmachemical plant

Further production of such plant in Ukraine was suspended due to insufficient funding. However, the accumulated experience of development, designing, manufacture and operation of plasmachemical equipment provides for a possibility to produce similar plasmachemical complexes mainly for decontamination of local flows of drinking and waste water including special periods of decontamination of centres bacterial pollution or destruction of the accumulated stocks of the bacteriological weapons. Such complexes can be applied for purification of local flows with homogeneous chemical composition of the extracted in mechanical engineering, hydrometallurgy etc. The author expresses deep gratitude to the workers of the above-mentioned institutions and enterprises the help rendered in the course of the research.

References [1] [2] [3] [4] [5] [6] [7] [8]

Viruses and bacteria in drinking water /V.I. Zadorognjaa, V.A. Bondarenko, V.V. Alekseenko etc. // Chemistry and technology of water -1993, -15, Nol.-P. 66-71. Wiedenmann A., Eleger B., Botrenhart R. Enterobactericeae as a criterion for the quality of drinking and swimmng-podwater//Zbl.Bacteriol/-1987. -B184, No 5.- P. 426. Kooij D. Properties Aeromonas and their occurrence hygienic significance in drinking water //Zbl.Bacteriol.-1988.- B.107..N91.-P. 1-17. Edge I.,Finch P. Observations on bacterial aftergrowth in water supply distribution systems, implications for disinfection strategics// J.Inst. Water Environ. Manag.-1987.-l,.Nol.-P.104-l 10. Lloud B. J.,Wheeler D. C.,Pardon M. The relationship between water-related discard and water quality with particular reference to urban water supply in developing country //Water Sci. And Technol.-1989.21,No6/7.-P. 579-591. Akin E.W. Occurrence of viruses in treated drinking water in the United States //Water Sci. And Technol.-1985.-17,No24/5.-P.689-700. Goldberg M.B.,Dicita V.J., Caldorwood S.B. Identification of an ironregulated virulence determinant in vibrio cholerae, using Tupho a mutagenesis//Infest,Immun.-1990.-59,.Nol.-55-60. Slade J.S. Viruses and bacteria and bacteria in a chick well //Water Sci. And Technol.-1985.-17,No 10.P.lll-125.

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[21] [22]

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Desinfection of advanced wastewater treatment influent by chlorine dioxide and ozone: Experiments using seeding poliovirus/ R.Warriner, K.D.Kostenbader, D.O.Cliver, W.-Ch. Ku// Water Res.-1985.19,No12. P.1515-1526. Lovzchevich E.L. Some aspects of inactivation enteroviruses process by chlorine // Hygiene and sanitation.-1973.- No 10.-P.11-15. Schulze E. Die virologische Uberwachung der Trink-wasserqualitat eine Ubersicht//Zbl.Microbiol.1990.-145,N22.-P.135-143. Magnetic immunesorption for selective concentrating of cholera embryos / S.A.Shaplo, E.I.Efimenko, N.I.Narbutovich etc. // Information of the problem-thematic of'Cholera' .-Rostov upon Don , 1989.-P.2728. Syrkina I.G., Uljankina G.S., Abramova V.I. Disinfectants. Review. Inform.series. Industry of chlorine. M. NIITECHIM -1986.-87 P. S.E., Speck M. L. Inactivation of microorganisms by electrohydraulic shock // Appl.microb.-1967.-l 5. P.1031-1037. Gilliland S.E., Speck M. L. Mechanism of the bactericidal action produced by electrohydraulic shock // Apple. Microb.- 1967.-15.P.1038-1044. Edebo L., Holme I., Selin I. Influence of the conductivity of the microbiological effect of transient electric arcs in aqueous systems .//Appl. Microb.-1969.- 17,.Nol.-P.59-62. Robinson J.W., Ham Mooyoung, Balaster A.N. Ultraviolet radiation from electrical discharges in water. // J .Appl. Phys. -1973. -44. P.73-75. Malik M.A., Chaffar A., Malik S.A. Water purification by electrical discharge. // Plasma Sources Sci. Technol. -2001,-10. P. 82-91. Pivovarov A.A., Sergejeva O.V. Physico-chemical transformations in water environments under action of non-equilibrium plasma of glow discharge. Problems of chemistry and chemical technology. Vol. 3, 1999. P. 48-50. Pivovarov A.A. The Advanced Technology of Extraction of Noble Metals from Industrial Waste in Circumstance of Market Economy. NATO Science Program. Advanced Research Workshop (ARW) "From Transitional Economy to Sustainable Development". Dnepropetrovsk, Ukraine, October 22-25, 2001. The instruction by definition of bactericide properties of new disinfectants./ Authorized by Ministry of Health of USSR No739-68. 06.05.1968. The methodical of recommendations by definition of preparations virulidic activity. /Authorised by Ministry of Health of USSR Nol 119-73. 06.09.1973.

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Antibacterial adsorption-filtering materials for individual protection means of organism 1. ANTIBACTERIAL ACTION OF METAL-CONTAINING CARBON ADSORBENTS N. KARTEL, A. GRIGORIEV, D. SHVETS, V. STRELKO Institute for Sorption and Problems of Endoecology, NAS of Ukraine, Kiev, Ukraine, e-mail: [email protected]

1. Introduction Questions of clearing of ecosystem objects (water, products of feed) from viruses, microorganisms and fungi, especially pathogenic for human organism, are exclusively important for today [1]. Their occurrence in products of feed and drinking water can be one of consequences of technogenic activity, infringement of sanitary norms in food industry, and also probable acts of bioterrorism directed on creation of complicated epidemiologic situations in concrete regions or objects. On the other hand, in medical practice the large importance is got by various materials with antibacterial activity for more effective treatment of contaminated wounds, ulcers and burns [2]. In this connection the search and creation of new perspective materials and technologies on their basis capable to keep and to render antibacterial (bactericidal, bacteriostatic) influence on microorganisms, is the major research problem of the experts of various areas - chemistry, material science, microbiology, medicine etc. To one of the fruitful approaches in creation of materials with antibacterial activity represents use of adsorption materials. Really, various adsorbents, having the developed porous structure and surface of interface are capable to keep crates of microorganisms at the expense of various intermolecular and ionic interactions of collective character with corresponding protein fragments of membrane structures of bacteria [3]. However the majority of adsorbents as a rule has no expressed antibacterial influence in relation to adsorbed on their surface microbial cells; that results in formation of conglomerates of activators with local infecting concentration [4, 5]. For maintenance bactericidal ability of adsorption materials the antibacterial means are introduced into their structure. The purpose of the present work was the study sorption-bactericidal action of carbon materials modified by ions of copper and zinc.

N. Kartel et al. /Antibacterial Action of Metal-Containing Carbon Adsorbents

227

2. Material and methods. In work it is used the 24-hour cultures of Staphylococcus aureus 209, Pseudomonas aeruginosa 103 and Escherichia coli 41, grown up on agarous nutritious media. The colonies of microorganisms were washed off from plates by isotonic solution of sodium chloride, twice washed and prepared a suspension, containing 109 microbial cells on 1 ml. The carbon adsorbents SCN and KAU represented accordingly spheres and grains of the size up to 0.5 mm. In experiments it was used carbons, having on a surface oxygen-containing functional groups, and also ions Cu2+ and Zn2+ with concentrations in the range of 2.5-0.025 meq per 1 g of a material. With the purpose of definition optimum of bactericidal concentration of metals included in adsorption material, previously by method of serial dilutions it was studied an action of various concentration (0.1-25 mg/ml) of copper and zinc salts (CuCl2, ZnCl2) in a physiological solution on viability of researched microorganisms. For definition of a level of microorganism adsorption on carbon materials the last were sterilized, packed on 0.5 cm3 in flasks, where 0.75 ml of microorganism suspension (109 microbial cells) was brought in. Adsorption was carried out at temperature 4 °C during I h at weak vibration of flasks. Further in flasks 10 ml of buffered physiological solution (pH 7.2) was brought in, shook up, and centrifugated during 10 min at 1000 cycles/min for deposition of adsorbent. Liquid phase was put on agarous nutritious media Endo and yolk-salt agar depending on a kind of microorganisms was taken in experiment. By practical consideration it was selected such concentration and volume of sowed material, that the number of evolved colonies did not exceed 200-300 on a plate. The control was served a similar volume of microorganism suspension without carbon material. The results were estimated, proceeding from differences of control and test sowings in recalculation on amount of adsorbed microbial cells in 1 cm3 of adsorption material by following formula: A = (K-O) / V where: A - sorption activity of carbon, (K-O) - amount of microbial cells adsorbed on carbon, V - volume of carbon material used in experiment.

Further the sorbents with keeping on it microbial cells were washed six-times by buffered physiological solution (pH 7.2), and elutes were sowed on agarous media. Bactericidal effect was observed at the following carrying washed adsorbent in meat-peptone broth (MPB). The infection ability of activators adsorbed on carbon materials was determined by a level of colonization of tissues on model of the isolated intestine fragment of white impure rats. The fragments were washed by a physiological solution, ligatured on the one side. Into a gleam it was quantitatively entered a suspension of carbon material SCN (size of particles up to 500 u) with definite amount of cells E. Coli 41 adsorbed on it. Then imposed second ligature. By control it was served the intestine fragments of similar length with entered in them suspensions E. coli of the same concentration as in experiment without adsorbent. To except the reproducing of microorganisms the experiments on adhesion were carried out at temperature 4 °C during 30 min. The ligature were removed, the fragments of intestine washed out by a buffered physiological solution (pH 7.2), homogenized, diluted to sowing concentration and sowed on plates of Endo' agar, differentiating thus adsorbed microorganisms

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from non-adsorbed ones on a surface of mucosa. The results were estimated on a difference between evolved colonies in test and control sowings in colony-formation units on 1 cm3 of a mucosa surface of intestine.

3. Results and Discussion Adhesion of microorganisms in metal-containing carbon sorbents. The study of influence of salts CuCl2 and ZnCb in decreasing concentration on growth of E. coli has shown that copper chloride in concentration of 0.4 mg/ml, and zinc chloride in concentration of 1.5 mg/ml do not render appreciable influence on reproducing broth culture E. coli 41. The appeared inhibition concentrations were: for salt of copper - 0.7-1.0 mg/ml, for salt of zinc - 4.0-15.0 mg/ml. The data of these experiments have formed the basis for introducing ions of copper and zinc in structure of adsorbents in a range of concentrations including threshold meanings for salt solutions - 80-0.6 mg/ml (2.5-0.25 meq per 1 g of sorbent). The study of adsorption of microorganisms has shown that oxidized carbon materials such as SCN, not modified by metals and widely used in medicine, substantially (from 5.4* 108 up to 7.1*108 microbial cells by 1 g of adsorbent) have adsorb E. coli and P. aeruginosa (Table 25). The level of adsorption S. aureus is rather low and makes 1.9*108 microbial cells by 1 g of adsorbent. It is necessary to note that carbons without oxygen-containing groups have greater adsorption activity concerning this type of microorganism - on the average 6.4* 108 microbial cells by 1 g of adsorbent. The introduction of copper ions in SCN in concentration 2.5 meq/g allows practically completely adsorb microbial cells from suspension (7.5*108 microbial cells by 1 g of adsorbent). It is possible to consider decreasing concentration of copper ions up to 0.025 meq/g proved for non-oxidized samples. On oxidized samples with the copper ions included in structure a level of adsorption of E. coli and P. aeruginosa is some less in comparison with samples of SCN, not containing copper ions. It is represented interesting that, as against oxidized copper-containing samples of SCN, having a rather low level of adsorption in the relation S. aureus, oxidized carbons with ions of zinc are characterized high adsorption activity concerning this microorganism (100% - at concentration Zn2+- 2.5 meq/g and 97.1% at concentration Zn2+- 0.25 meq/g). At the same time introduction in the carbon sample of SCN of zinc ions results in some decrease of a level of adsorption of E. coli and P. aeruginosa in comparison with a control sample, which has especially small concentration of metal (0.025 meq/g). Last years the increased attention is given to carbon sorption materials such as KAU (active carbon from fruit-stones), as to cheaper natural materials. The oxidized form of a sample of KAU, not modified by ions of metals, has quite high adsorption activity concerning researched microorganisms, reaching 7.5* 108 microbial cells by 1 g of adsorbent. Nonoxidized samples of KAU are characterized by a smaller level of adsorption in comparison with non-oxidized SCN and oxidized KAU. The updating fruit-stones carbons by copper ions provide high sorption-bactericidal effect at all samples taken in experiments. Thus as optimum concentration of copper ions it is possible to count 0.25 meq on 1 g of adsorbent, as the parameters of a level of adsorption in this case change in limits from 72 up to 100%. It is necessary to note that the copper ions in these samples are less effective in relation S. aureus, than ions of zinc. The sorption-bactericidal effect of fruit-stones carbons with concentration of ions of zinc 0.25 meq/g remains high for all taken in experiments microflora.

N. Kartel et al. / Antibacterial Action of Metal-Containing Carbon Adsorbents

229

The found out distinctions in levels of adsorption interaction at various samples with concrete cultures of microorganisms allow assuming an opportunity of the directed application of the modified carbon materials. All carbon materials containing ions of copper and zinc, as against unmodified, render more appeared destructive action on bacterial cells, and this proves to be true by absence of growth of microorganisms in sowings of elutes and at entering samples of carbons with adsorbed cells in meat-peptone broth. Table 25. Comparative sorption-bactericidal efficiency of carbon metal-containing adsorbents Sample of carbon materials SCN - initial - oxidized

Content of metal, meq/g

0

2.5 SCN-Cu2+ - initial - oxidized

0.25 0.025

2.5 SCN-Zn2+ - initial - oxidized

0.25 0.025

KAU - initial - oxidized

0.25 0.025

2.5 KAU-Zn2+ - initial - oxidized

(7.0+0.2)* 108 (7.1±0.4)*108 (7.4+0. 1)*108 (7.4+0.1)*108 (7.3+0.2)* 10s (7.0+0.2)* 108 (7.0+0.3)* 108 (4.6+0.4)* 108 (7.0+0.5)* 108 (6.5+0.2)* 108 (7.0+0.3)* 108 (5.9+0.1)*108 (5.0+0.7)*108 (2.8+0.7)* 10"

(6.4±0.6)*108 (1. 9+0.7)* 108 (7.5+0. 1)*108 (7.4+0. 1)*108 (5.7+0.3)* 108 (3.3+0.6)* 108 (5.6+0.6)*108 (2.9+0.2)* 108 (6.8+0.2)* 108 (7.5+0.1)*108 (6.5+0.6)* 108 (7.3+0.4)* 108 (5.1+0.6)*108 (7.2+0.3)* 108

(6.1+0.1)*108 (5.4±0.2)*108 (7.5+0. 1)*108 (7.5±0.1)*108 (7.5+0. 1)*108 (5. 1+0.5)* 108 (6.4+0. 1)*108 (3.4+0.4)* 108 (6.5+0.4)* 108 (6.1+0.1)*108 (6.2+0.3)* 108 (5.2+0.5)* 108 (3.8+0.9)* 108 (3.0±0.5)*108

(5.9±0.4)*108 (6.8±0.3)*108 (7.5+0.1)*108 (7.5+0.1)*108 (7.0±0.3)*108 (7.5+0. 1)*108 (5.8+0.5)* 108 (6.3+0.5)* 108 (7.2+0.3)* 108 (7.5+0.1)* 108 (6.4+0.4)* 108 (7.5+0. 1)*108 (3.5+0.7)* 108 (5.0+0.3)* 108

(4.9+0.2)* 108 (7.5+0.1)*108 (7.5+0. 1)*108 (7.5+0.1)*108 (5.4+0.3)* 108 (7.2+0.2)* 108 (5.5+0.4)* 108 (6.9+0.6)* 108 (7.5+0.1)* 108 (7.5+0. 1)*108 (7.5+0.1)*108 (7.2±0.3)*108 (5.3+0.1)* 108 (6.8+0.8)* 108

(5.9+0.2)* 108 (7.0+0.2)* 108 (7.5+0. 1)*108 (7.5+0. 1)*108 (7.4+0.2)* 108 (7.5+0. 1)*108 (5.7+0.8)* 108 (6.5+0.2)* 108 (7.5+0.1)*108 (7.5±0.1)*108 (6.6+0.2)* 108 (7.4+0.1)*108 (5.9+0.6)* 108 (5.2+0.4)* 108

0 2.5

KAU-Cu2+ - initial - oxidized

Sorption-bactericidal activity, microbial cells per 1 cm3 of adsorbents (M+m) P. aeruginosa S. aureus E. coli

0.25 0.025

Due to ability of carbon metal-containing adsorbents to destroy adsorbed microbial cells and simultaneously to adsorb their toxins, these materials can be used in medicine. For the proof of this thesis we carry out comparative study of a level of colonization of intestine mucosa of white rats by culture E. coli adsorbed on unmodified and copper-containing carbon such as SCN. The introduction of copper-containing carbon suspension with adsorbed cells of E. coli 41 in a cavity of living fragment of intestine strengthens a level of colonization in comparison with the control in 10-16 times. If to accept a level of colonization of intestine mucosa by bacteria E. coli, entered without adsorbent, for 1, the level of colonization for metal-containing carbons is equal 0, and for unmodified non-oxidized carbons SCN - 11+2. Thus, it is possible to assume that use of adsorption materials which are not having bactericidal properties, will promote adhesion process of activators on tissues are trope to them .

230

N. Kartel et al. / Antibacterial Action of Metal-Containing Carbon Adsorbents

Efficiency of adsorption of Cholera vibrionfrom water by copper-containing carbon fibrous adsorbents. Using the developed approaches on modification of carbon adsorbents by metal ions it was studied the efficiency of sorption removal of Cholera vibrion from water solutions. The structural - sorption characteristics of carbon materials and their modified by copper ions forms are illustrated in Table 26 It was found that the content of copper ions in sorbents in the range of 2-4 mg/g did not change essentially parameters of carbon fabric and felt but changed their surface chemistry. This is testified by the decreasing of pH zero of charge surface of carbons modified by copper ions (Table 26). The shape of the adsorption isotherm of trypsine on the carbon fabric in phosphate buffer testifies to the absence of influence at decreasing ratio of solid and liquid phases. The modification of carbon fabric by copper ions leads to increase the trypsine adsorption in 2.0-2.5 times. By analogy with this the influence of the modification was shown at study of the sorption activity of carbon materials to Cholera vibrion (Table 27). The influence of the nature of carbon materials is displayed at absence of microorganism on the carbon felt, and this correlated with its low structural-sorption characteristics and lower kinetic of the Cholera vibrion extraction in compare with the carbon fabric. It was established that using the ratio of solid and liquid phase 1:50, time of contact 5 min and contain of copper ions in the sorption material 2.3 mg/g it was observed 100% extraction of Cholera vibrion from sea water solution. With increase of the Cholera vibrion content to 106 microbial bodies was also founded 100% extraction of them from solutions by the modificated carbon fabric. So the modification of carbon materials by copper ions does not only increase their bactericidal properties but perhaps forms the copper (II) ion complexes with nitrogen- and oxygen groups of trypsine besides electrostatic interaction of carbon with polar molecules of albumen and the formation of H-bonds with hydroxyl and carboxyl carbon groups. Besides that, ions of copper on the surface of carbon sorbents can be able interact with spiral albumens and neutralize charge of membranes. Table 26. The structural - sorption characteristics of carbon materials Sorbents

Content of Cu2+; mg/g

Carbon fabric Carbon felt

2.3 3.9

Volume of sorption pores; cm3/g H2O C6H6 0.06 0.01 0.01 0.05 0.01 1.21 0.02 1.15

Adsorption of methylene blue, mg/g

pH zero of charge of surface

135 75 430 360

6.8 6.3 7.4 6.6

Table 27. Sorption ability of carbon materials to Cholera vibrion (the concentration of 104 m. b./ml). Sorbent

Content of Cu2+; mg/g

S:L

Time of contact; min

Sorption ability, %

Carbon fabric

1.0 1.3 2.3 4.0 -

1:50

5

1:100

5 60 5 60

60 70 95 100 100 0 0 75 100

Carbon felt

3.9


231

Figure 40. The influence of ratio Solid: Liquid on the adsorption of the trypsine by carbon fabric

4. Conclusions The carried out researches have shown that the copper and zinc ions are perspective at updating carbon materials with the purpose of giving them bactericidal properties. The received preliminary results of application of local sorption for treatment of an experimental purulent wound of white impure rats with use of metal-containing adsorbents have shown absence any general resorbtive toxic action of copper and zinc ions on organism of animals. As now there are various forms carbon adsorbents (from grains of various forms and thin powders up to fibrous structure), there is a real prospect of application of metal-containing adsorbents as for local adsorption in surgical practice, and for oral sorption in infectious intestine pathology. Besides the modifying of carbon materials is necessary and useful in case of their use for additional purification or additional disinfections of drinking water, which can be carried out even in household conditions.

References [1] [2] [3] [4] [5]

Grigor'eva, L.V., Kas'yanenko, A.M., Korchak, G.I. et al (1985) Sanitary microbiology of evtrofed water basins, Zdorov'ya, Kiev. Enterosorption/EA. Belyakov N.A. (1991), Tsentr Sorbtsionnyh Tekhnologiy, Leningrad. Iljin, L.A. (1987). Antimicrobial materials, Medicina, Moscow. Grigoriev, A.V., Zemskov, V.S., Shor-Chudnovsky, M.E., et al (1990) Interaction of metal-comtaining adsorbents with pathogenic microflora, Clinic Surgery, N3, pp. 42-44. Grigoriev, A.V., Znamensky, V.A., Bugaev, V.I., et al (1991) Adhesion of microorganisms in metalcontaining carbon sorbents, Microbiol. J., V. 53, N2, pp. 98-103.


232

Antibacterial adsorption-filtering materials for individual protection means of organism 2. FINE FILTERING MATERIALS ON THE BASE OF POLYPROPYLENE MICROFIBRES AND THEIR ANTIBACTERIAL PROPERTY M. TSEBRENKO, V. REZANOVA, I. TSEBRENKO, M. MAYBORODA, N. KARTEL* Kiev National University of Technologies and Design, Kiev, Ukraine, *Institute for Sorption and Problems of Endoecology ofNAS of Ukraine, 1. Introduction One of methods of effective protection of the human organism from harmful influence of colloid particles of air and water media, including crates of microorganisms and their toxins, is the microfiltration through fine fiber filters. In the Problem Laboratory of Synthetic Fibres at the Kiev National University of Technologies and Design the fundamental researches are executed in the field of physical chemistry of melts of polymer mixes allowed to create scientific bases of reception of ultrathin synthetic fibres [1,2]. The principal scheme of preparing ultrathin synthetic fibres as well as microphotographies of obtained bi-component product and texture of filtering material (after extraction of one of the component) are shown on Fig. 41.

X 1OOO

Figure 41. Scheme of ultrathin fibres formation and SEM microphotos of obtained product

By practical result of these researches was the development of the technology to prepare essentially new fine fibre filtering materials (FM) and filters, which are already

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produced by JSC "UKRFILTR" (Chernigov, Ukraine). Basic structural unit of developed FM are the ultra thin fibres with unique structure of a surface: each fibre of micron size is covered on all surface with most thin microfibrilles, departing from the basic fibre (see Fig. 42).

Figure 42. Microfibrillar structure of ultrathin polypropylene fibres

In result the extremely advanced surface turns out and it provids high adsorption ability and capacity. Such fibres are not present in a nature, and they can not be prepared on traditional technologies. The comparison of various filtering materials and membranes on selectivity testifies that we have created principally new FM of volume action. Its selectivity is higher, than traditional deep ones, and in the range of particles near 1 micron it separates as a membrane of a set type. This is possible to explain by several physico-chemical processes, which take place at fine filtration: adsorption, contacting effects, Brown diffision. The important role is played also an electrostatic interaction. Developed fine fibre polypropylene filtering materials, as against all other synthetic fibres, have a negative charge, which superiors a charge of a natural leather. It enables to catch microparticles and microdrops considerably smaller, than nominal size of pores of FM. By main characteristics the developed FM are at a level or surpass the filtering materials of such well-known producers, as "Millipor", "Pall", "Gelman" etc. To the present time the filters from polypropylene (PP) microfibres with a subtlety of clearing 1, 0.45, and 0.3 u are created and already widely used for a filtration of liquids. Use of PP gives to FM a chemical inertness and stability to aggressive media. By the results of toxicological tests the fine fibre PP filters are recommended for clearing drinking water in household conditions, salines and drugs for injection in organism. The carried out researches have shown that developed FM detains an oil fog (radius of particles 0.14-0.17 u) with efficiency of 99.99% (tests are spent in the L.Ya.Karpov Research Physical Chemistry Institute, Petryanov Laboratory of Filtration, Moscow), and at a filtration of technological air at the Sumy Biofactory and carbon dioxide at the Factory of Champaign Vines "Zolotaya Balka" (Sevastopol) the sterile filtered gaseous media are obtained. The filters combined with a carbon fabrics having, alongside with high selectivity, large adsorption activity are created also. 2. Improvement of drinking water indexes by fine fibre PP filters Together with the international association "Water and Health" the sanitary - chemical and microbiological researches were executed according to efficiency of clearing of drinking water in filter devices "VIN-5" and "Krynichka", in which the filters from PP microfibres, and also filters combined with a carbon fabric are used. The permission of Central Sanitary

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Epidemiologic Service at the Ministry of Public Health of Ukraine on clearing of drinking water by mentioned devices is received. It is shown (Table 28) that PP and combined filters, excepting actually filtering action, essentially improve quality of drinking water: in 3-3.5 times the contents of ammonia, nitrates, zinc, copper, iron, manganese decreases; residual chlorine and led are removed from water completely, in 2-10 times the contents of salts of heavy metals decreases. These are explained by various physico-chemical processes, which take place at precision filtration through fin fibre filters: adsorption, surface phenomena, Brown diffusion, electrostatic interaction. Table 28. Results of laboratory tests of filtering elements in FFF (fine fibre filters) N

Parameters

National Standard for Control Metods

Potable water (initial)

Potable water (after filtering)

Norm on National Standard GOST 2874-82 "Drinking Water"

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

Smell, un. Smack, un. Dimness, mg/dm3 Colourness, mg/dm3 Hardness, mg/dm3 N of nitrates, mg/dm3 N of nitrites, mg/dm3 Chlorides, mg/dm3 Sulfates, mg/dm3 Fluorine, mg/dm3 pH, un. Zinc, mg/dm3 Copper, mg/dm3 Cadmium, mg/dm3 Iron, mg/dm3 Residual Cl, mg/dm3 N ammonia, mg/dm3 Dry residue, mg/dm3

GOST 335 1-74 GOST 335 1-74 GOST 335 1-74 GOST 335 1-74 GOST 4151-72 GOST 18826-74 GOST 4 191-82 GOST 4245-72 GOST 4389-72 GOST 4386-81

1 0

PU3 > PU2. So the PU1-KAU composite adsorbent was determined as most effective due to the less screening of the pore structure. For comparison the homopolymers PVA, PVC forms the homogeneous dispergated system "active carbon-polymer". As the carbon filler and the polymer matrix have different thermal expansion coefficients there exist a possibility of destruction of the adhesive layer at the interface boundary due to shrinkage of the polymer matrix during the hardening of the composite. That is why the high elasticity and resistance against the deformation forces are necessary conditions for polymer matrix. Whereas the PVA and PVC form hard films (relative elongation of 10-50%, tensile strength of 46-54 MPa), it is expedience to use plasticizers. For comparison the relative elongation and tensile strength for polyurethane matrixes are the follows: PU1 and PU2: 350-600%, 12-25 MPa, PU3: 200%, 7 MPa. There are a lot of polar groups in polyurethane macrochain, such as -NH, -CO, -COC-, -COO-, available to interact with the surface of active carbon because of high valency unsaturation of the latter. According to IR-spectroscopy data of "urethane-active carbon" model system the interaction between nelectrons of active carbon and urethane carbonyl group may be supposed. It is likely the transformation of carbonyl bond of urethane group: >C=O; that is confirmed by slump of C=O bond intensity (1660 cm-1) and appearance of a new bond (1485 cm-1). The hydrophobic properties of CSM are conditioned by hydrophylicity of its polymer matrix. The values of the water absorbability for the initial polymers are: PVA - 138%, PVC - 0.006%, PU1 - 3.1%, PU2 - 3.71%, PU3 - 20.2%. Level of contamination for physiological solution (0.9% NaCl) after contacting with the initial carbon material and CSM has been determined. 0.5 g of material was put in a glass flask, 20 ml of a physiological solution and heparin (2500 iu) as surfactant were added, then the flask was shaken during 15 min and the contamination level was then studied using the electronic scanning microscope. Analysis of the microphotos confirms that the surface of the initial material is grainy and determines the weakly attached particles, which can be separated from the surface of carbon grain during the perfusion of the liquid. The CSMs have smoother surface and non-of weakly attached particles. The sorption ability of the initial carbon material and CSM was estimated using the methylene blue (319 a.u.m.) relates to a class of low molecular substances (100-500 a.u.m.), and vitamin B12 (1355 a.u.m.) presented a class of "medium" (500-5000 a.u.m.) molecules. The influence of the polymer immobilization of the active carbon on sorption-kinetic characteristics of sorbents was estimated by sorption-kinetic investigation in static conditions [5]. Comparing the coefficients of initial activity for studied materials, we calculated that immobilization by polymers leads to negligible decreasing of the sorption ability of both the

Y. Savelyev et al. / Composite Materials

246

"medium" and low molecular substances. However for the CSM with 5-7% polymer content these coefficients were decreased in 1.2 - 1.5 times. The analysis of results obtained for hard CSM showed the practically identical tendency of the pores frame parameters Ws and Ssp to diminution with the increase of the polymer content in CSM. Up to 10% of polymer content the changes in pores frame parameters have a linear dependence. The losses of sorption pores volume and specific surface area were estimated as polymer content in composite. At more than 10% of polymer content in CSM the significant decreasing of pores volume values is observed. According to conducted investigation the composite components are not inert to each other. So the character of pores volume value changes may be described by rectilinear low (compare with [6]):

where x - is the mass part of polymer in the mixture. The almost rectilinear site of Ws dependence in composite on polymer content may be expressed as follows:

In other words, the presence of polymer in CSM inactivates a carbon component by filling some part of pores. To reveal what exactly pores will be filled by polymer, a method of mercury porosimetry was used. It allows receiving a differential distribution of the pores volume on radiuses from 3 to 106 nm. The mercury porograms of the initial carbon and based composites with polymer content of 0.5 and 2 % are presented in Fig. 44 Quantitative data of obtained results are given in Table 38. Table 38. Composition and properties of CSM Example 1 2 3 4 5 6 7 8 9 10

Polymer content in CSM, % mass. 5 2 1 7 5 3 0.5 6.5 0.5 0.2

Ws, cm3/g

Ssp, m2/g

Dustiness, %

0.59 0.58 0.55 0.53 0.53 0.55 0.65 0.60 0.64 0.66

702 815 1063 437 830 977 1088 795 1080 1100

absence 0.1 0.15 absence 0.1 0.1 0.2 0.1 0.05 0.05

As is obvious, the changes in porous structure of carbon material took place in area of macro- and mesopores: their volumes and specific surfaces were decreased in 1.5-2 times at the low polymer content (about 2%) in composite. The Ws and Ssp indexes practically were not changed in this region. Tendency of augmentation of medial radius of macropores and decreasing of medial radius mesopores was also observed. It may be conclude, that increase of polymer content in CSM leads to a progressing degradation of transport system of active carbon with further retardation of kinetic processes of adsorption.

247


4.2. The "Elastic" type CSM Table 39. Composition and properties of the elastic CSM Example 1 2 3 4 5 6 7 8 9 10

Contents of components in the CSM (% mass.) carbon/polymer 23/77 33/67 41/59 47/53 50/50 55/45 60/40 23/77 33/67 55/45

Ws, cm3/g

S(sp.),m2/g

Dustiness,%

0.55 0.57 0.58 0.62 0.64 0.66 0.48 0.57 0.60 0.66

1086 1082 1065 1065 1080 1100 815 1006 1080 1100

absent absent absent absent absent absent absent absent absent absent

The analysis of obtained data shows, that parameters of porous frame (Ws and S sp ) of elastic CSM depend on the ratio of carbon and polymer in composite (%). At the carbon content more than 50%, CSM lose elasticity because of "extinguishing" the foam by carbon component. The optimum ratios of polymer-active carbon (dust) are established to be from 3050% to 70-50%. The Ws and Ssp values for CSM at these ratios a little differ from the same indexes of initial carbon and have practically identical tendency to change. Mercurial porograms CSM-1, CSM-2 (with the 33% content of a carbon material) and initial KAU demonstrate that the porous carbonic frame did not undergo essential changes. Estimating the effect of the filler on structure and properties of linked polyurethane's one should take into account the influence of the solid surface on chemical bonds network formation. Sorption effectiveness of the obtained materials seems to be achieved due to the formation of the network structure. It will allow finding a wide application for these materials in future. 4.3. Giving bactericidal activity to PU matrixes The compounds having biological activity interact with receptor by the certain part (biophore), mainly owing to formation of rather weak non-valent bonds. Among these interactions the main role Van-der-Vaals forces are acting. Alongside with its the large role play hydrogen bonds. Particularly, it is necessary to note the forces of the electrostatic interaction. The heteroatoms (N, O, S, P), heterocycles, groups -C=C-, aromatic structures as a whole can enter into composition of biophore. And if for monomeric compounds above mentioned is the determining factor in ensuring to it of the biological activity, for polymers it is necessary to take into account one more factor. The polymeric matrix, on the one hand, can promote availability of biophores, and with another - to block its. The structural modification of polyurethane's allows to adjust them supramolecular organization, latter allows to adjust both availability, and place of biophore's localization. Incorporation of the (macro)heterocyclic fragments into polyurethane macromolecule is one of the way to obtain of polymers with biological activity. (Macro)heterocyclic compounds are perspective agents from this point of view to formation of relatively weak non-valent bonds, which are answered the definite active centers of these compounds.

248


Linear polyurethane's of the general structure: H-[- CE - 1C - PE - 1C -]-EG were synthesized, where:

1C: aromatic or aliphatic diisocyanates; PE : oligoethers or oligoesters; Polyurethane foams were synthesized based on compounds I and II, oligoether and oligoester, aromatic diisocyanate and activators of the urethane formation process. • Linear polymers based on sulfonyl hydrazide of the crown ethers (I, HI) possessed of high fungicidal and bacteriostatic activity. • Introduction of quinoxaline (II) and thiazol (IV) derivatives into polyurethane macrochain intensify of polymer bactericidal activity. • Polyurethane foam based on compounds I, II are possessed of high biocompatibility and bactericidal activity. Bactericidal action of the polymers was studied as regards to Staphylococcus aureus, Pseudomonas aeruginisa, Bac. Subtilis, E. coli and some others. The polymers are possessed by bactericidal activity of prolonged action (at least 4 years) as regards to the microorganisms under study. Histotoxicity of the polymers were investigated by the tissue culture method. Histotoxic indexes of the studied polymers were in the range of 0.76-0.84, thus toxic action of the polymers on the tissue culture cells is absent. Haemolitic action of the polymers was studied. The polymers had been undergoing of the haemolysis on 0.9%, e.g. these polymers does not contain the haemolitic active compounds. Fungicidal activity of the polymers were investigated as regards to some kinds micromicetes as test-organisms: Aspergillus terreus Thom, Chaetomium varioti Bainier, Penicillium funiculosum Thom, Penicillium chrysogenum Thom, Penicillium cyclopium Westling, Trichoderma viride Pers. ex Fr.


249

5. Conclusion The new composite sorbents of two types, hard and elastic, were developed. It was shown that decreasing of adsorption properties of the initial carbon material is less than 7% at the polymer content in hard composite of 1-5%. The optimum ratio of polymer-active carbon (dust) in elastic composite is 30-50% to 70-50%. The main factor determined the high effectiveness of polyurethane application is the microphase separation in polymer and formation of hard and soft block regions. The association of hard blocks causes the motion of carbon to the interdomain area (region of the soft block) with subsequent redistribution of intermolecular bonds at the expense of screening the polymer polar groups by the active carbon and prevention of their interaction. The interaction between Ti-electrons of active carbon and urethane carbonyl group may be occurred. The primary factor of differing the sorption is the difference of the chain extender structures. Sorption efficiency of the elastic composite materials was achieved due to the formation of the network structure. For blockcomposite sorption materials the changes in porous frame of a carbon material took place in area of macro- and mesopores: their volumes and specific surfaces were decreased in 1.5-2 times at the small contents (about 2%) of polymer in a composite, where such indexes as Ws and Ssp practically did not change. Parameters of porous structure for elastic composites are not differed essentially from initial carbon at optimal "active carbon-polymer" ratio and have the same trend to change. The carried out researches have shown that introduction of various biological active compounds (biophores) in the PU matrixes during their synthesis open effective way to prepare both polymers and composites on their base with active carbons possessing definite bactericidal properties against pathogenic microorganisms and fungi.

References [1] [2] [3] [4]

[5] [6]

Lipatov, Yu.S. (1991) Physico-chemical bases of filling of polymers, Khimiya, Moscow. Savelyev, Yu.V. (2000) Polyurethane-urea ionomers based on macroheterocyclic polymers, Polymer & Polymer Composites, vol. 8, Nl, pp. 27-30. Savelyev, Yu.V., Veselov, V.Ya., and Grekov, A.P. (1991) Linear polymers based on hydrazine, in Polymer Films and Coverings", Leningrad, pp. 108-109. Savelyev, Yu.V., Veselov, V.Ya., and Grekov, A.P. (2000) Investigations of the reactions of 1,1dimethyl hydrazine with alkylhaloids and epoxy compounds, Ukr. Chem. J. (Russ), vol. 66, N12, pp. 110-114. Marushko, S.Z., Kartel, N.T., and Savelyev, Yu.V. (1996) Composite sorbents "Active CarbonePolymer" and their characteristics", in CARBON'96: Intern. Conf on Carbon (Newcastle), vol. 2, pp. 463-464. Fedorov, N.F., Ivakhnyuk, G.K., and Babkin, O.E. (1995) Modern problems of the theory of adsorption, in VIIIntern.Conf. "Theoretic Adsorption Aspects", Moscow, PAIMS, vol. 1, pp. 88-96.


251

List of Tables and Figures Table 1. Table 2. Table 3. Table 4. TableS. Table 6. Table7. Table 8. Table 9. Table 10. Table Table Table Table Table Table Table

11. 12. 13. 14. 15. 16. 17.

Table 18. Table 19. Table 20. Table 21. Table 22. Table 23. Table 24. Table 25. Table 26. Table 27. Table 28. Table 29. Table 30. Table 31. Table 32. Table 33.

Priorities for CDC's global infectious disease strategy, 2001-2002 Critical biological agents for public health preparedness List of infectious agents, adopted by Bulgarian Army Medical Service as possible biological agents Basic terms definitions Infectious diseases morbidity in the country ORM Threat Matrix Infected Personnel by Country Affected U.S. Conferees by German State Stages of Smallpox Infection (U.S. Army Office of the Surgeon General) Comparison between a conventional HAZMAT (HMI) incident and one involving a Weapon of Mass Destruction (WMD) Human Susceptibility to Brucella spp Examples of DoD Electronic Medical Surveillance Systems Components of JBAIDS Select Block I and II Agents Mass units associated with various bacterial spores and treatments Operational characteristics of three in vitro hybridoma culture methods Comparison of the three different cell culture methods examined in this study Inactivation of coliphages in water supply in dependence on time duration of GDP effect Inactivation of coliphages in drinking water depending on duration of GDP affect Dynamics of inactivation of the polio virus in drinking water under GDP influence Inactivation of coliphages in waste water in dependence on duration of GDP influence Dynamics of inactivation of the polio virus in the filtrate of native waste water under GDP influence Decontamination of water contaminated with S.aureus and Micobacterias under GDP influence Decontamination of water containing of polio virus under GDP influence Comparative sorption-bactericidal efficiency of carbon metal-containing adsorbents The structural - sorption characteristics of carbon materials Sorption ability of carbon materials to Cholera vibrion Results of laboratory tests of filtering elements in FFF (fine fibre filters) The characteristics of pathogenic microorganisms Change of microflora in drinking water and on FM after 10-15 days of operation The contents of microorganisms (col/ml) in filtrate The characteristics of the investigated samples The data on overgrowing samples of FM by microorganisms

19 27 48 48 49 95 115 116 118 148 163 181 187 187 193 200 200 219 219 220 220 221 221 221 229 230 230 234 235 235 236 237 237

252

Table 34. Table 35. Table 36. Table 37. Table 38. Table 39. Figure 1. Figure 2. Figure 3.

Figure 4. Figure 5. Figure 6. Figure 7. Figure 8. Figure 9. Figure 10. Figure 11 a. Figure lib. Figure lie. Figure lid. Figure 12. Figure 13. Figure 14. Figure 15 Figure 16. Figure 17. Figure 18. Figure 19. Figure 20. Figure 21. Figure 22. Figure 23. Figure 24. Figure 25. Figure 26. Figure 27. Figure 28. Figure 29. Figure 30. Figure 31. Figure 32.

Influence of the POM additives on microstructure of extrudates from mixes PP/PA of ratio 40/60 The features of the starting reagents Some features of foam polyurethane's Data of the specific surface area for sorbents Composition and properties of CSM Composition and properties of the elastic CSM

237 240 242 244 246 247

Microarray Technology 14 Epidemiology and Laboratory Capacity (ELC) program, fiscal years 1995 through 2000 20 Selected examples of how ELC program resources were used by states and large public health agencies to improve infectious disease surveillance and outbreak response 21 Estimated impact of PulseNet if the 1993 multistate food-borne outbreak of Escherichia coli O157:H7 had occurred in 1998 22 Conceptual diagram of the Laboratory Response Network 29 Algorithm developed for the rapid rule-out of Bacillus anthracis by clinical laboratories. 30 Environmental and clinical specimens processed by LRN laboratories 34 The Risk Management Paradigm 101 Conference Attendees by City in Germany 116 Smallpox Presentation and Progression over Time (SBCCOM) 118 1 st Generation Caseload 119 2nd Generation Caseload 120 3rd Generation Caseload 120 4th Generation Caseload 121 Infected Caseload - First 60 days for Baden-Wiirttemberg 121 Infected Caseload - First 60 days for Germany 122 Infected Caseload - First 60 days for Area of Responsibility 122 Effectiveness of Ring Vaccine (Baden- Wiirttemberg) 123 Effectiveness of Ring Vaccine (Germany) 124 Effectiveness of Ring Vaccine (Area of Responsibility) 124 Total Patient Caseload (Baden-Wiirttemberg) 125 Total Patient Caseload (Germany) 125 Total Patient Caseload (Area of Responsibility) 126 Projected Fatalities 126 Treatment Network (Baden-Wiirttemberg) 127 Treatment Network (Germany) 128 Treatment Network (Area of Responsibility) 128 Medical Personnel Requirements (Baden-Wiirttemberg) 129 Medical Personnel Requirements (Germany) 129 Medical Personnel Requirements (Area of Responsibility) 130 Key elements of WMD preparedness 153 Elements of a successful WMD Training Program. The arrows on the slide show the WMD prerequisite training classes 154 Relationship between Crisis Management and Consequence Management 155 Steps in an effective planning process 156 Effective elements of a WMD response - Plan, Train, Exercise, and Revise 157

253

Figure 33. FACS analysis showing specific binding of fluorescently-labeled mAbs to B. anthracis spores Figure 34. Schematic of antibody displaying filamentous phage particle Figure 35. ELISA detection of (A) MS2 virions and (B) recombinant MS2 coat protein using purified anti-MS2 Fab antibody Figure 36. Rep-PCR fingerprint of different Bacilli Figure 37. Method of protein identification using the Mascot search engine Figure 38. Super-imposed average gels non-induced versus induced Figure.39. Pilot plasmachemical plant Figure 40. The influence of ratio Solid: Liquid on the adsorption of the trypsine by carbon fabric Figure 41. Scheme of ultrathin fibres formation and SEM microphotos of obtained product Figure 42. Microfibrillar structure of ultrathin polypropylene fibres Figure 43. Dependence of the pores volume on benzene of the CSM and of the active carbon vs the polymer content in the CSM Figure 44. Data of the mercury porosimetry investigations for the CSM and KAU volume pore distribution on equivalent radii

192 196 197 208 210 213 224 231 232 233 243 244


255

Author Index AganB. Ahrens, J. T. Anderson, P.E. Asokliene, L. Bartoszcze, M. Bellenkes, A. H. Berencsi, G. Bray, D. Chomiczewski, K. Cosivi, O. Csohan, A. DelVecchio, V.G. Donlon, M.A. Elzer, P.H. Faludi, G. Finke, E. Fock, R. Grigoriev, A. Hanson, E. Kapustin, A Kartel, N. Kellogg, R. Khan, A.S. Kim, M. H. Kriz,B. Kutateladze, M. Kyncl, J. Lukens, D.C. Mayboroda, M. Meyer, R.F. Miller, M.J. Morse, S.A. Negut, M.

12 114 195 66 170 89 60 26 73 9 60 207 191 161 60 58 58 226 12 76 226,232,239 26 195 195 44 54 44 195 232 26 26 17,26 137

Netesov, S. Nichelson, D. Niedrig, M. Niemeyer, D. Niemeyer, D.M. Niemeyer, Debra M. O'Connell, K.P. Oyston, P.C.F. Park, J.T. Pavlin, J.A. Penkov, E. Perry, S.R. Pivovarov, A.A. Plochev, K. Price, R. Quinn-Doggett, K. Rezanova, V. Robota, L. Roweley, R. Savelyev, Yu. Savelyeva, O. Shvets, D. Stopa, P. J. Strelko, V. Thompson, R.G. Tibbetts, C. Tsebrenko, I. Tsebrenko, M. Twardowski, T. Valdes, J.J. Vigus, R.A. Woodall, J.

105 26 37,58 12 179 185 195 164 195 3 47 26 215 47 105 147 232 239 12 239 239 226 147 226,239 195 12 232 232 132 195 147 105,111

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