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Diseases: The Mouse in Biomedical Research
Diseases: The Mouse in Biomedical Research
Diseases: The Mouse in Biomedical Research
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Diseases: The Mouse in Biomedical Research

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The Mouse in Biomedical Research, Volume II: Diseases is a compilation of papers detailing infectious diseases of the mouse. This compilation deals with bacterial, mycotic, viral, protozoal, rickettsial, parasitic, non-neoplastic, and metabolic diseases of the mouse. Several papers describe the different diseases found in the digestive, respiratory, urogenital, integumentary, central nervous, lymphoreticular, musculoskeletal, cardiovascular, and endocrine systems of the mouse. This book lists the possible bacterial infections, as well as other miscellaneous infections such as those caused by aerosols, particles, and the air in the laboratory environment. This text also lists viruses that can affect the mouse such as the lactate dehydrogenase-elevating virus, mouse pox, polyomavirus, and the minute virus. This book describes the process of identification, diagnosis, epidemiology, treatment, control, prevention, and occurrence of these diseases. This text also reviews the diseases that can be transmitted from infected mice to humans, as well as through animal bites and allergic reactions. This book is suitable for researchers, clinical assistants, and scientists dealing with laboratory animals, particularly with mice as test animals. This book can also be helpful for veterinarians and doctors of infectious diseases transferred from animals.
LanguageEnglish
Release dateApr 22, 2014
ISBN9781483269139
Diseases: The Mouse in Biomedical Research

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    Diseases - Henry L. Foster

    Diseases

    The Mouse in Biomedical Research

    Henry L. Foster

    The Charles River Laboratories, Inc., Wilmington, Massachusetts

    J. David Small

    Veterinary Resources Branch, Small Animal Section, National Institutes of Health, Bethesda, Maryland

    James G. Fox

    Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, Massachusetts

    Table of Contents

    Cover image

    Title page

    AMERICAN COLLEGE OF LABORATORY ANIMAL MEDICINE SERIES

    Copyright

    List of Contributors

    Preface

    Introduction

    List of Reviewers for Chapters in This Volume

    Chapter 1: Bacterial and Mycotic Diseases of the Digestive System

    Publisher Summary

    I INTRODUCTION

    II BACTERIAL INFECTIONS

    III MYCOTIC INFECTIONS

    IV SUMMARY AND CONCLUDING REMARKS

    Chapter 2: Mycoplasmal and Other Bacterial Diseases of the Respiratory System

    Publisher Summary

    I INTRODUCTION

    II MURINE RESPIRATORY MYCOPLASMOSIS (MRM)

    III KLEBSIELLOSIS

    IV PASTEURELLOSIS

    V CHLAMYDIAL PNEUMONITIS

    VI CORYNEBACTERIOSIS

    VII MISCELLANEOUS INFECTIONS

    VIII CONCLUSIONS

    ACKNOWLEDGMENTS

    Chapter 3: Bacterial, Mycoplasmal, Mycotic, and Immune-Mediated Diseases of the Urogenital System

    Publisher Summary

    I INTRODUCTION

    II BACTERIAL DISEASES

    III MYCOPLASMAL INFECTIONS

    IV MYCOTIC INFECTIONS

    V GLOMERULONEPHRITIS

    Chapter 4: Bacterial and Mycotic Diseases of the Integumentary System

    Publisher Summary

    I INTRODUCTION

    II HAIRY INTEGUMENT

    III BACTERIAL DISEASES

    IV MYCOTIC DISEASES

    V NONINFECTIOUS SKIN DISORDERS

    ACKNOWLEDGMENTS

    Chapter 5: Bacterial, Mycoplasmal, and Mycotic Diseases of the Central Nervous System

    Publisher Summary

    I INTRODUCTION

    II MYCOPLASMA NEUROLYTICUM

    III MYCOPLASMA PULMONIS

    IV PSEUDOMONAS AERUGINOSA

    V CORYNEBACTERIUM KUTSCHERI

    VI PASTEURELLA PNEUMOTROPICA

    VII MYCOSES

    Chapter 6: Bacterial, Mycoplasmal, and Mycotic Diseases of the Lymphoreticular, Musculoskeletal, Cardiovascular, and Endocrine Systems

    Publisher Summary

    I INTRODUCTION

    II BACTERIAL INFECTIONS

    III MYCOPLASMAL INFECTIONS

    IV MYCOTIC INFECTIONS

    Chapter 7: Rickettsial and Chlamydial Diseases

    Publisher Summary

    I INTRODUCTION

    II RICKETTSIAL INFECTIONS

    III CHLAMYDIAL INFECTIONS

    Chapter 8: Viral Diseases of the Respiratory System

    Publisher Summary

    I INTRODUCTION

    II SENDAI VIRUS

    III PNEUMONIA VIRUS OF MICE (PVM)

    IV K VIRUS

    ADDENDUM

    Chapter 9: Viral Diseases of the Digestive System

    Publisher Summary

    I INTRODUCTION

    II EPIZOOTIC (EPIDEMIC) DIARRHEA OF INFANT MICE (EDIM), MOUSE ROTAVIRUS ENTERITIS

    III REOVIRUS 3 INFECTION (HEPATOENCEPHALOMYELITIS, ECHO 10 VIRUS INFECTION)

    IV MURINE (MOUSE) HEPATITIS VIRUS INFECTION (MHV)

    Chapter 10: Lactate Dehydrogenase-Elevating Virus

    Publisher Summary

    I INTRODUCTION

    II CHARACTERISTICS OF INFECTION

    III METHODS FOR DETECTING THE PRESENCE OF LDV

    IV REPLICATION IN TISSUE CULTURE

    V PROPERTIES OF THE VIRUS

    VI CONCLUSION

    ACKNOWLEDGMENT

    Chapter 11: Mousepox

    Publisher Summary

    I INTRODUCTION

    II VIRAL AGENT

    III SUMMARY: THE PRACTICAL PROBLEMS

    Chapter 12: Lymphocytic Choriomeningitis Virus

    Publisher Summary

    I INTRODUCTION

    II DEFINITIONS

    III PROPERTIES OF THE AGENT

    IV INFECTION OF MICE

    V EPIZOOTIOLOGY

    VI DIAGNOSIS

    VII CONTROL AND PREVENTION

    ACKNOWLEDGMENTS

    Chapter 13: Cytomegalovirus and Other Herpesviruses

    Publisher Summary

    I INTRODUCTION

    II MURINE CYTOMEGALOVIRUS

    III MOUSE THYMIC VIRUS

    Chapter 14: Polyomavirus

    Publisher Summary

    I INTRODUCTION

    II HISTORY

    III RELATED VIRUSES

    IV THE VIRUS PARTICLE

    V RESISTANCE OF THE VIRUS OR ITS DNA TO PHYSICAL AND CHEMICAL AGENTS

    VI CULTIVATION

    VII INFECTION OF MICE AND OTHER ANIMALS

    VIII SPREAD OF POLYOMAVIRUS AND METHODS FOR ITS PREVENTION

    IX CONCLUSIONS

    Chapter 15: Minute Virus of Mice

    Publisher Summary

    I INTRODUCTION

    II BIOLOGY OF MVM

    III STRUCTURE AND REPLICATION OF MVM

    Chapter 16: Mouse Adenovirus

    Publisher Summary

    I INTRODUCTION

    II ISOLATIONS OF MOUSE ADENOVIRUSES AND PATHOLOGICAL EFFECTS IN MICE

    III PHYSICAL CHARACTERISTICS

    IV SEROLOGICAL REACTIONS

    V EPIDEMIOLOGY AND PREVALENCE

    ACKNOWLEDGMENT

    Chapter 17: Mouse Encephalomyelitis Virus

    Publisher Summary

    I INTRODUCTION

    II VIRAL AGENT

    Chapter 18: Encephalomyocarditis Virus

    Publisher Summary

    I INTRODUCTION

    II HISTORY

    III ETIOLOGY

    IV EPIDEMIOLOGY

    V LABORATORY ANIMAL HOSTS

    ACKNOWLEDGMENT

    Chapter 19: Protozoa

    Publisher Summary

    I INTRODUCTION

    II PARASITES OF PARENTERAL SYSTEMS

    III PARASITES OF THE ALIMENTARY SYSTEM

    Chapter 20: Helminths

    Publisher Summary

    I INTRODUCTION

    II HELMINTHS OF MAJOR IMPORTANCE

    III HELMINTHS OF MINOR IMPORTANCE

    ACKNOWLEDGMENTS

    Chapter 21: Arthropods

    Publisher Summary

    I INTRODUCTION

    II ECTOPARASITIC ECOLOGY

    III DIAGNOSIS

    IV IDENTIFICATION

    V ARTHROPOD PARASITES OF LABORATORY MICE

    VI TREATMENT, CONTROL, AND PREVENTION

    Chapter 22: Zoonoses and Other Human Health Hazards

    Publisher Summary

    I INTRODUCTION

    II VIRAL DISEASES

    III RICKETTSIAL DISEASES

    IV BACTERIAL DISEASES

    V MYCOSES (RINGWORM)

    VI PROTOZOAN DISEASES (Entamoeba coli)

    VII HELMINTH DISEASES

    VIII ARTHROPOD INFESTATIONS

    IX BITES

    X ALLERGIC SENSITIVITIES

    XI CONCLUSION

    Chapter 23: Selected Nonneoplastic Diseases

    Publisher Summary

    I INTRODUCTION

    II OCCURRENCE

    Index

    AMERICAN COLLEGE OF LABORATORY ANIMAL MEDICINE SERIES

    Steven H. Weisbroth, Ronald E. Flatt, and Alan L. Kraus, eds.:

    The Biology of the Laboratory Rabbit, 1974

    Joseph E. Wagner and Patrick J. Manning, eds.:

    The Biology of the Guinea Pig, 1976

    Edwin J. Andrews, Billy C. Ward, and Norman H. Altman, eds.:

    Spontaneous Animal Models of Human Disease, Volume I, 1979; Volume II, 1979

    Henry J. Baker, J. Russell Lindsey, and Steven H. Weisbroth, eds.:

    The Laboratory Rat, Volume I: Biology and Diseases, 1979; Volume II: Research Applications, 1980

    Henry L. Foster, J. David Small, and James G. Fox, eds.:

    The Mouse in Biomedical Research, Volume I: History, Genetics and Wild Mice, 1981; Volume II: Diseases, 1982

    In preparation

    Henry L. Foster, J. David Small, and James G. Fox, eds.:

    The Mouse in Biomedical Research, Volume III: Husbandry

    Henry L. Foster, J. David Small, and James G. Fox, eds.:

    The Mouse in Biomedical Research, Volume IV: Experimental Biology

    Copyright

    COPYRIGHT © 1982, BY ACADEMIC PRESS, INC.

    ALL RIGHTS RESERVED.

    NO PART OF THIS PUBLICATION MAY BE REPRODUCED OR TRANSMITTED IN ANY FORM OR BY ANY MEANS, ELECTRONIC OR MECHANICAL, INCLUDING PHOTOCOPY, RECORDING, OR ANY INFORMATION STORAGE AND RETRIEVAL SYSTEM, WITHOUT PERMISSION IN WRITING FROM THE PUBLISHER.

    ACADEMIC PRESS, INC.

    111 Fifth Avenue, New York, New York 10003

    United Kingdom Edition published by

    ACADEMIC PRESS, INC. (LONDON) LTD.

    24/28 Oval Road, London NW1 7DX

    Library of Congress Cataloging in Publication Data Main entry under title:

    The Mouse in biomedical research.

    (American College of Laboratory Animal Medicine series)

    Includes index.

    Contents: v. 1. History genetics, and wild mice – v. 2. Diseases.

    1. Mice as laboratory animals. I. Foster, Henry L. II. Small, J. David. III. Fox, James G. IV. Series. [DNLM: 1. Mice. 2. Research. 3. Animals Laboratory.

    QY 60.R6 M932]

    QL737.R638M68 619′.93 80-70669

    ISBN 0-12-262502-1 (v.2) AACR2

    PRINTED IN THE UNITED STATES OF AMERICA

    82 83 84 85 9 8 7 6 5 4 3 2 1

    List of Contributors

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

    James B. Brayton(403),     Division of Comparative Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland 21205

    Margo A. Brinton(193),     The Wistar Institute, Philadelphia, Pennsylvania 19104

    J.D. Burek(425),     Toxicology Department, Health and Consumer Products, Dow Chemical Company, Indianapolis, Indiana 46268

    Harold W. Casey¹(43),     Department of Veterinary Pathology, Armed Forces Institute of Pathology, Washington, D.C. 20306

    Gail H. Cassell(21),     Departments of Comparative Medicine and of Microbiology, Schools of Medicine and Dentistry, University of Alabama in Birmingham, Birmingham, Alabama 35294

    Maureen K. Davidson(21),     Department of Comparative Medicine, Schools of Medicine and Dentistry, University of Alabama in Birmingham, and the Veterans Administration Hospital, Birmingham, Alabama 35294

    Wilbur G. Downs(341),     Yale Arbovirus Research Unit, Yale University School of Medicine, New Haven, Connecticut 06510

    Bernice E. Eddy²(293),     Experimental Virology Branch, Division of Virology, Bureau of Biologics, Food and Drug Administration, Bethesda, Maryland 20034

    Frank Fenner(209),     John Curtin School of Medical Research, Australian National University, Canberra, Australia

    Frederick G. Ferguson(83),     Laboratory Animal Resources, The Pennsylvania State University, University Park, Pennsylvania 16802

    James G. Fox(403),     Division of Comparative Medicine, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

    James R. Ganaway(1),     Comparative Pathology Section, Veterinary Resources Branch, DRS, National Institutes of Health, Bethesda, Maryland 20205

    John E. Harkness(83),     Laboratory Animal Resources, The Pennsylvania State University, University Park, Pennsylvania 16802

    Paul K. Hildebrandt(99),     Tracor Jitco, Rockville, Maryland 20852

    Chao-Kuang Hsu(359),     School of Medicine, University of Maryland, Baltimore, Maryland 21201

    George W. Irving, III³(43),     Department of Veterinary Pathology, Armed Forces Institute of Pathology, Washington, D.C. 20306

    Dennis F. Kohn(77),     Department of Comparative Medicine, University of Texas Medical School, Houston, Texas 77025

    Lisbeth M. Kraft(159),     National Aeronautics and Space Administration, Ames Research Center, Moffett Field, California 94035

    Fritz Lehmann-Grube(231),     Heinrich-Pette-Institut für Experimentelle Virologie und Immunologie and der Universität Hamburg, 2000 Hamburg 20, Federal Republic of Germany

    J. Russell Lindsey(21),     Department of Comparative Medicine, Schools of Medicine and Dentistry, University of Alabama in Birmingham, and the Veterans Administration Hospital, Birmingham, Alabama 35294

    J.A. Molello(425),     Toxicology Department, Health and Consumer Products, Dow Chemical Company, Indianapolis, Indiana 46268

    Thomas G. Murnane⁴(353),     U.S. Army Veterinary Corps, Office of the Surgeon General, Washington, D.C. 20310

    June E. Osborn(267),     Departments of Medical Microbiology and of Pediatrics, University of Wisconsin, Madison, Wisconsin 53706

    James A. Otten(335),     Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830

    John C. Parker(109),     Microbiological Associates, Bethesda, Maryland 20816

    Conrad B. Richter⁵(109),     Oak Ridge Associated Universities, Inc., Oak Ridge, Tennessee 37830

    Peter J. Tattersall(313),     Department of Human Genetics, Yale University School of Medicine, New Haven, Connecticut 06510

    Raymond W. Tennant⁶(335),     National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709

    Joseph E. Wagner(55),     College of Veterinary Medicine, University of Missouri, Columbia, Missouri 65201

    David C. Ward(313),     Department of Human Genetics, Yale University School of Medicine, New Haven, Connecticut 06510

    S.D. Warner(425),     Toxicology Department, Health and Consumer Products Department, Dow Chemical Company, Indianapolis, Indiana 46268

    Steven H. Weisbroth(385),     AnMed Laboratories, Inc., New Hyde Park, New York 11040

    Richard B. Wescott(373),     Department of Veterinary Microbiology and Pathology, Washington State University, Pullman, Washington 99164

    Cynthia Besch Williford(55),     College of Veterinary Medicine, University of Missouri, Columbia, Missouri 65201


    ¹Present address: Department of Pathology, Whittaker Toxigenics, Inc., Decatur, Illinois 62526

    ²Present address: 6722 Selkirk Court, Bethesda, Maryland 20817

    ³Present address: Directorate of Life Sciences, Air Force Office of Scientific Research, Boiling AFB, Washington, D.C. 20332.

    ⁴Present address: Apartado Postal 61-148, Mexico 6, D.F.

    ⁵Present address: Comparative Medicine Branch, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709

    ⁶Present address: Cellular and Genetic Toxicology Branch, National Toxicology Program, P.O. Box 12233, Research Triangle Park, North Carolina 27709

    Preface

    The American College of Laboratory Animal Medicine (ACLAM) was formed in 1957 in response to the need for specialists in laboratory animal medicine. The College has promoted high standards for laboratory animal medicine by providing a structure framework to achieve certification for professional competency and by stressing the need for scientific inquiry and exchange via progressive continuing education programs. The multivolume treatise, The Mouse in Biomedical Research, is a part of the College’s effort to fulfill those goals. It is one of a series of comprehensive texts on laboratory animals developed by ACLAM over the past decade: The Biology of the Laboratory Rabbit was published in 1974, The Biology of the Guinea Pig in 1976, and a two-volume work Biology of the Laboratory Rat in 1979 and 1980. Also, in 1979 the College published a two-volume text on Spontaneous Animal Models of Human Disease.

    The annual use of approximately 50 million mice worldwide attests to the importance of the mouse in experimental research. In no other species of animal has such a wealth of experimental data been utilized for scientific pursuits. Knowledge of the mouse that has been accumulated is, for the most part, scattered throughout a multitude of journals, monographs, and symposia. It has been fifteen years since the publication of the second edition of The Biology of the Laboratory Mouse edited by E. L. Green and the scientific staff of the Jackson Laboratories. It is not the intent of this work simply to update and duplicate this earlier effort, but to build upon its framework. We are indeed fortunate to have Dr. Green and many of his colleagues at the Jackson Laboratory as contributors to this treatise. It is the intended purpose of this text to assemble established scientific data emphasizing recent information on the biology and use of the laboratory mouse. Separation of the material into multiple volumes was essential because of the number of subject areas covered.

    The contents of Volume I are presented in fourteen chapters and provide information on taxonomy, nomenclature, breeding systems, and a historical perspective on the development and origins of the laboratory and wild mouse. Six chapters deal specifically with the ever-increasing diversity of inbred strains of mice, including coverage of methods of developing and the genetic monitoring and testing of these strains. The emphasis of this volume on genetics is also manifested by chapters discussing the H-2 complex, cytogenetics, radiation genetics, and pharmacogenetics.

    Because of the impact of spontaneous diseases on interpretation of, and potential for, complicating experimental research, it is of paramount importance for investigators to recognize these diseases and their effect on the mouse. Volume II, for the first time, compiles in one format a narrative detailing infectious diseases of the mouse; the chapters cover bacterial, mycotic, viral, protozoal, rickettsial, and parasitic diseases. Also, nonneoplastic and metabolic diseases are covered as well as the topic of zoonoses.

    Volume III provides comprehensive coverage of selected material related to normative biology and management and care of the laboratory mouse. Developmental, anatomical, nutritional, physiological, and biochemical parameters of the mouse are compiled in several chapters and will be of great interest and an important resource for normal biological profiles. A review of the histologic features was not included because of space constraints and the availability of this information in previous texts. Environmental monitoring and disease surveillance as well as management and design of animal facilities will be particularly useful for those individuals responsible for the management of mouse colonies. The chapters on gnotobiotics and gastrointestinal flora represent the state of the art in gnotobiology. The three chapters on selected aspects of immunology in the mouse serve to highlight the explosive progress being made in immunologic techniques and in strumentation and the underlying importance of genetic differentiation.

    The fourth volume includes selected applications of the mouse in research. Several chapters discuss the use of the mouse in infectious disease research, while others range from eye research to the use of the mouse in experimental embryology. The chapters devoted to the use of the mouse in oncological research follow a body system format. Research topics in other disciplines have not been included, but hopefully will be included in future editions.

    This treatise was conceived with the intent to offer information suitable to a wide cross section of the scientific community. It is hoped that it will serve as a standard reference source. Students embarking on scientific careers will benefit from the broad coverage of material presented in compendia format. Certainly, specialists in laboratory animal science will benefit from these volumes; technicians in both animal care and research will find topics on surgical techniques, management, and environmental monitoring of particular value.

    The editors wish to extend special appreciation to the contributors to these volumes. Authors were selected because of knowledge and expertise in their respective fields. Each individual contributed his or her time, expertise, and considerable effort to compile this resource treatise. In addition, the contributors and editors of this book, as with all volumes of the ACLAM series texts, have donated publication royalties to the American College of Laboratory Animal Medicine for the purpose of continuing education in laboratory animal science. This book could not have been completed without the full support and resources of the editors’ parent institutions which allowed time and freedom to assemble this text. A special thanks is also extended to the numerous reviewers of the edited work whose suggestions helped the authors and editors present the material in a meaningful and concise manner. We acknowledge and thank Rosanne Brown and Sara Spanos for their secretarial assistance. Also, the assistance provided to us by the staff of Academic Press was greatly appreciated.

    Finally, we especially acknowledge with deep appreciation the editorial assistance of Patricia Bergenheim, whose dedication and tireless commitment to this project were of immeasurable benefit to the editors in the completion of this text.

    Henry L. Foster, J. David Small and James G. Fox

    Introduction

    Although the past several decades have seen a dramatic change in the infectious disease experience of laboratory mice, with marked improvement in animal health and quality of research results, a detailed compilation of murine infectious diseases will be of great value to users of laboratory mice. With regard to my own experience in virus research, thirty years ago it was not unusual to receive shipments from commercial sources of Swiss mice decimated by Salmonella typhimurium. The impact of such shipments on an experimental program and on one’s own mouse stocks is easily envisioned. Ten years later Salmonella was no longer a problem, but when we began serologic screening of mouse colonies for indigenous viruses we found that some colonies, both commercial and institutional, contained virtually every known mouse virus while other colonies were free of almost all such agents. Not surprisingly, housing of animals from such diverse sources in common rooms led to serious morbidity in the clean stocks, particularly among infant animals. Contaminated animals have also been a serious source of introduction of extraneous agents into mouse-passage biological materials. Passenger agents in transplanted tumors and virus stocks, once accepted as an unavoidable component of complex in vivo systems, have become increasingly disruptive and hence unacceptable as studies of tumor biology have become more subtle and precise. Another unsavory by-product of using infested mice was the mistaken identification of a mouse virus, acquired during passage of a specimen through mouse tissues in vivo or even in vitro, as the etiologic agent of the disease being studied. The literature of the 1940s and 1950s is replete with such mistaken claims.

    In recent years subclinical infections with Sendai and minute viruses have been found to disrupt sensitive assays for various parameters of immune functions, and thus constitute serious threats to immunological research programs.

    Several events combined to bring about the vastly improved infectious disease experience with today’s mice. Most important was the development and application of techniques for deriving stocks by cesarean section and rearing them in strictly maintained quarantine. This technology was made possible by the husbandry techniques and barrier procedures developed by the pioneers of germfree techniques, and much credit is due to them as well as to the few farsighted commercial mouse breeders who led the way in their application. A second important factor was the development and widespread application of serologic monitoring procedures for indigenous agents, combined with increased knowledge of their natural history. Definition of those agents present in a mouse colony is in many respects more important than elimination of every agent of minimal pathogenicity. Further, awareness of the problems and the wide availability of clean, defined mice have contributed greatly to improving the quality and standards of laboratory mice.

    It is of the greatest importance to recognize that elimination of agents from mouse colonies increases, rather than reduces, the need to be informed and aware of the agents. In their natural occurrence in breeding colonies, most viruses of mice cause little disease; they infect young animals passively protected by maternal antibody, producing subclinical infections that provide immune protection for the infected animal as well as its offspring. In the absence of indigenous immunizing infection, a number of agents can induce epizootic disease with high morbidity and mortality rates. Our clean colonies are thus highly vulnerable, particularly to ectromelia, hepatitis, and Sendai virus infections. Awareness, quarantine procedures, monitoring programs, and knowledge of the natural biology of indigenous murine agents will long be required of all users of mice.

    Another area in which knowledge of indigenous infections of mice is of major importance to infectious disease research is in their value as model systems that represent naturally occurring host-parasite relationships. Antimicrobial defense mechanisms, the interplay of infectious agents with the immune system, and mechanisms of pathogenesis of various infectious diseases can be studied in mouse systems with a scale and preciseness unmatched in other host systems. The importance of naturally occurring mouse infections as models is illustrated by the fact that the original discoveries of an astounding number of major virus groups were made with the members that occur in mice. Polyomaviruses (polyoma- and K viruses), coronaviruses (mouse hepatitis), rotaviruses (epidemic diarrhea of infant mice), parainfluenza viruses (Sendai), cytomegaloviruses (MSGV), B-type retroviruses (mammary tumor virus), arenaviruses (LCM), pneumovirus (PVM), and picornaviruses (Theiler’s) all fall into this category. In addition, mice carry viruses belonging to at least five other major families (C-type retrovirus, reo-, adeno-, pox-, and parvoviruses). This diversity of viral flora of the mouse provides natural models for a wide spectrum of viral diseases.

    The surgical derivation and barrier-rearing techniques have also drammatically reduced bacterial and parasitic infections. However, they remain important problems, particularly in conventional colonies containing large numbers of mice from mixed sources. Mycoplasmal infection remains a common, serious health problem, and hyperplastic colitis due to Citrobacter freundii can also seriously threaten a colony. Pinworms and ectoparasites are by no means a thing of the past.

    The detailed information on infectious agents of the mouse which has been assembled in this volume is a unique compilation that should be of much use in dealing with the practical problems of animal care and in exploiting the rich opportunities that mouse models offer for the study of the biology of infectious disease.

    Retroviruses—the murine leukemia viruses, mammary tumor viruses, and A particles—are among the major model systems in viral carcinogenesis. The complexity and experimental and biochemical detail of these agents would require a book of their own. Several review books on this subject have appeared in the past few years and another major volume is in preparation.

    Wallace P. Rowe,     Laboratory of Viral Diseases, National Institute of Allergy and Infectious Diseases, Bethesda, Maryland 20014

    List of Reviewers for Chapters in This Volume

    Henry J. Baker,     University of Alabama

    Stephen W. Barthold,     Yale University School of Medicine

    Patricia C. Brennan,     Argonne National Laboratory

    J. Roger Broderson,     Center for Disease Control

    Gail H. Cassell,     University of Alabama

    Gerald A. Cole,     The Johns Hopkins University

    John E. Craighead,     University of Vermont

    Clyde J. Dawe,     National Institutes of Health

    Robert J. Flynn,     Argonne National Laboratory

    James R. Ganaway,     National Institutes of Health

    F.M. Garner,     Rockville, Maryland

    M. David Hoggan,     National Institutes of Health

    Carel F. Hollander,     Institute for Experimental Gerontology TNO

    John Hotchin,     Department of Health, State of New York

    Lawrence Kilham,     Dartmouth Medical School

    Lisbeth M. Kraft,     National Aeronautics and Space Administration

    Norman D. Levine,     University of Illinois

    Howard Lipton,     Northwestern University

    Leonard Marcus,     State Diagnostic Laboratory, Massachusetts

    Donald N. Medearis,     Massachusetts General Hospital

    George Migaki,     Armed Forces Institute of Pathology

    Cedric A. Mims,     Guy’s Hospital Medical School

    Abner L. Notkins,     National Institutes of Health

    John C. Parker,     Microbiological Associates, Inc.

    Peter G.W. Plagemann,     University of Minnesota

    Fred R. Quimby,     Cornell University Medical College

    Conrad B. Richter,     Oak Ridge Associated Universities

    David M. Robinson,     Walter Reed Army Institute of Research

    Wallace Rowe,     National Institutes of Health

    Joseph G. Tully,     National Institutes of Health

    Steven H. Weisbroth,     AnMed Laboratories

    G.N. Woode,     Iowa State University

    Conrad E. Yunker,     National Institutes of Health

    Chapter 1

    Bacterial and Mycotic Diseases of the Digestive System

    James R. Ganaway

    Publisher Summary

    This chapter discusses the various bacterial infections affecting the digestive system of mice: Salmonella, Bacillus, Citrobacter, Pseudomonas, Escherichia, Clostridium, and Klebsiella. Salmonellosis of mice is one of the most studied diseases of animals. Spontaneous salmonellosis in mice has been a universal problem in conventionally maintained mouse production colonies. The disease in mice has been studied experimentally because it is an excellent model of a similar disease in man—typhoid fever. The chapter presents the various factors that influence the induction and course of naturally occurring and experimentally induced Salmonella infection in mice. The disease caused by the Bacillus infection—Tyzzer’s disease—results from a number of factors such as overcrowding, high temperature and humidity, feeding moist food, immunosuppressive therapy, and corticosteroid drugs. Knowledge of the spontaneous or naturally occurring diseases of laboratory animals is essential primarily because of the need to assess the impact of the intercurrent disease during the conduct of biomedical research. Such knowledge is also valuable from a comparative medicine standpoint in providing animal models for studying similar disease conditions of man and other animals.

    I. Introduction

    II. Bacterial Infections

    A. Salmonella

    B. Bacillus

    C. Citrobacter

    D. Pseudomonas

    E. Escherichia

    F. Clostridium

    G. Klebsiella

    III. Mycotic Infections

    IV. Summary and Concluding Remarks

    References

    Related References

    I INTRODUCTION

    The determination of which microbial agents to include under the broad subject Bacterial and mycotic diseases of the digestive tract of the mouse was not easily resolved. There are probably very few microbial pathogens of man or lower animals that colonize and cause pathology in the digestive tract only. Furthermore, it appears untenable to consider possible portal of entry as the criterion because the gastrointestinal tract is exposed to an infinite variety of microorganisms. Indeed, the microbiology of the gastrointestinal tract of conventional animals is extremely complex, so much so that its true character is yet to be determined. The present coverage, with the concurrence of the Editorial Committee, is an attempt to include recognized pathogens which are known to colonize the gastrointestinal tract of mice.

    The biological characterization and methods for identification of recognized microbial agents are not necessarily included, as these can be readily found in numerous textbooks and Bergey’s Manual of Determinative Bacteriology. Only selected references are cited in the text. A list of related references is also included.

    It is hoped that constructive and critical review will impress the reader not with how much we know, but with how little we know. New findings will help to fill this void and provide a basis for correction of existing interpretations.

    II BACTERIAL INFECTIONS

    A Salmonella

    Salmonellosis of mice is one of the most studied diseases of animals. Historically, the mouse has been used in greater numbers than any other laboratory animal, due, in part, to the comparative low cost and the ease of production and maintenance in the laboratory. Spontaneous salmonellosis in mice has been a universal problem in conventionally maintained mouse production colonies. The disease in mice has been studied experimentally because it is an excellent model of a similar disease in man, typhoid fever. Consequently, the pathogenesis and mechanism of acquired immunity have received much attention. The mouse is also used for testing the potency of vaccines destined for use in the protection of man against typhoid fever. Indeed, this enteric and progressive systemic disease in mice is seemingly so well understood that it has been used as a model to study herd immunity (Greenwood et al., 1931) and the effects of nutrition (Schneider, 1956) and heredity (Gowen, 1948) upon resistance to disease.

    The increasing sophistication of biomedical research has stimulated a demand for mice (and other laboratory animals) that are free of pathogenic microbiota. This is because latent or subclinical infections are often activated by experimental stresses, such as irradiation, and by the administration of corticosteroid and cytotoxic drugs and anti-lymphocyte serum. Intercurrent disease can complicate or nullify the results of experiments. Because of the recognized futility of eradicating Salmonella spp. (as well as a host of other agents) from conventionally maintained populations of infected mice (discussed in more detail later), a new approach was clearly needed. The cesarean derivation and production of mice under strict barrier-maintained conditions is a fairly recent and highly successful means of providing mice free of most indigenous pathogenic microbiota, including salmonellas. The disease-free state of such mice can be extended throughout the conduct of research, but only if adequate protection of the environment is maintained so as to exclude the introduction of contaminants. One must also constantly be aware of that ever-present source, the human factor. This is especially true with a zoonotic disease such as salmonellosis. The success of the cesarean-derived, barrier-maintained mouse colony is so widely acclaimed and the technology so universally practiced that there is a tendency to suggest that salmonellosis of mice no longer poses a problem. This suggestion gains favor when one considers the fact that a ready and reliable means of detection of the infection is available, and reports of spontaneous salmonellosis in mice have diminished in recent years. On the other hand, it is unrealistic to rely upon the frequency of reported occurrences in the literature as a basis for the assessment of the prevalence of a disease so common and well understood as salmonellosis. Speculation aside, the prevalence of salmonellosis in mice used in biomedical research in the United States today remains unknown. Certainly, the salmonellas are ubiquitous in nature and are encountered in research laboratories, where many species of animals, many of which are potential carriers (Fox and Beaucage, 1979), are maintained under conventional colony conditions. The only means of making this determination is to monitor for the presence of Salmonella spp. by isolation and identification in the laboratory.

    The classification of Salmonella is complex and, scientifically, the present methods of nomenclature are unsatisfactory (LeMinor and Rohde, 1974). There are approximately 1600 recognized serotypes (Smith, 1977). A recent recommendation (Edwards and Ewing, 1972) would reduce the number of recognized species to three: Salmonella cholerae-suis, S. typhi, and S. enteritidis. All other previously recognized species are designated as serotypes of S. enteritidis. Accordingly, the most commonly isolated salmonella from mice is S. enteritidis ser Typhimurium (Edwards et al., 1948); other reported serotypes are Poona (Franklin and Richter, 1968), Paratyphi A, Infantis, Montevideo, Oranienburg, Blockley, California, Anatum (Margard and Litchfield, 1963), Tennessee, Senftenberg (Haberman and Williams, 1958), Binza, and Bredeney (Wetmore and Hoag, 1960). Salmonellae are gram-negative, nonspore-forming, usually motile rods which do not hydrolyze urea and usually do not ferment lactose. Bergey’s Manual of Determinative Bacteriology should be consulted for common biochemical characters. Selective media of value in isolation of salmonellae for diagnostic purposes will be discussed later.

    Salmonellae are primarily intestinal parasites of vertebrates and are frequently isolated from sewage, river and sea water, and certain foods. They can survive for varying lengths of time under such conditions, but it is doubtful if they can exist indefinitely in any environment outside the animal body (Wilson and Miles, 1975). Contaminated feed and bedding are considered the usual sources of infection for mice (Carlton and Hunt, 1978; Haberman and Williams, 1958). In a study in the United Kingdom (Stott et al., 1975), Salmonella spp. were found in 19% of the animal feed samples tested and were associated with meat and bone meal. It was found that pelleting of the feed resulted in a 1000-fold reduction in the numbers of Enterobacteriaceae. Commercially available laboratory animal feeds in the United States are normally pelleted and are pasteurized or sterilized by autoclaving when fed to barrier-maintained mice. Though the usual route of infection is by ingestion, experimentally the conjunctival route is more effective than the oral route, requiring fewer organisms to establish an infection (Darlow et al., 1961; Tannock and Smith, 1971; Bate and James, 1958). In the research setting, where many species of animals from varied sources are commonly maintained using conventional husbandry practice, the carrier animal is a likely source of infection to other animals in the area. In the closed or barrier-maintained colony, where food and bedding are autoclaved and vermin, birds, and feral animals are excluded, the human carrier should be considered as a possible source of infection.

    Many factors strongly influence the induction and course of naturally occurring and experimentally induced Salmonella infection in mice: virulence, route of infection, and dose of the Salmonella organism; age, sex, and inheritance factors of mice which favor resistance; nutrition of the mouse; intercurrent disease, both naturally occurring and experimentally induced; other experimental stresses which suppress immunity, such as irradiation and corticosteroid drug administration; environmental factors, such as temperature fluctuation; alteration of normal gastrointestinal motility, caused by withholding food or by morphine administration; and alteration of normal gastrointestinal microflora, e.g., by the oral administration of antibiotics. Some of these factors have been studied extensively and were worthy of further comment.

    Strains of Salmonella differ in their virulence for mice. Salmonella gallinarium, though related antigenically to S. enteritidis, is almost avirulent for the mouse (Collins et al., 1966). Carter and Collins (1974a) studied five serotypes of Salmonella in three strains of mice and found that S. paratyphi A, S. paratyphi B, and S. typhi grew very poorly in mice following intravenous inoculation even after 20 serial passages. On the other hand, S. paratyphi C and an S. typhityphimurium hybrid produced progressive systemic infection in C57BL mice with a mean lethal dose of < 10 organisms. Tannock et al. (1975) have shown that mutant strains as well as the wild type of S. typhimurium can associate with and invade the intestinal mucosa of infected gnotobiotic mice; neither O antigen, flagella, nor pili appeared to be essential for the association with the mucosal surface of the mouse ileum. Jenkin (1962) and Bohme (1970) suggest that susceptibility of the mouse to S. typhimurium infection may be affected by an antigenic relationship between host and parasite. The shared antigen(s) appears to be absent or is masked in avirulent strains, whereas the virulent strain shares antigen(s) with the host, which is therefore unable to produce antibody because of self recognition. Others (Furness and Ferreira, 1959; Mackenzie et al., 1940; Pike and Mackenzie, 1940) suggest that virulence of a strain is dependent upon the ability of the salmonella to multiply intracellularly; virulent strains do so, whereas avirulent strains fail to do so.

    When considering that the natural route of infection in mice is by ingestion, it is noteworthy that a dose ≥ 10⁶ virulent S. enteritidis organisms is required to infect mice which have a normal gastrointestinal microflora (Miller and Bohnhoff, 1962; Collins and Carter, 1978). Experimentally, the LD50 of virulent S. enteritidis for conventional TRU:ICR mice was 2.5 × 10³ by the intraperitoneal route, 1 × 10⁴ by the intravenous route, and 5 × 10⁶ by the oral route; the comparable LD50 values for germ-free mice by similar routes were 4 × 10³, 2 × 10³, and 3-5, respectively (Collins and Carter, 1978). The very small number of organisms required to initiate a lethal infection in germ-free mice by the oral route is in sharp contrast to that required in the conventional counterpart and supports the suggestion of others (Miller and Bohnhoff, 1963, Margard et al., 1963; Savage, 1972) that the normal intestinal microflora exerts an inhibitory effect upon the establishment of Salmonella infection by the oral route.

    Inheritance markedly affects the resistance of mice to salmonellosis (Webster, 1937; Gowen, 1948, 1960; Collins, 1972; Bohme, 1970; Bohme et al., 1959; Darlow et al., 1961; Groschel et al., 1970; Robson and Vas, 1972; Oakberg, 1946; Plant and Glynn, 1974, 1976). Certain studies (Robson and Vas, 1972; Plant and Glynn, 1976) indicate that BALB/c and C57BL mice are very susceptible, whereas A/J mice are quite resistant. Recognition of this inherited resistance or susceptibility of strains of mice presents a problem in the critical interpretation of the literature because many of the earlier studies used noninbred Swiss white mice, which behave similarly to the A/J strain in native and vaccine-elicited resistance to infection with S. typhimurium (Robson and Vas, 1972; Collins, 1972). Inherited resistance factors should not be confused with acquired immunity, as Gowen (1948) demonstrated that genetic resistant strains became increasingly resistant when immunized. The nature of inherited resistance to salmonellosis is unclear; it may be associated with glycogen metabolism in the liver (Oakberg, 1946) and/or related to the ability of the mouse to produce a good delayed type hypersensitivity reaction (Plant and Glynn, 1976; Mackaness, 1967). Apart from the factors mentioned above, weanling mice are more susceptible than older mice (Tannock and Smith, 1971), and in a single undesignated strain of mice, females were found to be more susceptible than males (Gowen, 1960). Though commercially prepared feed is readily available, universally used today, and apparently adequate, Schneider and Webster (1945; Schneider, 1956) found a resistance factor in wheat, malted barley, and dried egg white which was dializable, alcohol extractable, and not stored in the body. The resistance factor is neither a vitamin nor an antibiotic but is categorized as a member of a class of ecological ectocrines called pacifarins (Schneider, 1967). Nutritional iron deficiency has an attenuating effect upon S. typhimurium infection of mice (Puschmann and Ganzoni, 1977), whereas iron overload states appear to promote bacterial growth and inhance the virulence of S. typhimurium (Jones et al., 1977).

    Gastrointestinal motility appears to have a marked effect upon the establishment of Salmonella infection in the intestine (Miller and Bohnhoff, 1962; Carter and Collins, 1974b; Ruitenberg et al., 1971). Miller and Bohnhoff (1962) suggested that the reason large inocula (≥ 10⁶ virulent S. enteritidis) are required to infect orally mice having a normal gastrointestinal microflora is motility of the gastrointestinal tract. Using a dye marker, oral inoculum appeared in the cecum within 30-60 min and in the feces within 6 hr. Decreased resistance of mice was noted when morphine sulfate (250 mg/kg subcutaneously) was given or food was withheld for 24 hr. The ill-defined reduction in host resistance following 24-hr starvation was not related to the presence of food in the stomach at the time of experimental inoculation.

    Several studies (Bohnhoff et al., 1954; Miller et al., 1954, 1956; Miller, 1959; Bohnhoff and Miller, 1962; Miller and Bohnhoff, 1963; Ushiba et al., 1955; Meynell, 1955) indicate that alteration of the normal gastrointestinal microflora by oral administration of antibiotics markedly reduces the resistance of mice infected experimentally with S. enteritidis by the oral route. As previously mentioned, ≥ 10⁶ virulent S. enteritidis by mouth is required to initiate infection in 50% of the mice. When similar mice were given streptomycin (50 mg by mouth) within 24 hr before challenge, < 10 S. enteritidis organisms were required (Bohnhoff and Miller, 1962). A similar effect was noted with penicillin and, to a lesser degree, with Oxytetracycline and bacitracin. Thus the extremely small inoculum (< 10 organisms) required to initiate infection in germ-free mice by the oral route (Collins and Carter, 1978) is comparable to that required to infect conventional mice following the oral administration of antibiotics and strongly favors an inhibitory role of the normal gastrointestinal microflora of mice in establishment of Salmonella infection in the intestine.

    Considering the suggested high frequency of salmonellosis in conventionally maintained colonies of mice, it is remarkable that so few descriptions of spontaneous disease and of the epizootiology of salmonellosis in mice appear in the literature. In the absence of evidence to the contrary, it may be that high morbidity and mortality are not observed as frequently in colonies of infected mice as are commonly observed in colonies of infected guinea pigs (Ganaway, 1976). Having examined approximately 26,000 mice from various production colonies in the United States for evidence of Salmonella infection, Margard et al. (1963) suggested that obvious or clinically apparent salmonellosis was the exception rather than the rule. In an experimental study (Miller and Bohnhoff, 1962) wherein mice received 10⁶ virulent S. enteritidis intragastrically, less than 3% of the infected mice died (usually within 7–10 days of inoculation) and infected mice (bacteremic) rarely showed signs of illness. According to Rabstein (1958), however, when salmonellosis is enzootic in the mouse production colony, there are periods of quiescence and periods of high mortality, diarrhea, anorexia, loss of weight, roughened hair coat, and small litters, both in number and in size. Conjunctivitis (Carlton and Hunt, 1978) and impaired gluconeogenesis (Moore et al., 1977; Berry and Smythe, 1960) have also been observed.

    The incubation period is variable depending upon one or more of the previously mentioned predisposing factors but is usually 3-6 days (Haberman and Williams, 1958). Experimental studies (Carter and Collins, 1974b) indicate that the primary site of penetration of the intestine by S. enteritidis administered to mice intragastrically is the distal ileum. Most of the challenge dose (99%) is excreted in the feces; only a few bacteria pass across the mucosa and invade the Peyer’s patches. Here they multiply and spread to the draining mesenteric lymph nodes. The bacteria continue to multiply and spread by the lymphatics to other lymph nodes, liver, and spleen; a bacteremia ensues. After further multiplication in the liver, they pass via the bile to the intestine, where further multiplication and reinvasion of the mucosa occur. In chronic infections, bacteria are shed intermittently in the feces for months, resulting in a carrier state. Approximately 5% of such mice become carriers (Margard et al., 1963; Rabstein, 1958).

    Lesions seen at necropsy are highly variable depending upon whether the disease is acute or chronic (Carlton and Hunt, 1978; Carter and Collins, 1974b; Jones, 1967; Haberman and Williams, 1958). In acute cases, there may be no obvious lesions; the mucosa of the distal ileum may appear hyperemic, and the lower intestine may be empty or contain a small amount of fluid. If the infection is prolonged, yellowish-white miliary foci may be seen in the liver (Fig. 1), spleen, and lymph nodes, and varying degrees of necrosis and hemorrhage may be seen in the lower intestine. The spleen is usually enlarged (Fig. 1). In experimentally infected, germ-free mice (Collins and Carter, 1978), all mice died within 10 days of a progressive systemic infection involving the lower intestine (severe diarrhea), liver, spleen, lymph nodes, and lung.

    Fig. 1 Outbred Swiss mouse 7 days after peritoneal inoculation with Salmonella enteritidis. The spleen is enlarged, and there are small white foci in the liver. (Courtesy of Dr. Alice O’Brien, Uniformed Services University for Health Sciences.)

    Microscopically, lesions are found wherever there is bacterial invasion and colonization; the extent of the lesion is proportional to the course of the disease and the number of bacteria present in the tissue (Collins and Carter, 1978; Carter and Collins, 1974b; Bakken and Vogelsang, 1950; Miller and Bohnhoff, 1962). Necrotic foci may be seen in the intestine, mesenteric lymph nodes, liver (Fig. 2), and spleen. Focal acacquired cumulations of polymorphonuclear leukocytes and histiocytes are found in the lymphoid follicles of the ileum, spleen, and lymph nodes. Reticuloendothelial cell hyperplasia may be so marked in the spleen as to give it a bloodless appearance. In the liver, there is venous thrombosis. Bacteria are commonly present at the periphery of the granulomatous lesions.

    Fig. 2 N.NIH(s) mouse 7 days after intravenous inoculation with Salmonella enteritidis. There is focal necrosis of the liver and invasion with polymorphonuclear leukocytes. H&E stain. ×220.

    Probably the most intensively studied aspect of salmonellosis of mice is that which is concerned with the mechanism of immunity. The principal motivation has been the need to develop an effective and safe vaccine to protect man against systemic Salmonella infection, especially S. typhi, the cause of typhoid fever. The mouse has been used not only to test the effectiveness of the numerous vaccine preparations but also as a model for studying and understanding the immune response mechanisms. It is beyond the intention of this chapter to review critically these numerous studies. However, no discussion of salmonellosis in mice would be complete without mention of acquired immunity. Two schools of thought have been actively pursued: One suggests that killed vaccines do not afford protection and that immunity is mediated by cellular rather than humoral mechanisms (Mackaness et al., 1966; Blanden et al., 1966; Collins et al., 1966; Collins, 1968a,b, 1969; Collins and Mackaness, 1968; Collins and Carter, 1972; Mitsuhashi et al., 1961; Sato et al., 1962; Germanier, 1972; Hobson, 1957a; Macleod, 1954; Howard, 1961; Venneman et al., 1970; Venneman and Berry, 1971a,b); the other suggests that killed vaccines are effective and that opsonic or humoral antibody plays an important role in acquired immunity (Jenkin and Rowley, 1963, 1965; Jenkin et al., 1964; Kenney and Herzberg, 1967, 1968; Turner et al., 1964; Rowley et al., 1964, 1968; Robson and Vas, 1972; Ushiba, 1965; Dimache et al., 1976). Rowley et al. (1968) and Ushiba (1965) suggest that the two mechanisms are not mutually exclusive and that both opsonic antibody and phagocytic cells play vital roles. Possibly, an important point in summary for anyone contemplating the use of one of these preparations for immunizing or protecting mice from spontaneous disease in a production colony or in a research laboratory setting is that in no instance was the carrier state eliminated. If the immunogen was a live virulent or attenuated strain, the carrier state remained; if the immunogen was not alive, the challenge was alive and it persisted, producing a carrier. The results of these studies are in agreement with our understanding of the naturally occurring disease; active infection is universally recognized as the most effective means of producing immunity known, and yet, carriers persist. As immunity is a relative state, the question for users of mice in the laboratory is not one of how to protect mice from progressive systemic salmonellosis; rather, it is how to avoid the complicating effects of infection, and this is accomplished by maintaining the Salmonella-free status of the mice.

    Diagnosis is based upon isolation and identification of Salmonella organisms. Isolation is readily accomplished from any of the affected tissues or blood during the septicemic stage. In mice which have experienced systemic disease, bacteria may persist in the liver and spleen for several weeks.

    An important aspect of quality control of mice destined for use in biomedical research today is the detection of subclinical salmonellosis in the carrier animal. Confidence in the validity of the monitoring program is greatly facilitated by access to records of the diagnostic laboratory responsible for monitoring the health status of the production colony, whereby the cause of any illness or death in the colony is known. Lacking such information, as when purchasing mice from a vendor, one must resort to sampling procedures when the mice arrive at the using facility. Assuming a carrier rate of 5% and a desire to detect at least one infected mouse at the 95% confidence level, examination of a sample size of 58 mice would be required.* Furthermore, if a determination of the Salmonella-free status of the supplier is a consideration, there may be reason to question whether the sample is truly representative of the production colony population.

    The detection of carriers by culture of fecal samples has been studied extensively (Banwart and Ayres, 1953; Dixon, 1961; Margard and Litchfield, 1961; Margard et al., 1963). The best results of isolation from mouse feces were obtained by selective enrichment in selenite F cystine broth (18–24 hr), followed by plating on brilliant green agar and choosing suspect colonies for transfer to triple sugar iron-urea (Margard et al., 1963). Confirmation is determined serologically. The investigators suggest that samples from individual mice are more effective in detection of the carrier than are pooled feces from several mice, due presumably to the presence of inhibitors in the feces of some mice. This could be an important consideration when attempting to determine the incidence of shedding within a population, as pointed out by Margard et al. (1963). However, to determine only whether Salmonella is present in a given population, use of the pooled sample may have merit, especially in consideration of the expected small carrier rate (5%) and the large sample size required to detect shedding in one mouse. Further study is needed.

    Since antibody is not always detectable by the agglutination test in culturally positive mice (Hobson, 1957b; Tannock and Smith, 1971) and serological cross-reactivity is so common among bacteria, even of different genera, the detection of Salmonella-infected mice (see Otis, Volume III), past and present, by serological means does not appear to be useful. However, Morello et al. (1964) suggest the use of a hemolytic test to detect chronic salmonellosis in mice. Further study is needed.

    Salmonella-infected mice are not acceptable tools for the conduct of biomedical research. Furthermore, such mice can be a source of infection for other animals and man. Salmonellosis of mice is preventable by production methods utilizing a barrier system (see Chapter 16, this volume). The Salmonella-free state can be maintained in the research laboratory by adhering to strict husbandry practices (see Chapter 15, this volume, and Lang, Volume III). Neither antibiotic treatment (Seligmann and Wassermann, 1949; Hobson, 1956; Haberman and Williams, 1958; Slanetz, 1946, 1948; Rabstein, 1958) nor vaccination with a variety of preparations (Mitsuhashi et al., 1958, 1959; Blanden et al., 1966; Mackaness et al., 1966; Rowley et al., 1964; Hobson, 1957c; Hashimoto et al., 1961; Germanier, 1970; Germanier and Furer, 1971; Collins and Carter, 1972; Wray et al., 1977) will prevent the development of the carrier state and is therefore not recommended.

    B Bacillus

    Ernest Tyzzer (1917) originally described this disease condition, which destroyed his colony of Japanese waltzing mice. If it seems difficult to conceive that a microbial agent would kill an entire population, consider the known characteristics of the etiologic agent: a gram-negative rod 0.5 × 8–10 μm, motile by peritrichous flagella, spore-forming, pleomorphic, obligate intracellular parasite, very fastidious in selection of cells for metabolism and growth (epithelial cells of lower intestinal mucosa, smooth and cardiac muscle cells, nerve cells, and hepatocytes), unclassified but referred to as Bacillus piliformis (see reviews in Ganaway et al., 1971a; Fujiwara, 1978). The literature is replete with descriptions of unsuccessful attempts to culture this interesting parasite in cell-free media. Although two reports of isolation in such media have appeared (Kanazawa and Imai, 1959; Simon, 1977), the evidence is not convincing. Other than by passage in a vertebrate host, the only presently known means of propagating B. piliformis is by the inoculation of embryonated hens’ eggs via the yolk sac route (Craigie, 1966a; Ganaway et al., 1971b; Fries, 1977a).

    Tyzzer’s disease was recognized in mice only until 1965, when Allen and associates (1965) described the condition in laboratory rabbits. The disease occurs worldwide, and fatal infections have since been described in a wide variety of laboratory animals, in free-living animals, and in horses (Ganaway et al., 1976). In the United Kingdom, Tyzzer’s disease is thought to be the greatest cause of ruined cancer research studies (Anonymous, 1961) and one of the four most important diseases of laboratory mice (Tuffery, 1956). The importance, and indeed the prevalence, of the disease in mice in the United States remain unknown.

    The source of infection for colonies of mice remains unknown. Since the vegetative phase of B. piliformis outside the host cell is so unstable (Craigie, 1966a; Ganaway et al., 1971a,b; Fujiwara, 1978), it would appear that the spore represents the interepizootic survival mechanism and the means of spread of the infection between animals. Bedding soiled by animals which die of Tyzzer’s disease remains a source of infection for other animals for extended periods of time (Tyzzer, 1917; Allen et al., 1965; Ganaway et al., 1971a). Thus, the ingestion of feces-contaminated food seems to be the most likely means of acquiring infection. The use of animal feed that is not sterilized might explain the introduction of B. piliformis into cesarean-derived, barrier-type colonies of mice (Mullink, 1968; Hunter, 1971; Ganaway et al., 1976). Fries (1978) recently reported infection of the mouse fetus in utero following an infective intravenous challenge of the pregnant dam. Such studies need to be extended to explore the possibility of vertical transmission.

    Several factors that could predispose mice to Tyzzer’s disease have been suggested: overcrowding (Tyzzer, 1917; Rights et al., 1947), high temperature and humidity (Gard, 1944), feeding moist food (Gard, 1944), immunosuppressive therapy such as X irradiation (Tuffery, 1956) and corticosteroid drugs (Kaneko et al., 1960; Fujiwara, 1978; Ganaway et al., 1971a; Craigie, 1966b), and inherited susceptibility (Tyzzer, 1917; Gowen and Schott, 1933). Overcrowding, high humidity, and feeding moist food which is easily contaminated with feces may contribute to the buildup of spores in the environment to the extent that clinical disease becomes manifest. In the United Kingdom, the mortality due to Tyzzer’s disease in noncompromised, noninbred mice is believed to be low (2-3%), but when a group of mice were X-irradiated, 47% died as a result of B. piliformis infection (Tuffery, 1956). The use of corticosteroid drugs has been a valuable aid in experimental studies whereby B. piliformis can be predictably maintained in passage in mice inoculated intravenously with suspensions of infected liver (Fujiwara, 1978).

    The first sign of Tyzzer’s disease is usually the occurrence of sudden deaths. Close examination of cagemates may reveal varying degrees of diarrhea, which, if present, is of short duration (Tyzzer, 1917; Gard, 1944; Rights et al., 1947). At necropsy, conspicuous lesions are usually seen in the liver as miliary, white to yellowish-gray foci scattered throughout the parenchyma (Fig. 3). The lower intestinal tract appears normal or is slightly reddened. No other tissues appear to be affected grossly. Histologically, the miliary foci in the liver appear as areas of coagulation necrosis and are located generally in the immediate vicinity to a branch of the portal vein, indicating an embolic origin from the gastrointestinal tract. Variable numbers of neutrophils and lymphocytes surround the necrotic areas. Bundles of slender, sticklike rods, B. piliformis,* appear randomly arranged in the cytoplasm of apparently viable hepatocytes at the border of the necrotic and normal tissue (Figs. 4 and 5). Varying stages and degrees of infection may be seen in sections of the same liver. The bacilli are numerous in early stages and absent in late stages. The bacilli are also found in the epithelial cells of the intestinal mucosa of the terminal ileum, cecum, and proximal colon. Here, as in the liver, the ease of demonstrating the bacilli depends upon the degree and stage of infection and the use of proper staining technique.

    Fig. 3 CBA/N mouse 3 days after intravenous inoculation with Bacillus piliformis (rabbit origin). Numerous white foci are scattered throughout the liver.

    Fig. 4 Note the border of a focal necrotic lesion in the liver of a mouse with Tyzzer’s disease. Bacillus piliformis organisms are not demonstrated with H&E stain. ×330.

    Fig. 5 Bacillus piliformis organisms are readily demonstrated in hepatic cells at the border of a focal necrotic lesion in the liver of a mouse with Tyzzer’s disease using the Warthin-Starry silver impregnation technique. ×330.

    The diagnosis of mice examined within a reasonably short time after death or killed during a stage of acute disease is distinctive and easily accomplished. It is based upon the demonstration of typical bacilli within the cytoplasm of hepatocytes or intestinal epithelial cells of the mucosa.

    As expected, the number of subclinical infections appears to exceed greatly the number of clinical infections. The indirect immunofluorescence antibody technique (FAT) appears to be considerably more sensitive than the complement fixation (CF) test for determining previous infection. Using the CF test, Fujiwara (1967) found antibody in individual sera of 4-10% of the mice from colonies in which Tyzzer’s disease was known to be enzootic. Using FAT, Fries (1977b) found antibody in 36-83% of the mice from known infected colonies. The higher prevalence rates seem more probable for a fecal-oral-transmitted disease. Confirmation and extension of this effort are needed to provide prevalence data and to better our knowledge of the poorly understood B. piliformis parasite. High prevalence rates of subclinical disease may indicate that a vaccine would be useful in protecting mice (and other animals). Fujiwara et al. (1965) reported that formalinized infected mouse liver suspension was antigenic and, when used as a vaccine, would protect mice against a subsequent, otherwise lethal challenge (intravenous route). However, it did not prevent the development of liver lesions.

    The means to prevent or control Tyzzer’s disease have not been adequately studied. The natural history of Tyzzer’s disease, prevalence rates of infection in various animal species, the possibility of reservoirs and carrier states, and the actual mechanisms of spread of the disease among animals remain areas of speculation. As previously mentioned, either nonsterilized animal feed or vertical transmission might be the means of introducing B. piliformis into caesarian-derived, barrier-maintained colonies of mice. Also, as previously mentioned, a buildup of spores in the environment and/or immunosuppression might be expected to result in fatal infections. In certain instances, antibiotics might be helpful (Craigie, 1966b; Ganaway et al., 1971a,b; Fujiwara, 1978). Since the diagnosis of Tyzzer’s disease is based upon histopathological findings, antibiotic treatment of individual animals is not a consideration. It is conceivable that an epizootic might be averted by the oral administration of tetracycline, as reported by Hunter (1971). It is unlikely, however, that a spore-forming bacterium can be eliminated from a large population of animals by the use of antibiotics. A vaccine might be helpful but is yet to be developed and tested. Until convincing evidence of vertical transmission is provided, the cesarean-derived, barrier-maintained colony should offer the best means of providing Tyzzer’s disease-free mice. Efforts to maintain this state in the laboratory during the conduct of research are appropriate, especially if the mice are to be used in studies involving immunosuppression.

    C Citrobacter

    Colitis, characterized by marked mucosal hyperplasia and varying degrees of inflammation, has been observed as a naturally occurring epizootic disease of mice associated with a variant of Citrobacter freundii. Various terms have been used to describe the condition: neoplasia (Pullinger and Iverson, 1960), colitis cystica (Brynjolfsson and Lombard, 1969), catarrhal enterocolitis (Brennan et al., 1965), colitis with rectal prolapse (Ediger et al., 1974; Bieniek and Tober-Meyer, 1976), and transmissible murine colonic hyperplasia (Barthold et al., 1976; Silverman et al., 1979).

    Citrobacter freundii, the type species of this genus, is placed in group I of the family Enterobacteriaceae (Sedlak, 1974). A normal inhabitant of the intestine of man, C. freundii is found in water, food, feces, and urine. It is a gram-negative rod which grows readily on ordinary media. Though most strains are motile, ferment lactose, and utilize citrate as the sole source of carbon, the atypical strains that have been associated with colonic hyperplasia of mice (Brennan et al., 1965; Ediger et al., 1974; Barthold et al., 1977) are nonmotile and either fail to utilize citrate or do so only marginally.

    Citrobacter freundii has been a cause of significant nosocomial infections associated with contamination of intravenous fluids and is an opportunistic pathogen associated with urinary tract, respiratory tract, wound, and cutaneous infections in man (Hodges et al., 1978). The prevalence of typical or atypical C. freundii in colonies of mice, in the absence of clinical disease, remains unknown, as field survey studies have not been performed. Ediger et al. (1974) isolated atypical C. freundii from 4361 clinically normal mice from a conventional colony. Brennan et al. (1965) isolated atypical C. freundii from 43% (65 of 151) newly arrived, unaffected mice from commercial breeders. Likewise, the source of infection for the mouse colony remains unknown. For the microbiologically monitored, barrier-type colony, man might be considered as a possible source of infection.

    Diet, inherited resistance and age of the mouse, and virulence of C. freundii strains have marked predisposing influences on the induction and severity of disease (Barthold et al., 1977). In experimental studies using an atypical strain of C. freundii, each of four strains of mice examined (NIH Swiss, DBA/2J, C57BL/6J, and C3H/HeJ) developed colonic hyperplasia, but the degree of hyperplasia varied significantly with the mouse strain. Mortality was low or absent in DBA/2J, NIH Swiss, and C57BL/6J mice but reached 45% in C3H/HeJ mice between 2 and 3 weeks postinoculation. Both suckling mice and adults were susceptible, but moderate mortality was seen in younger mice and was rare in adults (Barthold et al., 1978). On the other hand, Silverman et al. (1979) reported a natural outbreak in a group of 210 adult A/J mice in which they observed 50% morbidity and 25% mortality. Bieniek and Tober-Meyer (1976) observed high morbidity (37%) in a breeding colony of Han:NMRI mice, in which young adults (5-7 weeks of age) were primarily affected. Significant differences in the degree of colonic hyperplasia in affected mice were noted between groups fed four commercially available diets (Barthold et al., 1977). The responsible factor(s) in the diet remains unknown. In an experimental study designed to determine the infectivity of various isolants of C. freundii, each of 19 isolants obtained from other animal species, including man, failed to colonize the lower intestinal tract or to cause colonic hyperplasia in otherwise susceptible mice (Barthold et al., 1977).

    In natural outbreaks, retarded growth, ruffled fur, soft feces, occasional prolapse of the rectum (Fig. 6), and moderate mortality in late suckling and early weanling-age mice may be seen (Brennan et al., 1965; Ediger et al., 1974; Barthold et al., 1978). Brennan et al. (1965) noted diarrhea with pasting or soiling of the perineum as a constant

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