A laboratory manual for the isolation identification and characterization of avian pathogens 5th (2008)

A LABORATORY MANUAL FOR THE

ISOLATION,
IDENTIFICATION, AND
CHARACTERIZATION
OF AVIAN
-

UH*

**

w/

«

Fifth Edition

THE AMERICAN ASSOCIATION OF AVIAN PATHOLOGISTS



A LABORATORY MANUAL FOR THE

ISOLATION, IDENTIFICATION
AND CHARACTERIZATION
OF AVIAN PATHOGENS

Fifth Edition

American Association of Avian Pathologists

Editorial Committee
Louise Dufour-Zavala, Editor-in-Chief
David E. Swayne
John R. Glisson
Janies E. Pearson
Willie M. Reed
Mark W. Jackwood
Peter R. Woolcock

Copies Available from:
American Association of Avian Pathologists
953 College Station Road
Athens, GA 30602-4875


Frederic J. Hoerr

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1

3

4

2

7

5
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6

Cover:
1. Direct fluorescent antibody test on primary chicken embryo kidney cells showing
syncytia formed by ILT virus in primary chick embryo kidney cells. (Rey Resurreccion,
GPLN, Oakwood, GA) 2. Ninety well serum plate prepared for ELISA testing for the
detection of AE antibodies. (Len Chappell, GPLN, Oakwood, GA). 3. Bacterial culture on
a TSI slant. (Doug Waltman, GPLN, Oakwood, GA). 4. Reverse transcriptase-polymerase
chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP) analysis if
the spike (SI) glycoprotein gene of the Arkansas strain of infectious bronchitis virus
(IBV). Lanes 1 and 2 are molecular weight markers. Lane 3 is the IBV amplicon digested
with BstYI, lane 4 is the IBV amplicon digested with Haein, and lane 5 is the IBV
amplicon digested with XcmI. (Mark Jackwood, PDRC, Athens, GA). 5. Ten-fold
dilution series of avian influenza virus RNA run with the USDA type A influenza M gene
real-time RT-PCR test on the Applied Biosystems 7500 FAST system. (Erica Spackman,
SEPRL, Athens, GA). 6. Microphotograph of Mycoplasma gallisepticum colonies on agar
(35x) (Stan Kleven, PDRC, Athens, GA). 7. Bio Merieux API 20 system for bacteria
identification using a series of biochemical tests. (Doug Waltman, GPLN, Oakwood, GA).
8. Embryonated egg candling to locate the chorio-allantoic sac for inoculation of a virus
isolation sample. (Rey Resurreccion, GPLN, Oakwood, GA). 9. Wet mount of Aspergillus
sp. conidiofores after culture in Sabouraud's dextrose agar (100X). The isolate was
obtained from a case of systemic aspergillosis in 9-wk-old broiler breeder pullets.
(Guillermo Zavala, PDRC, Athens, GA).
Pictures 2,3,7,8 and Page design by HMM Photography (Heidi Migalla).

Copyright © 1975, 1980, 1989,1998,2008 by American Association of Avian Pathologists, Inc. Athens, Georgia

All rights reserved
Copyright is not claimed for Chapters 22, 36

Chapters 16,29, 35,40,44, 49: US Government copyrighted, user rights reserved, printed by permission
Chapters 30, 31: (British) Crown copyrighted and (British) Crown user rights reserved, printed by permission

Chapter 6: (Australian) Crown copyrighted and (Australian) Crown user rights reserved, printed by permission
Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the author, the supporting
institutions or the American Association of Avian Pathologists and does not imply its approval to the exclusion of other products that also
may be suitable

Fifth edition 2008
Previously entitled Isolation and Identification ofAvian Pathogens
Library of Congress Catalog Card Number: 2008924452

ISBN 978-0-9789163-2-9

All rights reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means,
electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the copyright owner.
Printed in the United States of America

10

987654321

Printed by
OmniPress, Inc., Madison, Wisconsin 53704

ii


A Laboratory Manual for the Isolation, Identification and Characterization
of Avian Pathogens

TABLE OF CONTENTS
Diagnostic Principles. Frederic J. Hoerr...................................................................................................................................1
Salmonellosis. W. Douglas Waltman and Richard K. Gast.................................................................................................... 3
Colibacillosis. Margie D. Lee and Lisa K. Nolan..................................................................................................................10
Pasteurellosis, Avibacteriosis, Gallibacteriosis, Riemerellosis, and Pseudotuberculosis.
John R. Glisson, Tirath S. Sandhu, and Charles L. Hofacre....................................................................................................12
5. Bordetellosis. Mark W. Jackwood........................................................................................................................................ 20
6. Infectious Coryza. Pat J. Blackall........................................................................................................................................ 22
7. Campylobacter in Poultry. Jaap A. Wagenaar and Wilma F. Jacobs- Reitsma..................................................................... 27
8. Spirochetosis. Stephen R. Collett........................................................................................................................................... 31
9. Erysipelas. George L. Cooper and Arthur A. Bickford........................................................................................................ 36
10. Listerosis. George L. Cooper and Arthur A. Bickford........................................................................................................ 39
11. Staphylococcosis. Stephan G. Thayer and W. Douglas Waltman.........................................................................................42
12. Streptococcosis and Enterococcosis. Stephan G. Thayer and W. Douglas Waltman............................................................ 44
13. Clostridial Diseases. Stephan G. Thayer and David A. Miller............................................................................................. 47
14. Tuberculosis. Susan Sanchez and Richard M. Fulton.......................................................................................................... 53
15. Mycoplasmosis. Stanley H. Kleven......................................................................................................................................59
16. Chlamydiosis. Arthur A. Andersen and Daisy Vanrompay................................................................................................. 65
17. Omithobacteriosis. Richard P. Chin and Bruce R. Charlton................................................................................................ 75
18. Mycoses and Mycotoxicoses. R.D. Wyatt............................................................................................................................ 77
19. Adenovirus. Brian M. Adair and J. Brian McFerran.............................................................................................................84
20. Hemorrhagic Enteritis of Turkeys Marble Spleen Disease of Pheasants. F. William Pierson and Sctott D. Fitzgerald....... 90
21. Infectious Laryngotracheitis. Deoki N. Tripathy and Maricarmen Garcia........................................................................... 94
22. Marek’s Disease. Patricia S. Wakenell and Jagdev M. Sharma........................................................................................... 99
23. Duck Virus Enteritis. Peter R. Woolcock............................................................................................................................ 106
24. Herpesviruses of Free-Living and Pet Birds. Erhard F. Kaleta........................................................................................... 110
25. Pox. Deoki N. Tripathy and Willie M. Reed....................................................................................................................... 116
26. Budgerigar Fledgling Disease and Other Avian Polyomavirus Infections. Branson W. Ritchie and Phil D. Lukert........ 120
27. Psittacine Beak and Feather Disease. Branson W. Ritchie and Phil D. Lukert................................................................... 122

28. Chicken Anemia Virus. M. Stewart McNulty and Daniel Todd......................................................................................... 124
29. Avian Influenza. David E. Swayne, Dennis A. Senne and David L. Suarez....................................................................... 128
30. Newcastle Disease Virus and Other Avian Paramyxoviruses. Dennis J. Alexander and Dennis A. Senne.........................135
31. Avian Metapneumovirus. Richard E. Gough and Janice C. Pedersen................................................................................. 142
32. Infectious Bronchitis. Jack Gelb, Jr. and Mark W. Jackwood............................................................................................. 146
33. Turkey Coronavirus. Mark W. Jackwood and James S. Guy.............................................................................................. 150
34. Enteric Viruses. Don Reynolds and Ching Ching Wu.................................................................................................... 153
35. Oncornaviruses, Leukosis/Sarcomas and Reticuloendotheliosis. Aly M. Fadly, Richard L. Witter, and Henry D. Hunt.. 164
36. Avian Encephalomyelitis. Louis van der Heide................................................................................................................... 173
37. Duck Hepatitis. Peter R. Woolcock............................................................................................................................
175
38. Turkey Viral Hepatitis. Willie M. Reed.............................................................................................................................. 179
39. Viral Arthritis/Tenosynovitis and Other Reovirus Infections. John K. Rosenberger and Erica Spackman.........................181
40. Arbovirus Infection. Eileen N. Ostlund and James E. Pearson........................................................................................... 184
41. Infectious Bursal Disease. John K. Rosenberger, Y.M. Saif, and Daral J. Jackwood......................................................... 188
42. Parvovirus of Waterfowl. Richard E. Gough...................................................................................................................... 191
43. Cell-Culture Methods. Karel A. Schat and Holly S. Sellers................................................................................................ 195
44. Virus Propagation in Embryonating Eggs. Dennis A. Senne..............................................................................................204
45. Virus Identification and Classification. Pedro Villegas and Ivan Alvarado........................................................................209
46. Titration of Biological Suspensions. Pedro Villegas........................................................................................................... 217
47. Serologic Procedures. Stephan G. Thayer and Charles W. Beard.......................................................................................222
48. Molecular Identification Procedures. Daral J. Jackwood and Mark W. Jackwood..............................................................230
49. Antigen Detection Systems. Mary J. Pantin-Jackwood and Sandra S. Rosenberger.......................................................... 233
Appendix of Abbreviations and Acronyms used in the Text...................................................................................................... 241
Quick Reference Diagnostic Chart.............................................................................................................................................. 244
Index............................................................................................................................................................................................ 247

1.
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4.

iii


CONTRIBUTING AUTHORS
Brian Adair
Veterinary Sciences Division
Agri-Food and Biosciences Institute
Stoney Road
Stormont
Belfast BT4 3SD
Northern Ireland
E-mail:
FAX: +44(0)2890525773

Richard P. Chin
California Veterinary Diagnostic Laboratory System
Fresno Branch
School of Veterinary Medicine
University of California - Davis
2789 S. Orange Ave.
Fresno, California 93725
E-mail:
FAX: (559) 485-8097

Dennis J. Alexander
Central Veterinaiy Laboratory (Weybridge)
New Haw, Addlestone
Surrey KT15 3NB

United Kingdom
E-mail:
FAX: +44-1932-357856

Stephen R. Collett
Department of Population Health
College of Veterinary Medicine
The University of Georgia
953 College Stations Road
Athens, Georgia 30602-4875
E-mail:
FAX: (706) 542-5630

Ivan R. Alvarado
120 Spring Lake Pointe
Athens, GA 30605
E-mail:
FAX: (404) 506-9013

George L. Cooper
School of Veterinary Medicine
University of California-Davis
Calif. Vet. Diagnostic Lab. System
Turlock Branch
P.O. Box P
Turlock, California 95381
E-mail: .
FAX: (209) 667-4261

Dr. Arthur A. Andersen

USDA-ARS
Former Address:
National Animal Disease Center
P.O.Box 70, Ames, IA 50010
Email:

Charles W. Beard
130 Red Fox Run
Athens, GA 30605
E-mail:
FAX: (706) 548-6410

Aly M. Fadly
USDA- Agricultural Research Service
Avian Disease and Oncology Laboratory (ADOL)
3606 East Mount Hope Road
East Lansing, Michigan 48823
E-mail:
FAX: (517) 337-6776

Arthur A. Bickford
University of California-Davis
California Veterinaiy Diagnostic Laboratory System
Turlock Branch
1650 Simon Drive
Turlock, California 95382
E-mail:
FAX: (209) 632-4258

Scott D. Fitzgerald

Diagnostic Center for Population and Animal Health
College of Veterinary Medicine
Michigan State University
PO Box 30076
Lansing, Michigan 48909-7576
E-mail:
FAX: (517)355-2152

Pat J. Blackall
Animal Research Institute
Locked Mail Bag No. 4
Moorooka, Queensland 4105
Australia
E-mail:
FAX: +61-7-33629-429

Richard M. Fulton
Diagnostic Center for Population and Animal Health
Michigan State University
PO Box 30076
Lansing, Michigan 48909
E-mail:
FAX: (517)355-2152

Bruce R. Charlton
California Veterinary Diagnostic Laboratory System
Turlock Branch
School of Veterinary Medicine
University of California - Davis
3327 Chicharra Way

Coulterville, California 95311
E-mail:
FAX: (209) 667-4267

Maricarmen Garcia
Department of Population Health
College of Veterinary Medicine
The University of Georgia
953 College Station Rd.
Athens, Georgia 30602-4875
E-mail:
FAX: (706) 542-5630

iv


Richard K. Gast
USDA, ARS
Russell Research Center
950 College Station Road
Athens, GA 30605
E-mail:
FAX: (706) 546-3035

Daral J. Jackwood
Food Animal Health Research Program
Ohio Agricultural Research and Development Center
The Ohio State University
1680 Madison Ave
Wooster, Ohio 44691

E-mail:
FAX: (330) 263-3760

Jack Gelb, Jr.
Department of Animal and Food Sciences
College of Agricultural Sciences
531 South College Avenue
University of Delaware
Newark, Delaware 19716-2150
E-mail:
FAX: (302)831-2822

Mark W. Jackwood
Department of Population Health
College of Veterinary Medicine
The University of Georgia
953 College Station Road
Athens, Georgia 30602-4875
E-mail:
FAX: (706) 542-5630

John R. Glisson
Department of Population Health
College of Veterinary Medicine
The University of Georgia
953 College Station Rd.
Athens, Georgia 30602-4875
E-mail:
FAX: (706) 542-5630

Wilma F. Jacobs-Reitsma
RIKILT Institute of Food Safety
Bomsesteeg 45
6708 PD Wageningen
The Netherlands
E-mail:
FAX: +31-317-417717

Richard E. Gough
Central Veterinary Laboratory (Weybridge)
New Haw, Addlestone
Surrey KT15 3NB
United Kingdom
E-mail:
FAX: +44-1932-357856

Erhard F. Kaleta
Institute for Avian and Reptile Medicine
Justus-Liebig-University
Frankfurter Strabe 91, D-35392 Giefien
Germany
E-mail:
FAX: +49-641-201548

Janies S. Guy
North Carolina State University
College of Veterinary Medicine
Department of Population Health & Pathobiology
4700 Hillsborough Street
Raleigh, North Carolina 27606

E-mail: Jim
FAX: (919)513-6464

Stanley H. Kleven
Department of Population Health
College of Veterinary Medicine
University of Georgia
953 College Station Rd
Athens, Georgia 30602-4875
E-mail:
FAX: (706) 542-5630

Frederic J. Hoerr
Thomas Bishop Sparks
State Diagnostic Laboratory
P. O. Box 2209
Auburn, Alabama 36831-2209
E-mail:
FAX: (334) 8244-7206

Margie D. Lee
Department of Population Health
Poultry Diagnostic and Research Center
The University of Georgia
953 College Station Road
Athens, Georgia 30602-4875
E-mail:
FAX: (706) 542-5771

Charles Hofacre

Department of Population Health
College of Veterinary Medicine
The University of Georgia
953 College Station Road
Athens, Georgia 30602-4875
E-mail:
FAX: (706) 542-5630

Phil D. Lukert
Department of Medical Microbiology
College of Veterinary Medicine
University of Georgia
Box 207
Colbert, Georgia 30628
E-mail:
FAX: (706) 542-5771

Henry Hunt
USDA- Agricultural Research Service
Avian Disease and Oncology Laboratory (ADOL)
3606 East Mount Hope Road
East Lansing, Michigan 48823
E-mail:
FAX: (517) 337-6776

J. Brian McFerran
19 Knocktem Gardens
Belfast BT4 3LZ
Northern Ireland
FAX: +44-1232-658040

v


M. Stewart McNulty
Department of Agriculture
Veterinary Sciences Division
Stormont, Belfast BT4 3SD
Northern Ireland
FAX: +44-1232-525773
E-mail: m-mcnulty @utvintemet. com

Willie M. Reed
Purdue University
Dean, School of Veterinary Medicine
Lynn Hall Room 1176
West Lafayette, Indiana 47907-2026
E-mail:
FAX: (765) 496-1261

David A. Miller
National Veterinary Services Laboratories
USDA-APHIS-VS
1800 Dayton Road
Ames, Iowa 50010
E-mail:
FAX: (515) 239-8569

Donald L. Reynolds
2520 Veterinary Administration

Iowa State University
1802 Elwood Drive
Ames, Iowa 50011
E-mail:
FAX: (515) 294-8956

Lisa K, Nolan
Dept, of Vet Microbiology and Preventive Medicine
College of Veterinary Medicine
1802 Elwood Dr VMRI #2
Iowa State University
Ames, LA 50011
E-mail:
FAX: (515) 233-2136

Branson W. Ritchie
Department of Medical Microbiology
College of Veterinary Medicine
University of Georgia
Athens, Georgia 30602
FAX: (706) 542-6460
Email:
John K. Rosenberger
Aviserve, LLC
Delaware Technology Park
1 Innovation Way, Suite 100
Newark, DE 19711
E-mail:
FAX: (302) 368-2975

Eileen N. Ostlund
Head Equine Ovine Viruses Section
Diagnostic Virology
NVSL
Ρ,Ο,Βοχ 844
Ames, IA 50010
E-mail:
FAX: (515) 663-7348

Sandra S. Rosenberger
Aviserve, LLC
Delaware Technology Park
1 Innovation Way, Suite 100
Newark, DE 19711
E-mail:
FAX: (302) 368-2975

Mary J. Pantin-Jackwood
Southeast Poultry Research Laboratory USDA-ARS
934 College Station Road
Athens, Georgia 30605
E-mail:
FAX: (706) 546-3161

Y. M. Saif
Food Animal Health Research Program
Ohio Agricultural Research and Development Center
The Ohio State University
1680 Madison Avenue
Wooster, Ohio 44691

E-mail:
FAX: (330) 263-3677

Janies E. Pearson
4016 Phoenix
Ames, Iowa 50014
(515) 292-9435
E-mail:
Janice C. Pedersen
Avian Section, Diagnostic Veterinary Services Laboratory
NVSL
1437 270th Street
Madrid, Iowa 50156
E-mail:
FAX: (515) 663-7348

Susan Sanchez
Athens Diagnostic Laboratory
0103 Athens V.M. Diag Lab
501 D.E. Brooks Dr
Athens, GA 30602
E-mail:
FAX: (706)542-5568

Frank W. Pierson
Center for Molecular Medicine and Infectious Diseases
Virginia-Maryland Reg. College of Vet. Medicine
Virginia Polytechnic Institute and State University
Blacksburg, Virginia 24061
E-mail:

FAX: (540) 231-3426

Tirath S. Sandhu
Cornell University Duck Research Laboratory
37 Howell Place
P.O. Box 427
Speonk, New York 11972
E-mail:

vi


Stephen G. Thayer
Department of Population Health
College of Veterinary Medicine
The University of Georgia
953 College Station Road
Athens, Georgia 30602-4875
E-mail:
FAX: (706) 542-0252

Karel A. Schat
Unit of Avian Health
College of Veterinary Medicine
Cornell University
Ithaca, New York 14853
E-mail:
FAX: (607) 253-3384

Daniel Todd

Dept of Agriculture and Rural Development from N. Ireland
Veterinary Sciences Division
Stormont, Belfast BT4 3SD
UK
E-mail:
Tel: +44 2890 525773

Holly Sellers
Department of Population Health
Poultry Diagnostic and Research Center
The University of Georgia
953 College Station Road
Athens, GA 30602-4875
E-mail:
FAX: (706)542-5630

Deoki N. Tripathy
Department of Veterinary Pathobiology
College of Veterinary Medicine
University of Illinois
104 West McHenry
Urbana, Illinois 61801
E-mail:
FAX: (217) 244-7421

Dennis A. Senne
National Veterinary Services Laboratories
Veterinary Services
Animal and Plant Health Inspection Service
United States Department of Agriculture

1800 Dayton Road
Ames, Iowa 50010
E-mail:
FAX: (515) 663-7348

Daisy Vanrompay
Ghent University
Faculty of Bioscience engineering
Department of molecular biotechnology
Coupure Links 653
9000 Ghent, Belgium
E-mail:
FAX: +32 09 2646219

Jagdev M. Sharma
Department of Veterinary PathoBiology
258 Veterinary Science Building
College of Veterinary Medicine
University of Minnesota
1971 Commonwealth Avenue
St. Paul, Minnesota 55108
E-mail: sharmOO 1 @umn.edu
FAX: (612) 625-5203

Louis Van Der Heide
Dept. Of Pathobiology U-89
PO Box 37
12 Yeomans Road
Columbia, Connecticut 06237
E-mail:

FAX: (860) 486-2794

Erica Spackman
USDA-Agriculture Research Service
Southeast Poultry Research Laboratory
934 College Station Road
Athens, GA 30605
E-mail:
FAX: (706)546-3161

Pedro Villegas
Department of Population Health
College of Veterinary Medicine
University of Georgia
953 College Station Road
Athens, Georgia 30602-4875
E-mail:
FAX: (706) 542-5630

David L. Suarez
USDA-Agriculture Research Service
Southeast Poultry Research Laboratory
934 College Station Road
Athens, GA 30605
E-mail:
FAX: (706)546-3161

Jaap A. Wagenaar
Department of Infectious Diseases and Immunology
OIE Reference Laboratory for Campylobacteriosis

and WHO Collaboration Centre for Campylobacter
Faculty of Veterinary Medicine
Utrecht University
P.O. Box 80.165
3508 TD Utrecht
The Netherlands
E-mail: i
FAX: +31 (0)30-2533199

David E. Swayne
USDA-Agricultural Research Service
Southeast Poultry Research Laboratory
934 College Station Road
Athens, Georgia 30605
E-mail:
FAX: (706) 546-3161

vii


Patricia S. Wakenell
Department of Veterinary Medicine
Population Health & Reproduction
University of California
Davis, California 95616
E-mail: pswakenell@ucdavis. edu
FAX: (530) 752-5845

Peter R. Woolcock
California Veterinary Diagnostic Laboratory System

Fresno Branch
School of Veterinary Medicine
University of California - Davis
2789 South Orange Ave
Fresno, California 93725
E-mail:
FAX: (559) 485-8097

W. Douglas Waltman
Georgia Poultry Laboratory
P.O. Box 20
4457 Oakwood Road
Oakwood, Georgia 30566
E-mail:
FAX: (770) 535-1948

Ching-Ching Wu
Purdue University
Animal Disease Diagnostic Laboratory
406 South University Street
West Lafayette, Indiana 47907-2065
E-mail:
FAX: (765) 497-1405

Richard L. Witter
USDA- Agricultural Research Service
Avian Disease and Oncology Laboratory
3606 East Mount Hope Road
East Lansing, Michigan 48823
E-mail:

FAX: (517) 349-0817

Roger Wyatt
195 Edgewood Dr. SW
Athens, GA 30606
(706) 548-2297
E-mail:

viii


PREFACE
This manual has its origins in the need for a book to codify standardized method for testing and evaluating poultry vaccines. The
National Academy of Sciences sponsored the original publication entitled Methods for the Examination of Poultry Biologies, and completed
two successive revisions. In 1975, the American Association of Avian Pathologists (AAAP) accepted responsibility for the publication, but
because the need for standardizing testing of vaccines had been met, the scope and purpose of the manual was broadened to be a resource for
laboratory procedures for the isolation and identification of disease-causing agents. The title was changed to Isolation and Identification of
Avian Pathogens for the 1st edition and to A Laboratory Manual for the Isolation and Identification of Avian Pathogens for the 3rd edition.
The manual was intended as a bench resource for daily use in the diagnostic laboratory.

With the 4th edition, the focus had shifted from a manual of procedures for isolation and identification of pathogens to a more
encompassing manual for diagnosis of the disease and isolation and/or demonstration of the pathogen. This change was needed because
many clinical specimens are presented as unknowns for determination of etiology and some pathogens are difficult to isolate, but can be
demonstrated with newer molecular or immunologic techniques.
With the 5th edition, the AAAP appointed Dr. Louise Dufour-Zavala as Editor-in-Chief. David Swayne served as advisor to the Editorin-Chief and section editor. John Glisson, Willie M. Reed, Mark W. Jackwood and James E. Pearson continued as section editors for their
expertise in bacteriology, veterinary diagnostics, molecular biology, and virology. Peter Woolcock was newly appointed to support the
virology section.

The 5th edition has a new title to reflect the molecular advances and modem testing procedures allowing us to characterize, type,
speciate and serotype several avian pathogens. It also has a new chapter on Turkey Coronavirus. A color plate with tissue culture images is

included. Improvements to individual chapters include updated terminology and information on molecular techniques.
In the appendix section, we removed the appendix of sources and the appendix of reference antisera as they are now readily available on
the Internet.

The editorial committee thanks all of the 67 contributors who prepared new chapters or revised existing chapters for the 5th edition. We
also thank Crissie Boyd, Georgia Poultry Laboratory Network, for her tremendous clerical support in formatting the Manual. We thank Heidi
Migalla, HMM photography, for the design of the cover and Aaron Nord at Omni Press for the printing services.
Editorial Committee:
Louise Dufour-Zavala, Editor-in-Chief
David E. Swayne
John R. Glisson
Mark W. Jackwood
James £. Pearson
Willie M. Reed
Peter Woolcock

ix


X


1
Diagnostic Principles
Frederic J. Hoerr
In avian diagnostic medicine the usual immediate challenge is
making a definitive diagnosis for the presenting problem of
morbidity or mortality. When the case is probed deeper, however,
evidence may emerge of a concurrent or preceding infectious
disease, management or nutritional problem, or other condition that

contributes to the presenting problem. In food supply medicine
involving a population of animals, the challenge is to evaluate one
or more diagnoses for priority of response. In practice, the
diagnostic results are considered along with animal welfare,
production economics, and public health in the development of
strategies for treatment, prevention, and control of the disease.
Three factors influence the expression of an infectious disease: the
virulence of the causative organism, the level of exposure or dose of
the inoculum, and the susceptibility of the host. Within a poultry
flock, each individual reacts according the net influence of these
factors. The uniformity of exposure and resistance among the
individuals in the flock will eventually influence flock performance
and possibly food safety.

pneumovirus, infectious bronchitis, infectious laryngotracheitis,
infectious bursal disease, chicken infectious anemia, reovirus,
hemorrhagic enteritis virus, mycoplasmosis, infectious coryza,
pasteurellosis, and others. For example, isolation of infectious
bronchitis virus is an important first step in understanding a
respiratory disease. If the virus is determined to be a new serotype,
a significant change in vaccine virus serotype will be required to
control the disease. If the isolated virus is indistinguishable from a
vaccine strain administered at an earlier age, it may indicate the
need for improvements in vaccine administration, or point to
immunosuppression from infectious bursal disease, chicken
infectious anemia, or other diseases.
Some vaccine viruses are readily isolated for several days post­
administration. Extended periods of virus shedding in a flock may
reflect variable immunity existing at the time of vaccine
administration. For respiratory viruses, isolation of a vaccine virus

for a prolonged period after administration can be indicative of socalled rolling reactions. In this situation, poor uniformity in flock
immunity will result in some chicks being resistant to virus
infection at the time of vaccine administration, then becoming
susceptible during the end of the shedding period of a pen mate.
The attenuated virus infection spreads from bird-to-bird resulting in
vaccination reactions of prolonged duration. Attenuated vaccine
viruses may re-acquire virulence characteristics during this process.
Rolling reactions and lengthened periods of vaccine virus isolation
also occur with uneven or ineffective vaccine administration
technique. Vaccine virus shed from infected pen mates eventually
infects susceptible chicks causing uneven and sometimes harsh
reactions within the flock. Differentiating vaccine strains from wild­
type or field-challenge viruses or bacteria in the laboratory may
require molecular genetic sequencing and analysis.
The presence of co-pathogens can increase the severity of a viral
disease such as infectious bronchitis or the respiratory form of
Newcastle disease. Mycoplasma infections and elevated
concentrations of ammonia and airborne dust increase the severity
of respiratory viral disease. In these situations, the actual cause of
death or economic loss in the form of condemnations at slaughter
from respiratory compromise or septicemia caused by Escherichia
coli. While the laboratory effort may focus on the viral infection,
the cumulative effects of the co-pathogens must also take into
consideration for effective treatment and control. This situation
requires a comprehensive approach to diagnostics, including
virology, bacteriology, serology, and pathology.
Specific-pathogen-free sentinel birds are useful in identifying
specific primary infectious pathogens. These are typically used
when interference from overwhelming co-pathogens or vaccine
strains obscures the isolation effort of primary pathogen. Sentinel

birds can be placed in a flock for a specific time and then brought
into the laboratory for pathogen isolation and identification
procedures. Selective immunization of the sentinels focuses the
susceptibility and thereby increases the efficiency of identifying the
challenge strain.
The relative significance of an isolate also depends on whether it
has food safety or public health significance. For example,
Salmonella, Escherichia, and Campylobacter can be pathogenic for
poultry but even in the apparent absence of disease they can be
significant contaminants on processed poultry or poultry products.
Avian influenza virus has strain-variable virulence among different
poultry species, and some strains have considerable public health
significance.

HISTORICAL PERSPECTIVE

The very existence of this book makes a statement about the
importance of infectious diseases that affect avian species.
Detection and characterization of infectious pathogens have
advanced substantially in recent decades, building on classical
methods for cultivation, isolation, characterization, and
immunological assay. Procedures based on the specific molecular
genetic characteristics of disease agents are now extensively applied
for the detection and characterization of infectious pathogens.
Diagnostic technology today uses a variety of sensitive and specific
methods that may not require the actual cultivation and isolation of
an organism for a diagnosis. Isolation of an agent still has an
important place in the investigative process. Isolates are important
in assessing virulence and pathogenesis, and in the development of
vaccines. Detection technologies offer significant gains and

substantial advantages in the number of samples that can be tested
in a short time, and in the overall efficiency of testing. Enzymelinked immunological detection (antigen capture ELISA) is used for
viral and bacterial diseases. The polymerase chain reaction and
nucleotide probes offer many approaches for identification of
fragments of genetic code specific to a species or biotype of
organism. A panel or multiplex of serological assays can reveal the
spectrum of infectious agents that have infected an individual bird.
Simultaneous statistical analysis of ELISA titers for various
infectious pathogens helps to assess the frequency and distribution
of the infection or vaccine-induced humoral immunity within the
flock. Advances in diagnostic expertise that improve sensitivity and
specificity, and reduce the time required for the test generally lead
to improvements in disease prevention and control. This is essential
to safeguarding poultry health in the ever larger size of poultry
flocks under the care of food supply medicine.

SIGNIFICANCE OF IDENTIFIED PATHOGENS
An isolated or detected disease agent should be evaluated for
diagnostic significance relative to the case history, clinical disease,
and lesions. Poultry in commercial flocks may experience
sequential or simultaneous disease challenges, as well as exposure
to agents in attenuated live vaccines. In a diagnostic investigation,
both field challenge agents and attenuated live agents may be
detected and require further characterization and differentiation.
Examples of live vaccines include those for Newcastle disease

1


Frederic J. Hoerr

microorganisms. Maintenance and calibration of equipment should
include but not be limited to scales, precision pipettes, incubators,
refrigerators, freezers, autoclaves, spectrophotometers and other
visualization apparatuses, and electrophoresis equipment.
Recording, documentation and review of laboratory procedures
should be routine. Commercial test kits should be used according to
manufacturer’s recommendations. Inventories of reagents and test
kits should be managed with respect to expiration dates.

DISEASE REPORTING

The isolation and identification of certain pathogens carries the
responsibility of reporting to state/provincial or federal regulatory
officials, who in turn have responsibilities to report internationally
to the World Organization for Animal Health (OIE) (1). The OIE
website maintains a current list of internationally reportable
diseases which includes Newcastle disease, turkey rhinotracheitis,
any H5 or H7 avian influenza, Pullorum disease, and others.
Reporting regulations vary among countries, states, and provinces,
as do the definitions of reportable strains of an agent. Laboratory
diagnosticians generally report to the next level of authority in their
organization. Question about the responsibility to report a disease
should be directed to the state or regional veterinary regulatory
health official. Trade, regulatory and other legal issues may
surround the isolation and accurate identification of certain
pathogens, or serological evidence thereof. Erroneous reporting,
misidentification or failure to identify an agent may carry serious
consequences. In the United States, the USDA National Veterinary
Services Laboratory is the agency that should be consulted about

agent identification, and in other countries, the appropriate central
reference laboratory.

LABORATORY SAFETY

The avian diagnostic laboratory today should operate under
standard biosafety criteria (4). Although some avian procedures can
be conducted at biosafety level 1, biosafety level 2 is desirable
because some avian pathogens are agents of moderate potential
hazard to personnel. Biosafety level 3 may be required for viruses
and bacteria that may cause serious or potentially lethal disease as a
result of exposure by the inhalation route. Salmonella,
Campylobacter, Chlamydia, Erysipelas, Escherichia and Newcastle
disease can infect persons causing clinical disease from mild
conjunctivitis to systemic illness. Certain strains of highly
pathogenic influenza virus may cause human fatalities. Fungal
cultures carelessly handled can result in massive release of spores
into the laboratory environment. Laboratory workers should be
informed about these risks and trained in proper procedures for
routine and specialized aspects of laboratory duties. Laboratories
should be kept clean and orderly, and surfaces frequently cleaned
and disinfected. Warning notices should be displayed by
laboratories which contain hazardous substances or conditions.
Biosafety cabinets of adequate rating should be maintained and
fully functional. Eating, drinking, and smoking should be prohibited
in laboratory work areas. Personal protective equipment including
appropriate laboratory clothing is always in order, and respiratory
masks and protective eye glasses should be worn when zoonotic
pathogens are suspected.

QUALITY MANAGEMENT
A quality management program to include standard operating
procedures for the isolation and identification of infectious
pathogens improves the overall consistency and quality of the
laboratory effort. A quality management program evaluates
laboratory procedures for the yield of relevant and timely data. It
involves quality assessment by specifying performance parameters
and setting limits for acceptable performance, and quality
improvement by correcting problems and preventing their
reoccurrence.
Quality management helps to ensure that the
laboratory information is accurate, reliable and reproducible, with
the goal of eliminating or reducing test variation within and
between laboratories. The elements of a quality program comprise
written procedure manuals, record keeping, documentation, and
retention, training, education and evaluation of personnel,
proficiency testing, laboratoiy safety, and maintenance and
monitoring of equipment.
The International Standards Organization (ISO) is a developer and
publisher of international standards for laboratoiy quality
management (2). ISO is a non-governmental organizational network
composed of the national standards institutes that currently
represent 157 countries. ISO document 17025 is the main standard
for diagnostic laboratories worldwide, including the USDA
National Veterinary Services Laboratory. These standards are
reflected in the essential requirements for accreditation by the
American Association of Veterinary Laboratoiy Diagnosticians
(AAVLD), which accredits publically-supported diagnostic
laboratories in the United States and Canada (3). The AAVLD
defines laboratoiy quality management to involve validation of test

methods, among other criteria. Validation of a test requires ongoing
documentation of internal or inter-laboratory performance using
known reference standard(s) for the species and/or diagnostic
specimen(s) of interest, and one or more of the following: the test is
endorsed or published by reputable technical organization
(including the American Association of Avian Pathologists
Isolation and Identification of Avian Pathogens); published in a
peer-reviewed journal with sufficient documentation to establish
diagnostic performance and interpretation of results; or
documentation of an internal or inter-laboratory comparison to an
accepted methodology or protocol.
In the avian diagnostic laboratory, quality management may
include the utilization of check tests, reference sera, and reference
strains of infectious agents, and maintenance of stock cultures of

STORAGE AND ARCHIVING OF ISOLATES
A diagnostic laboratory has a wealth of information and valuable
material pass through it. The agents isolated from commercial
poultry represent a selection process involving large homogeneous
animal populations under performance rearing conditions. These
isolates have value for investigations of virulence and pathogenesis;
epidemiology of emerging agents; evaluation of new drugs and
vaccines, and potentially as new vaccines. Isolates from
noncommercial poultry have value in comparative studies and in
epidemiology. All viruses and most bacteria require ultracold (-70
F or lower) storage that can be achieved with specialty freezers or
liquid nitrogen storage. Some bacteria and fungi can be stored in
special media at room temperature. Specific information about
storage and archiving of samples is provided in the text.

References
1. OIE: World Organization for Animal Health,
classification2007.htm?eld7.
2. International Organization for Standardization,
/>3. American Association of Veterinary Laboratory Diagnosticians.
AAVLD Essential Requirements for An Accredited Veterinary
Medical Diagnostic Laboratory.
/>d=aavld.
4. Centers for Disease Control and Prevention. BMBL Section ΙΠ:
Laboratoiy Biosafety Criteria.
http: //www.cdc. gov/od/ohs/biosfty/bmbl4/bmbl4 s3 .htm.

2


2
SALMONELLOSIS
W. Douglas Waltman and Richard K. Gast

SUMMARY. Avian Salmonella infections are important as both a cause of clinical disease in poultry and as a source of food-bome
transmission of disease to humans. Host-adapted salmonellae (5. pullorum and S. gallinarum) are responsible for severe systemic diseases,
whereas numerous serotypes of non-host-adapted paratyphoid salmonellae are often carried subclinically by poultry and thereby may
contaminate poultry products. Crowding, mal-nutrition, and other stressful conditions as well as unsanitary surroundings can exacerbate
mortality and performance losses due to salmonellosis, especially in young birds.
Agent Identification. Salmonella infections in poultry flocks are diagnosed principally by the isolation of the bacteria from clinical tissue
samples, such as pooled internal organs and sections of the intestinal tract. Egg contents may also be cultured to detect S. enteritidis.
Preliminary identification of infected or colonized flocks is often obtained by culturing poultry house or hatchery environmental samples,
such as drag swabs, litter, dust, feed, and hatch residue. Clinical and environmental samples are generally subjected to selective enrichment,
with nonselective preenrichment sometimes employed when salmonellae are present in very small numbers or are potentially injured.
Delayed secondary enrichment often improves Salmonella recovery from many sample types. Samples are plated on selective agar and

bacterial colonies are identified as Salmonella by further biochemical and serological testing.
Serologic Detection in the Host. Serologic identification of infected poultry plays an important role in programs for controlling the spread
of Salmonella in commercial flocks, especially in regard to S. pullorum and 5. gallinarum. Agglutination tests, particularly the rapid whole­
blood plate test, are used for verification of the Salmonella status of flocks participating in the National Poultry Improvement Plan.

measures be followed any time individuals go onto a farm or into a
hatchery.

INTRODUCTION
Avian Salmonella infections are important as both a cause of
clinical disease in poultry and as a source of food-bome
transmission of disease to humans. Host-adapted salmonellae are
responsible for pullorum disease (S. pullorum) and fowl typhoid
(S. gallinarum), which are severe systemic diseases that have
become relatively rare in countries with testing and eradication
programs (13). Infections with non-host-adapted (paratyphoid)
salmonellae are common in all types of birds (9,10), although some
serotypes are found predominantly in a fairly limited range of hosts
(e.g., S. arizonae is isolated mostly from turkeys). Crowding, mal­
nutrition, and other stressful conditions as well as unsanitary
surroundings can exacerbate mortality and performance losses due
to salmonellosis. Paratyphoid salmonellae can also cause human
illness, and food-bome Salmonella outbreaks can lead to severe
economic losses to poultry producers as a result of regulatory
actions, market restrictions, or reduced consumption of poultry
products.

Clinical Cases
Samples taken from the internal organs of the birds with systemic
salmonellosis are typically free of competing bacteria. In such

cases, isolation is usually relatively simple, and may only require
direct plating on nonselective media. Liver, spleen, heart, heart
blood, ovary or yolk sac, synovial fluid, eye, and brain (if torticollis
is observed) are excellent sources for recovery. Sterile cotton-tipped
swabs are preferred to wire inoculating loops, except for small
organs in young birds, because swabs transfer more tissue. Several
tissues should be cultured from each bird, as tissues with lesions do
not always yield salmonellae. Portions of several tissues may be
pooled and added to selective enrichment broth to increase the
possibility of isolation (Fig. 2.1). Sections of the intestinal tract,
especially the ceca and cecal tonsils, may be cultured by inoculation
into selective enrichment broth.
Serologic Reactors
Because of the potential consequences involved in culturing
serologic reactors, especially pullorum-typhoid, these cases should
be cultured more intensively than routine clinical cases. Also, in
some situations reactor birds may be asymptomatic, the salmonellae
may be present in smaller numbers than in clinical cases, or they
may be more localized. Recent use of antimicrobial agents may
prevent the recovery salmonellae, although they may not
completely clear the organism from the bird.
Reactors should be evaluated by both direct and selective culture
procedures (Fig. 2.1) (15,17). Tissues showing any pathologic
lesions should be sampled using a sterile cotton-tipped swab and
inoculated onto a nonselective plating medium. If desired the swab
may be broken off into a tube of nonselective broth, incubated at
35-37 C for 24 hr, and plated onto nonselective agar.
Portions of the liver, spleen, heart, gall bladder, ovaiy and oviduct
should be pooled (10-15 g total) and then minced, blended, or
stomached. Selective enrichment broth is added to the tissue (one

part tissue homogenate to 10 parts broth) and incubated at 35-37 C
for 24 hr, and if negative they are incubated for an additional 24 hr.
After the internal organ samples have been removed, the intestinal
samples may be taken. Portions of the ceca and the cecal tonsils
(and other sections of the intestinal tract including the crop may
added) should be pooled and minced, blended, or stomached.
Selective enrichment broth is added to the sample (one part sample
to 10 parts broth), incubated typically at 41 C for 24 hr, and plated
on selective plating media.

CLINICAL DISEASE
Most salmonellae are transmitted horizontally, but some serotypes
(such as S. pullorum and S. enteritidis) can be transmitted vertically
and often produce highly persistent flock infections. Pullorum
disease primarily affects chickens, turkeys, and other fowls during
the first few weeks of life. Fowl typhoid is also observed in mature
poultry. These diseases are uncommon in pet birds species and
pigeons. Paratyphoid salmonellae, particularly some strains of
S. enteritidis, can occasionally cause morbidity or mortality in
young birds of diverse species.
Histopathologic lesions associated with avian salmonellosis include
fibrinopurulent perihepatitis and pericarditis; purulent synovitis;
focal fibmoid necrosis, lymphocytic infiltrations, and small
granulomae in various visceral organs; and serositis of the
pericardium, the pleuroperitoneum, and the serosa of the intestinal
tract and mesentery (9,10,13). Infections with S. arizonae can
produce encephalitis and hypopyon in young turkeys and chickens.
SAMPLE COLLECTION

Both clinical tissue and environmental samples should be collected

as aseptically as possible to prevent cross-contamination. This
includes the use of sterile sampling materials (e.g., swabs, scoops,
bags) and disposable gloves. It is critical that strict biosecurity
3


W. Douglas Waltman and Richard K. Gast

levels above 0.85 appear to promote the environmental survival and
multiplication of salmonellae.
The frequency and extent of sampling often varies according to the
purpose of the monitoring. For example, the NPIP has established
guidelines for testing breeding flocks for salmonellae (15).
Environmental samples pose a challenge to the detection of the
salmonellae, because salmonellae are often present in low numbers,
salmonellae may be present but injured, and large populations of
other bacteria are often present. Therefore, highly selective
enrichment and plating media must be used.
Drag Swabs. Flock infection or contamination can be conveniently
detected by use of drag swabs. Properly assembled and sterilized 3or 4-square inch gauze pads moistened with DSSM are drawn by 34-ft lengths of cord across the surface of the floor litter or dropping
pit for 15-20 min. The full length of the occupied building is
traversed one or more times. The swabs are placed in individual
sterile plastic bags at the farm and transported as soon as possible to
the laboratory on ice packs. Selective enrichment broth is added
directly to the bags containing the swabs (15). An alternative to the
gauze pad is a commercially available sponge. Caution is advised to
make sure these sponges are specially made for culturing purposes,
because many household-type sponges contain bacteriocidal or
bacteriostatic substances.
In studies with broiler chickens, results from several unpooled drag

swabs closely reflected intestinal excretion rates of salmonellae in
the flock (7). Such sampling circumvents more cumbersome
methods of environmental monitoring (5).
Floor Litter. Samples should be collected from representative dry
areas of floor litter; wet and caked ateas, including those around
waterers and feeders, should be avoided because salmonellae
survival may be poor in such areas. A sterile wooden tongue
depressors or similar device is used to collect 5 g of dry litter from
the upper 2.5-5.0 cm (1-2 inches) of floor litter into a sterile plastic
bag from five sites in the pen or house. Additional sample pools
should be collected from other areas of the house or pen to obtain
the following ratio of samples to birds per pen: fewer than 500
birds, 5 pooled samples; 500-2500 birds, 10 pooled samples; 2500
birds or more, 15 pooled samples (15). The sample numbers
suggested above are necessary for reasonable dependability in
monitoring semimature or mature flocks in which excretion rates
may be very low.
Nest Boxes or Egg Belts. Loose litter in nests is a preferred sample
site after breeder hens have been in lay 2-4 wk. Following the same
procedure for the nest litter as given for the floor litter, collect fine
material from the bottoms of at least five nests for each pooled
sample. Samples need not be weighed or measured after some
experience is acquired, but weight should not exceed 10-15 g per
pooled sample.
Many companies no longer use wood shavings in their nest boxes
because of automated egg collection. In lieu of sampling nest
shavings, swabbing the inside of an equal number of nest boxes
using DSSM moistened gauze pads has been shown to be effective.
Each pad may be used to sample 5-10 nest boxes and then placed
into a sterile bag for culture. It is a good practice to combine

sampling the floor, by using either litter or drag swabs, with nest
sampling.
In lieu of swabbing the nest boxes in houses with mechanical nests,
the egg belts may be swabbed with DSSM-moistened swabs,
usually at the front of the house.
Dust. In some regions, dust has been a more dependable source for
isolation of salmonellae than litter. Local experience should be a
guide for determining the most dependable source. Samples are
collected by DSSM moistened gauze pads or by wooden tongue
depressors from 15 or more sites of dust accumulation per pen.
Depending on the volume collected, five samples may be pooled
into one. Use cotton-tipped swabs only for hard-to-reach areas.

One-Day-old Hatchlings
It is often important to determine the presence of Salmonella
contamination or infection in 1-day-old chicks or poults. Four
methods have been used. The first method, approved by the
National Poultry Plan(NPIP), takes 25 1-day-old chicks (in groups
of 5) and pools the internal organs, yolk sac, and intestinal tracts
from each group in three separate sterile plastic bags (16). Selective
enrichment broth is added to all five groups of pooled samples.
A second method cultures chick meconium collected during chick
handling for sexing. Pools of about 5 g of meconium are collected
from the chicks into sterile plastic bags and inoculated with
selective enrichment broth.
A third method cultures papers that line the boxes the chicks are
transported in from the hatcheiy to the farm. The surface of these
chick papers may be swabbed with double-strength skim milk
(DSSM)-moistened gauze pads or by directly placing pieces of the
paper into a sterile plastic bag and adding selective enrichment

broth.
A fourth method, with good sensitivity, uses 50-100 chicks that
have been held for 48-72 hr with only clean water available for
consumption. This promotes widespread dissemination of infection
among boxmates and increases the likelihood of detection by
culturing the paper liner or even by cloacal swabbing of the birds in
some cases. However, an important difficulty in using this method
is that the chicks must be maintained without cross contamination
from external sources of Salmonella.
Cloacal Swabs
Cloacal swabs have been shown to be an unreliable means of
detecting salmonellae from birds (4). Generally, salmonellae are
excreted intermittently, and often in low numbers. Furthermore
cloacal swabbing of birds requires laborious culture of 300-500
birds to provide dependable results.
Egg Culture
The surface of intact eggs can be sampled by immersion in 30-50
ml of selective enrichment broth in a plastic bag, with soaking or
manual rubbing for at least 30 sec before removal of the egg.
Eggshells can also be manually crushed in selective enrichment
broth to allow more complete access of the media to internal
surfaces. After disinfection of eggshells (generally in 70% ethanol
or 2% tincture of iodine), egg contents can be cultured at a standard
1:10 ratio in selective or nonselective media.
The contents of 10-30 eggs are often pooled for culturing because
of the usual low incidence and level of Salmonella contamination.
To allow salmonellae to multiply to easily detectable levels (and to
minimize media consumption), pools of egg contents should be
incubated for at least 1 day at 37 C or 3 days at 25 C before
subculturing into broth media. Incubated egg pools can be

transferred directly onto selective agar, but significantly greater
detection sensitivity can be attained by using one or more
enrichment steps.
Environmental Monitoring
Environmental monitoring or surveillance has become a useful
method for predicting potential infection or colonization of flocks
with the paratyphoid salmonellae. Environmental sampling has been
shown to be effective and less invasive than other sampling
methods. Although environmental monitoring may show good
predictive evidence of salmonellae in a flock, it is an indirect
indicator and flocks should not be diagnosed as infected based
solely on the environmental sampling.
The probability of detecting salmonellae in environmental samples
depends on the number of salmonellae excreted into the
environment by the flock, the survival of salmonellae in the sample
locations, the intensity of sampling, and the culture methods used.
Fecal shedding of salmonellae is often greatest during the first few
weeks of life, when the susceptibility of chicks to infection is
greatest. Litter surface water activity (equilibrium relative humidity)
4


Chapter 2

Cage Housing. The best methods of sampling for cage houses have
not been fully determined. Methods in current use include sterile
gauze pads moistened with DSSM to aseptically collect material
from egg transport belts and elevators, rollers, diverters, and manure
scrapers or cables. All segments of the house should be sampled.
The drag swab method is also used with layer cages that allow

droppings to accumulate under cages.
Hatcheries. The area most likely to yield salmonellae is the
hatcher during or following hatching. Selective Agar plates, opened
and exposed for 5-10 min to the air in various parts of the hatcheiy
building, have been used to monitor hatcheries. However, failure to
find salmonellae on such plates is not a reliable indication of
freedom from hatchery contamination.
A sterile tongue depressor may be used to collect at least one
heaping tablespoonful of fluff and dust from each hatcher. This
sample is placed into a sterile plastic bag with selective enrichment
broth.
The most reliable sample has been the hatch residue (2). About 1015 g of eggshells and other hatch residue remaining after the chicks
are removed from the hatch trays is placed into a sterile plastic bag
to which selective enrichment broth is added. Alternatively, several
hatch trays may be swabbed with gauze pads and pooled.
Feed and Feed Ingredients. About 100 g of feed or feed
ingredients should be collected into a sterile plastic bag representing
multiple sites in a lot of feed. Care should be taken to prevent cross
contamination of the samples.
The Association of Official Analytical Chemists (AOAC)
International method is the most widely accepted procedure for
culturing feed and feed ingredients (1). Twenty-five grams of feed
is mixed with 225 ml of lactose broth. The mixture is allowed to
stand for 1 hr and the pH is adjusted to 6.8 + 0.2. The culture is
incubated at 35 C for 24 hr. One milliliter of the culture is
transferred into 10 ml of tetrathionate (TT) broth and 0.1 ml is
transferred into Rappaport-Vassiliadis (RV) broth. The two
enrichment broths are incubated at 43 C and 42 C, respectively.
After 24 hr, the cultures are inoculated onto bismuth sulfite,
Hektoen enteric (HE), and xylose lysine desoxycholate (XLD)

agars.
Some investigators have suggested that this procedure be modified
by using universal preenrichment or buffered peptone water (BPW)
as the preenrichment broth; by reducing the selective enrichment
incubation temperatures to 41 C; and by replacing the plating media
with more selective agars, such as brilliant green supplemented with
novobiocin (BGN) and xylose lysine tergitol 4 (XLT4) agars.

Salmonellosis

PREFERRED CULTURE MEDIA AND SUBSTRATES

Recommended Steps, Media, and Incubation Times and
Temperatures.
The isolation and identification flow chart in Fig. 2.1 details the
selection and use of preferred media in a variety of testing
situations. Selective enrichment broths are typically inoculated at a
1:10 ratio of sample to broth, except for RV enrichment, which is
inoculated at a 1:100 ratio from a pre-enrichment broth.
Typically, internal organ samples and samples having lower
background levels of bacteria may be incubated at 35-37 C.
Intestinal and environmental samples having higher levels of
background bacteria are commonly incubated at 40-43 C. Because
of potential incubator problems and the sensitivity of some strains
to higher temperatures, the preferred incubation temperature is
41+0.5 C. Routine monitoring of the temperature of incubators is
critical when using the higher temperatures.
Selective enrichment broths should be incubated 20-24 hr and
plated. Use of a delayed secondary enrichment (DSE) procedure is
highly recommended (see below), but if not, the enrichment broths

should at least be incubated an additional 24 hr and replated.
Precautions for Environmental and Intestinal Samples
Isolation of salmonellae from environmental and intestinal
monitoring samples is much more demanding than isolation from
samples collected from bacteremic (clinically affected) or carrier
(serologic reactor) birds. In bacteremic or carrier birds, salmonellae
populations are often high, particularly with respect to bacterial
competitors. In contrast, salmonellae levels are ordinarily low in
environmental or intestinal samples where competing bacteria are
found in high levels. These competitors, if not controlled, seriously
impair media detection efficiency, resulting in false negatives.
The dependable isolation of salmonellae from environmental and
intestinal samples is, therefore, significantly improved by
incubation of enrichment broths at 41 + 0.5 C for a full 24-36-hr
period, use of novobiocin- or tergitol (niaproof) 4-supplemented
media, DSE of primary selective enrichment broths, and use of a
plate-streaking technique that produces well-separated colonies.

Non-Selective Media
Salmonellae grow well on such nonselective broth media as veal
infusion, brain-heart infusion, or nutrient broth. Nonselective agars
that may be used include blood agar and nutrient agar. MacConkey
agar, although mildly selective, is frequently used as a nonselective
medium.
These media are preferred for tissues from clinical cases and
serologic reactors in which the likelihood of competing bacteria is
low. Use of nonselective media is important when S. pullorum or S.
gallinarum are suspected, as some strains may be inhibited by
selective media.

Shipping and Storing Samples
Holding Media. Studies have shown that the best moistening agent
and holding media for environmental swabs is DSSM (12). This
medium is prepared by dissolving 200 g Bacto Skim Milk (BD
Diagnostics Systems, Sparks, MD) in 1 liter distilled or deionized
water in a large flask and autoclaving. The DSSM may be stored in
the refrigerator.
Sample Storage Times and Temperatures. Specimens from
clinical diagnostic consignments and serologic reactors should be
cultured immediately upon collection and no more that 24 hr after
collection even if they are refrigerated. Meconium, freshly voided
feces, and water should be refrigerated at Ί-b C as soon as possible
and inoculated into appropriate culture media within 24 hr. Drag
swabs and other swabs containing DSSM may be refrigerated for 3
days or frozen for 7 days before inoculation of selective enrichment
broth (12). Because the survival rate of salmonellae in floor litter
samples varies considerably, even under refrigeration, it is best to
culture floor litter within 2 days of collection. This is advisable
despite the fact that some dry environmental samples have yielded
salmonellae when held for several months at room temperature and
low humidity.

Pre-enrichment
Preenrichment is the process of inoculating nonselective broth
media with the sample and incubating it for 24 hr at 35-37 C.
Typically, 1.0 ml of the preenriched culture is transferred to 10 ml
of a TT enrichment broth or 0.1 ml is transferred to 10 ml of RV
enrichment broth. The purpose of preenrichment is to revive injured
salmonellae that may be present in some samples. Typical
preenrichment media include lactose broth and BPW.

Selective Enrichment Broths
Tetrathionate Enrichments. Tetrathionate broths are the preferred
selective enrichments for salmonellae isolation. Older formulations,
such as Mueller-Kauffrnann TT brilliant green broth, require the
addition of both iodine and brilliant green solutions to the broth
immediately before use. However, newer formulations, such as TT
broth, Hajna, or TT, Hajna and Damon (BD Diagnostic Systems,

5


W. Douglas Waltman and Richard K. Gast

Sparks, MD) are supplied already containing brilliant green and
require only the addition of an iodine solution to the broth base. The
newer TT formulations contain more nutrients and buffers. The
iodine solution differs for the individual TT formulations, so care
must be taken to ensure the appropriate one is used and that it is
prepared and stored correctly.
Selenite Enrichments. The use of selenite enrichments are no
longer recommended because they have a short shelf life, are not as
effective at higher incubation temperatures (3), potentially
mutagenic (11), and in many geographic areas are considered to be
a hazardous waste.
Rappaport-Vassiliadis Enrichments. A selective enrichment that
is gaining acceptance and appears to be replacing selenite
enrichments is RV broth. The RV enrichment procedure begins with
preenrichment of the sample in BPW and then inoculation into RV
broth at a ratio of 1:100. The RV broth should be incubated at 4142 C.

Xylose Lysine Desoxycholate, XLD Supplemented with
Novobiocin, and XLT4 Agars. The differential characteristics of
these media include the lysine decarboxylation and H2S-producing
abilities of most salmonellae. Hydrogen sulfide-positive
salmonellae colonies are nearly or fully jet black. Proteus (inhibited
on XLDN and XLT4) colonies may appear blackish, but the color is
less brilliant, tending to have a gray or green cast as the colonies
age. Colonies of Citrobacter freundii ordinarily have black centers
and prominent creamy-colored borders on these media.
Other Plating Media. Bismuth sulfite and Hektoen enteric agars,
although widely used in human clinical situations and advocated by
the Food and Drug Administration (FDA) and AOAC International
for food and feed, lack the sensitivity and specificity of BGN and
XLT4. Modified lysine iron agar (MLIA) incorporates novobiocin
and is similar in recovery to BGN or XLDN. Rambach agar, a new
chromogenic agar, appears to be effective, but it is more expensive
than other options. Recently, several other chromogenic agars have
been developed for the isolation of Salmonella. The most
thoroughly studied of these, Miller-Mallinson (MM) media, has
been found to be very effective.

Delayed Secondary Enrichment (DSE)
After the 24-hr incubation of the TT and plating onto BGN and
XLT4 agars, the TT culture is left at room temperature for 5-7 days.
If the original plating was negative for salmonellae, 0.5-1.0 ml of
the original TT broth is transferred into 10 ml of fresh TT broth and
incubated at 37 C for 24 hr. The new TT broth is plated and
processed as before (16). Studies have shown that DSE increases
the recovery rates of salmonellae from most sample types (18).

Rapid Salmonella Detection Techniques
A number of rapid tests have been approved for detecting
salmonellae in various samples. Typically these have been approved
based on comparison to AOAC International culture procedures (1).
The rapid tests have several advantages over conventional culture,
such as obtaining results in less than 48 hr, being less labor
intensive, and having the possibility of automation. For many
sample types, especially clinical, processing plant, and food
samples, these rapid tests appear to be veiy effective. However,
some sensitivity and specificity problems are apparent with feed
and environmental samples.
A variety of rapid detection systems are commercially available,
including antigen-capture enzyme-linked immunosorbent assay
(AC-ELISA) systems, DNA probes, polymerase chain reaction
systems, immunodiffusion, immunofluorescence, magnetic bead
systems, bioluminescence, and other novel applications. A listing of
these tests may be found at the FDA-BAM website. Currently,
NPIP has approved a rapid ruthenium-labeled sandwich
immunoassay and two PCR systems for detecting Salmonella in
environmental samples (15). As technology advances the sensitivity
and specificity of rapid systems will surely increase and allow more
widespread use.

Selective Plating Media
Rationale for Using. Because of the high levels of nonsalmonellae
bacteria in most intestinal and environmental samples, selective
plating media must be used to assist the selective enrichment broth
in inhibiting other bacteria. Conventional salmonellae plating media
lacks the selectivity necessary for these samples. Many media can
inhibit most coliforms, however, the primary problems are with

species of Proteus, Providencia, Morganella, and Pseudomonas.
These bacteria resemble salmonellae on some plating media and
must be screened, resulting in high percentages of false-positive
cultures. The addition of 20 pg of novobiocin (N1628 Sigma
Chemical Co., St. Louis, Mo.) per ml of plating media, especially
brilliant green (BG) (14) and XLD agars, results in the inhibition of
Proteus. Another plating medium that has proven to be effective is
XLT4 (BD Diagnostic Systems, Sparks, MD). This medium inhibits
Proteus and Pseudomonas aeruginosa. A combination of two
plating media that are predicated on different selective and
differential characteristics is advocated. BGN and XLT4 are two
good choices (8). The use of BGN agar, which demonstrates the
positive hydrogen sulfide (H2S) production characteristic, increases
the likelihood of detecting atypical strains. Hydrogen sulfide­
negative strains undetected on XLT4 should be obvious on BGN,
because they usually remain lactose-negative. Conversely, lactose­
positive strains (eg., S. arizonae and others) undetected on BGN
should be obvious on XLT4 because they usually remain MS­
positive.
Brilliant Green (BG) Supplemented with Sulfapyridine or
Sulfadiazine (BGS), and BGN Agars. Brilliant green agar has
been supplemented with antimicrobial compounds to make it more
selective. The purpose of adding sulfapyridine or sulfadiazine and
novobiocin was primarily to inhibit Proteus species. Salmonellae
colonies on these media are usually transparent pink to deep
fuchsia, surrounded by a reddish medium. These colonies may lose
this characteristic appearance if there is a heavy growth of lactosefermenting colonies or if the colonies are not well separated. Some
nonsalmonellae bacteria produce colonies similar in appearance to
salmonellae and must be screened further. Host-adapted S. pullorum
and 5. gallinarum colonies are smaller and grow slower than nonhost-adapted salmonellae. Consequently, all plates should be

incubated for 48 hr.

AGENT IDENTIFICATION
Basic Identification Screening Media
The combined use of TSI and LI agar slants is generally sufficient
for presumptive identification of most salmonellae-suspect colonies.
At least three well-separated colonies are selected for transfer to
each set of TSI and LI agar slants. Each colony is stabbed into the
butt of the TSI and LI agars and streaked across the slants. The
tubes are read after incubation for 24 hr at 37 C. The use of more
selective plating media (BGN and XLT4) to screen these
salmonellae suspect colonies results in fewer false positives
(nonsalmonellae bacteria).
The presence of multiple serotypes may be more likely in some
samples than in others. Also, in situations where S. enteritidis, S.
pullorum, qt S. typhimurium are being monitored, and other
salmonellae are present, screening many more colonies may be
necessary to ensure the absence of S. enteritidis, S. pullorum, or S.
typhimurium serotypes. The Colony Lift Immunoassay (Synbiotics
Corp., San Diego, CA) may be useful in detecting group D
salmonellae in these situations (6).
Triple Sugar Iron (TSI) Agar. Most salmonellae produce an
alkaline (red) slant and acid (yellow) butt, with gas bubbles in the
agar and a blackening due to H2S production that often obscures the

6


Chapter 2

acid reaction in the butt of the tube. Salmonella gallinarum does not
form gas in TSI, whereas S. pullorum may show weak gas
production. Both of these Salmonella may or may not show H2S
production. Some paratyphoid strains may be H2S-negative in this
medium.
Lysine Iron (LI) Agar. Salmonellae will show lysine
decarboxylation, with a deeper purple (alkaline) slant and alkaline
or neutral butt with slight blackening due to H2S production, with
the exceptions noted in the previous paragraph. LI agar is useful in
differentiating common intestinal flora such as Proteus and
Citrobacter from further consideration. Proteus (also Providencia
and Morganella) produces a reddish or port-wine colored slant,
indicating lysine deamination, and a yellow (acid) butt on LI agar
medium. Citrobacter gives a purple (alkaline) slant and a yellow
(acid) butt, with some H2S production. Rarely, both slant and butt
are yellow.

Salmonellosis

SEROLOGIC DETECTION IN THE HOST

Serologic Testing of Poultry Flocks
Several serologic tests have been developed for detecting
antibodies to salmonellae. The most commonly used tests for
pullorum-typhoid include the whole blood plate (WBP) (not
approved for use in turkeys), rapid serum plate, standard
macroscopic tube agglutination, microagglutination, and
microantiglobulin tests (9,15). Possibly the most widely used of
these tests is the WBP test, which can be performed in the field
using commercially available antigens. A drop of fresh blood is

mixed with a drop of antigen on a plate, mixed, and observed for
agglutination within 2 min.
The tube agglutination test is also commonly used. It requires sera
for testing with an antigen that may be obtained from the NVSL. In
the macroscopic tube agglutination test the sera are diluted 1:25 to
1:50 for chickens or 1:25 for turkeys. The sera are mixed with the
antigen, incubated at 37 C for 20-24 hr, and observed for
agglutination. Because the WBP test is not approved for turkeys, the
tube agglutination or rapid serum agglutination tests are typically
used.
Agglutination tests have also been used for detecting antibodies to
various paratyphoid salmonellae, especially S. typhimurium, S.
arizonae, and S. enteritidis. These tests have met with varying
degrees of success due to sensitivity and specificity problems and
thus have not found widespread commercial application. Because S.
enteritidis and S. pullorum share somatic antigens, pullorumtyphoid antigen preparations have sometimes been applied for
detecting antibodies to S. enteritidis.
Enzyme-linked immunosorbent assays (ELISAs) have been
developed for detecting antibodies to various salmonellae. The most
widely used ELISAs have been developed for detecting antibodies
to S. enteritidis. These ELISAs have been used with some success,
particularly in Europe, but concerns about their specificity still
persist.
The various serologic tests for detecting antibodies to salmonellae,
especially the agglutination tests, are subject to false positive
reactions. Confirmation of all positive serologic tests by culturing
the bird for salmonellae is important.

Biochemical and Serological Confirmation
Biochemical Identification. Further biochemical tests may be

necessary to confirm an isolate as Salmonella. Table 2.1 lists
several typical biochemical reactions of salmonellae. Key tests for
differentiating salmonellae from other bacteria include those for
urea {Proteus and most Providencia and Morganella are urease
positive), beta-galactocidase (salmonellae with the exception of
S. arizonae, are negative whereas C. freundii and other coliforms
are positive), and indole (Escherichia coli and Escherichia tarda
produce indole, salmonellae do not).
Commercially available identification systems may also be very
useful in confirming isolates. They use a number of biochemical
tests in a simple to use format.
Serological Typing. The presumptive salmonellae colonies
identified by the TSI and LI agar reactions should be serologically
typed. The first phase of serologic typing is to serogroup the isolates
based on their somatic O-group antigens using commercially
available polyvalent somatic antisera. Individual polyvalent antisera
are tested against each isolate in a simple plate agglutination assay.
Bacterial growth from an agar plate is emulsified in a small amount
of physiologic saline to form a milky suspension, and one drop of
polyvalent O antisera is mixed with it on a slide or plate. The
agglutination reaction is read within 60 sec. After determining
which polyvalent antiserum agglutinates with the isolate, additional
testing is performed using individual single factor O-group antisera
that comprised the polyvalent antiserum. In this maimer, each
isolate is typed to a specific serogroup.
The next step, which is often performed at a reference laboratory,
is to determine the serotype. The serotype is based on the flagellar
antigen present on almost all salmonellae, except 5. pullorum and
S. gallinarum. Commercially produced antisera are available for
serotyping most isolates (BD Diagnostic Systems, Sparks, MD).

The serotyping procedure involves an antigen extraction step with
formalin, followed by a microtube agglutination test.

DIFFERENTIATION FROM CLOSELY RELATED AGENTS
Young birds with generalized Salmonella infections may show
signs and lesions identical to any bacteremia. In severe outbreaks of
salmonellosis, fibrinous liver and heart lesions may be very similar
to those seen with air sac disease (colibacillosis). The heavy
yellowish white cheesy exudate covering the retina of turkey poults
with S. arizonae infection and occasionally with other paratyphoid
infections can be confused with signs of aspergillosis. Nervous
signs associated with infection of the brain of fowl may resemble
those of Newcastle disease or other diseases affecting the central
nervous system. Joint involvement may be mistaken for synovitis or
bursitis due to other infectious agents.

7


W. Douglas Waltman and Richard K. Gast

Figure 2.1 General Salmonella /solation and identification

A Hajna TT or Mueller-Kauffmann tetrathionate enrichment broths.
For RV broth, follow special inoculation and preenrichment instructions of manufacturer
B Beef extract, veal infusion, or comparable non-selective media.
A broth detect can help detect low Salmonella levels in live birds.
C BGN in combination with XLT4 is preferred (refer to text)
D Colony lift immunoassays can significantly increase the reliability detecting Group D Salmonella
(S. enteritidis, S. pullorum, etc. on plating agars (refer to text)

E If combined results with TSI and LI agars, additional identification media, and O-group screening
procedures are inconclusive, restreak original colony onto selective plating agar to check for purity.
F Reevaluate if epidemiologic, necropsy or other information strongly suggests the presence of an unusual
strain of Salmonella.

8


Chapter 2
Table 2.1 Typical biochemical reactions of Salmonella K
Paratyphoid
S. pullorum
S. gallinarum
salmonellae
Dextrose
A
A(G)b
AG
Lactose
Sucrose
Mannitol
A
A(G)
AG
Maltose
A
AGe
(-)D
Dulcitol
A

AG
Malonate
Urea
+
Motility
-

Media

Salmonellosis

4. Fanelli, M J., W. W. Sadler, C. E. Franti, and J. R. Brownell.
Localization of salmonellae within the intestinal tract of chickens. Avian
Dis. 15:366-375. 1971.
5. Kingston, D. J. A comparison of culturing drag swabs and litter for
identification of infections with Salmonella spp. in commercial chicken
flocks. Avian Dis. 25:513-516. 1981.
6. Lamichhane, C. M, S. W. Joseph, W. D. Waltman, T. Secott, E. M Odor,
J. deGraft-Hanson, E. T. Mallinson, V. Vo, and M Blankford. Rapid
detection of Salmonella in poultry using the colony lift assay. In:
Proceedings of the Southern Poultry Science Society. Atlanta, Ga. Poult.
Sci. (Suppl. 1)74:198. 1995.
7. Mallinson, E. T., C. R. Tate, R. G. Miller, B. Bennett, and E. RussekCohen. Monitoring poultry farms for Salmonella by drag swabs sampling
and antigen capture immunoassay. Avian Dis. 33:684—690. 1989.
8. Miller, R. G., C. R. Tate, E. T. Mallinson, and J. A. Scherrer. Xylose
lysine tergitol 4: an improved selective agar medium for the isolation of
Salmonella. Poult. Sci. 70:2429-2432. 1991.
9. Gast, R. K. Paratyphoid infections. In: Diseases of poultry, 11th ed. Y. M
Saif, H. J. Barnes, J. R. Glisson, A M Fadly, L. R. McDougald, and D. E.
Swayne, eds. Iowa State University Press, Ames, Iowa. pp. 583-613. 2003.

10. Nagaraja, K. V., B. S. Pomeroy, and J. E. Williams. Arizonosis. In:
Diseases of poultry, 9th ed. B. W. Calnek, H. J. Bames, C. W. Beard, W. M
Reid, and H. W. Yoder, Jr., eds. Iowa State University Press, Ames, Iowa,
pp. 130-137. 1991.
11. Noda, Μ, T. Takano, and H. Sakurai. Mutagenic activity of selenium
compounds. Mutat. Res. 66:175-179. 1979.
12. Opara, Ο. O., L. E. Carr, C. R. Tate, R. G. Miller, E. T. Mallinson, L. E.
Stewart, and S. W. Joseph. Evaluation of possible alternatives to double­
strength skim milk used to saturate drag swabs for Salmonella detection.
Avian Dis. 38:293-296. 1994.
13. Shivaprasad, Η. N. Pullorum Disease and Fowl Typhoid. In: Diseases of
poultry, 11th ed. Y.M Saif, H. J. Bames, J; R. Glisson, A. M Fadly, L. R.
McDougald, and D. E. Swayne, eds. Iowa State University Press, Ames,
Iowa. pp. 568-582. 2003.
14. Tate, C. R., R. G. Miller, E. T. Mallinson, and L. W. Douglass. The
isolation of salmonellae from poultry environmental samples by several
enrichment procedures using plating media with and without novobiocin.
Poultry Sci. 69:721-726. 1990.
15. United States Department of Agriculture (USDA). National Poultry
Improvement Plan and Auxiliary Provisions. Animal and Plant Health
Inspection Service, 91-55-063. USDA, Washington, D.C. Feb 2004.
16. Waltman, W. D., A. M Home, C. Pirkle, and T. G. Dickson. Use of
delayed secondary enrichment for the isolation of Salmonella in poultry and
poultry environments. Avian Dis. 35:88-92. 1991.
17. Waltman, W. D., and A. M Home. Isolation of Salmonella from
chickens reacting in the pullorum-typhoid agglutination test. Avian Dis.
37:805-810. 1993.
18. Waltman, W. D., A. M Home, and C. Pirkle. Influence of enrichment
incubation time on the isolation of Salmonella. Avian Dis. 37:884—887.
1993.

S. arizonae

AG
(-)C
AG
AG
+F
+

A = acid produced; G = gas produced; 0 = variable reaction
B Blood agar is generally used as the non-selective agar plate. If desired, the
swabs or pieces of tissue may be inoculated into a non-selective broth, such
cas brain-heart infusion or nutrient broth.
Brilliant green agar supplemented with novobiocin (BGN) in combination
with xylose-lysine-tergitol (niaproof) 4 (XLT4) agar are preferred (refer to
text). If S. pullorum is suspected, MacConkey (MAC) agar may be
substituted for XLT4 agar).
D Colony lift immunoassays may increase the reliability of detecting Group
D Salmonella (e.g. S. enteritidis, S. pullorum, etc) on plating media (refer to
text).
E Salmonella typhimurium var. Copenhagen, as isolated from pigeons,
frequently forms no acid in maltose broth and can be confused with S.
pullorum. However, it is motile.
F Turns deep Prussian blue within 24 hr.

ACKNOWLEDGMENT

The guidance supplied by Edward T. Mallinson is very gratefully
acknowledged

REFERENCES

1. Association of Official Analytical Chemists (AOAC) International.
Official methods of analysis, 16th ed. P. Cunniff, ed. AOAC International,
Gaithersburg, Md. pp. 55-94. 1996.
2. Bailey, J. S., N. A. Cox, and Μ E. Berrang. Hatchery-acquired
salmonellae in broiler chicks. Poult. Sci. 73:1153-1157. 1994.
3. Carlson, V. L., and G. H. Snoeyenbos. Comparative efficacies of selenite
and tetrathionate enrichment broths for the isolation of Salmonella
serotypes. Am. J. Vet. Res. 35:711-718. 1974.

9


3
COLIBACILLOSIS
Margie D. Lee and and Lisa K. Nolan
SUMMARY. Escherichia coli is the causative agent of colibacillosis in poultry. The disease results from a systemic infection involving the
blood, joints, and/or air sacs of birds. Young birds (4-8 wk old) may die of acute septicemia that is preceded by only a brief period of
anorexia and depression. At necropsy, lesions are sparse but may include swollen, dark-colored liver and spleen and increased fluid in the
body cavities. Birds surviving the acute phase may develop fibrinopurulent airsacculitis, pericarditis or arthritis. Whether lesion-associated
isolates are primary pathogens or whether environmental factors are responsible is unknown at this time.
Agent Identification. Isolation of E. coli is significant if made from the internal organs or blood from fresh carcasses. MacConkey agar is
selective and differential for E. coli and is preferred for primary isolation. Presumptive positive colonies (pink on MacConkey that produce
A/A reaction on triple sugar iron agar and that are oxidase-negative, gram-negative rods should be confirmed as E. coli by a positive indole
test and the inability to produce H2S. But some isolates do not form pink colonies on MacConkey, therefore multiple colonies should be
separately inoculated on triple sugar iron agar to detect these pathogens.
Serologic Detection in the Host. Host serology is not useful for diagnosis because many birds have antibody to normal intestinal flora
isolates of E. coli.

fibrinopurulent lesions suggest subacute colibacillosis, swab
samples of exudate should be collected from the pericardial sac, air
sacs, and joints. Lesions present more than 1 wk are often sterile.
When postmortem changes are obvious, bone marrow samples may
be useful because they are less likely than other tissues to contain
intestinal E. coli. Escherichia coli isolates survive well on sealed
agar slants for storage and shipping. For long-term storage, mix E.
coli broth culture with sterile glycerol 1:1 and store at -20 to -60 C.

INTRODUCTION
Colibacillosis of poultry is a common systemic infection caused by
avian pathogenic Escherichia coli (APEC).
The disease is
economically important to poultry production worldwide.
Colibacillosis occurs in many forms.
It may occur as
colisepticemia, which typically leads to death. However, some
birds fully recover from colisepticemia, and others may recover
with sequelae (2). Colibacillosis may also be localized, manifesting
as omphalitis, yolk sac infection, cellulitis, swollen head syndrome,
enteritis, acute vaginitis, salpingitis, or peritonitis (2,12). For E. coli
infections to become clinically apparent, adverse environmental
factors or other infectious agents are usually required (2, 3). A
foodbome link between human disease and APEC has not been
established. However, recent reports of similarities between the
virulence attributes of APEC and human extraintestinal pathogenic
E. coli, suggest that some APEC may be capable of causing disease
in human beings (4,10).

PREFERRED CULTURE MEDIA AND SUBSTRATES

Escherichia coli grows well in most commonly used culture
media, but differential media are useful for primary isolation.
MacConkey's agar is a selective and differential medium for the
isolation of enteric organisms. Tryptose blood agar with 5% bovine
blood can be used as a primary culture medium to support growth of
E. coli and other bacterial pathogens.
Several differential
biochemical characteristics can be obtained through use of Kligler’s
iron agar or triple sugar iron agar slants. All the above media are
readily available from BD Diagnostics (Sparks, Md.).
Microbiology identification kits such as the API 20E (bioMerieux
Vitek, Hazelwood, Mo.) and Enterotube (Becton Dickinson
Microbiology Systems) are useful for performing the biochemical
tests and are available commercially.

CLINICAL DISEASE
Clinical signs of colibacillosis are nonspecific and vaiy with the
age of bird, duration of infection, organs involved, and concurrent
disease conditions. In young (4 to 8-wk-old) broilers and poults
dying of acute septicemia, death is preceded by a brief period of
anorexia, inactivity, and somnolence. At necropsy, lesions are
sparse except for swollen, dark-colored liver and spleen and
increased fluid in all body cavities. Birds surviving the septicemia
phase of the disease are unthrifty and develop subacute
fibrinopurulent airsacculitis, pericarditis, perihepatitis, and
lymphocyte depletion in the bursa and thymus. Airsacculitis,a
classic lesion of colibacillosis, occurs following respiratory
exposure to large numbers of E. coli, but also occurs as a sequel to
bacteremia. Other less common lesions are arthritis, osteomyelitis,

salpingitis, and pneumonia (1,3,6). Cellulitis, responsible for
substantial economic losses for the poultry industry, has also been
associated with E. coli infection (12).

AGENT IDENTIFICATION

Colony Morphology and Biochemical Features
Blood samples should be diluted 1:10 in brain-heart infusion broth
then inoculated directly onto MacConkey’s plates. Swabs from
lesions can be used to streak MacConkey’s plates. MacConkey’s
agar should be incubated aerobically for 18-24 hr at 37 C for the
primary isolation of E. coli. Escherichia coli, Enterobacter, and
Klebsiella ferment lactose and can be distinguished from the other
enteric organisms. On MacConkey's agar, most E. coli and some
Enterobacter isolates produce 1-2-mm-diameter hot-pink colonies
(lactose positive) whereas Klebsiella and Enterobacter aerogenes
form large mucoid pink colonies (8). However slow lactosefermenting E. coli are frequently isolated from cases of
colibacillosis and these may not form pink colonies. If primary
cultures reveal large numbers of a predominant colony type
suggestive of E. coli, pick several of these colonies and use them to
separately inoculate triple sugar iron agar (or Kligler’s iron agar),
sulfide-indole-motility (SIM) medium, and blood agar plates. If the
clinical signs suggest colibacillosis infection, and no hot-pink
colonies are seen on the MacConkey’s plates, pick a couple of non­
pink colonies to inoculate blood agar, triple sugar iron agar and
SIM. A Gram stain and the oxidase reaction can be performed on
colonies from blood agar plates. Escherichia coli, Enterobacter,

SAMPLE COLLECTION
Only internal organs or blood, not feces or intestine, are useful

samples. Because normal intestinal flora E. coli readily invade
other tissues after death, specimens from fresh carcasses are
necessary. When acute colisepticemia is suspected, heart blood and
liver should be sampled aseptically. One ml of blood collected by
needle and syringe can be used to inoculate broth media (1:10),
which is used to streak agar plates. Sterile culture swabs or
inoculation loops can be stabbed into the liver parenchyma after
searing the capsule with a flamed scalpel or spatula. When

10


Chapter 3 Colibacillosis

and Klebsiella are oxidase-negative, gram-negative rods. On triple
sugar iron agar (or Kligler’s iron agar), these organisms produce
acid (even the slow lactose-fermenting E. coli) and gas but not H2S.
On SIM medium, E. coli is positive for the indole reaction, positive
or negative for motility, and negative for H2S, whereas
Enterobacter and Klebsiella are usually indole-negative (Fig. 3.1).
The site of sample collection, condition of the carcass, and nature
of the lesion are important when deciding whether isolation of E.
coli is relevant. The isolation of a pure culture of E. coli from the
organs or blood of moribund birds or freshly dead carcasses is
indicative of colibacillosis. Pathogenicity of E. coli isolates has
traditionally been established by inoculating young (less than 3-wkold) chicks or poults parenterally with 0.1 ml of overnight broth
culture. Pathogenic isolates should produce death or characteristic
lesions of colibacillosis within 3 days. Alternately, inoculation of
embryonated eggs may be used to establish the pathogenicity of E.
coli isolates (7). Also, many APEC share a complex of plasmidassociated virulence genes, whose presence may be helpful in

distinguishing APEC from non-pathogenic strains (9). Hence,
multiplex PCR, targeting these common genes, may have value in
confirming the pathogenic nature of E. coli isolates (11).

Antimicrobial Susceptibility
APEC isolates are frequently resistant to more than one antibiotic.
Seventy to 90% of isolates are resistant to sulfa drugs, tetracyclines,
streptomycin, and gentamicin. It is not uncommon to find isolates
that are multi-resistant to greater than 3 antibiotics. Resistance to
fluoroquinolones is less frequently detected; a recent study (13)
reported that 84% of isolates were susceptible to enrofloxacin.

SEROLOGIC DETECTION IN THE HOST
Serology is not commonly used to detect E. coli infection.

DIFFERENTIATION FROM CLOSELY RELATED AGENTS
Colibacillosis should be distinguished from other bacterial
infections causing fatal septicemia or fibrinopurulent inflammation
of air sacs, pericardium, joints, and other viscera. Diseases to be
considered in the differential diagnosis include mycoplasmosis,
salmonellosis, pasteurellosis, pseudotuberculosis, erysipelas,
chlamydiosis, and staphylococcosis. Colibacillosis is a common
complication of concurrent viral respiratory or enteric infections.
REFERENCES

1. Arp, L.H.

Pathology of spleen and liver in turkeys inoculated with

Escherichia coli. Avian Pathol. 11:263-279. 1982.

2. Barnes, H.J., J.-P. Vaillancourt, and W.B. Gross. Colibacillosis. In:
Diseases of poultry, 11th ed Y.M Saif, H. J. Bames, J.R. Glisson, A.M
Fadly, L.R. McDougald, and D.E. Swayne, eds. Iowa State University
Press, Ames, Iowa. pp. 631-652. 2003.
3. Gross, W.B. Colibacillosis. In: Diseases of poultry, 9th ed. B.W.
Calnek, H.J. Bames, C.W. Beard, W.M Reid, and H.W. Yoder, eds. Iowa
State University Press, Ames, Iowa. pp. 138-144. 1991.
4. Johnson, J.R., A.C. Murray, A. Gajewski, M Sullivan, P. Snippes, MA.
Kuskowski, and K.E. Smith. Isolation and Molecular Characterization of
nalidixic acid-resistant extraintestinal pathogenic Escherichia coli from
retail chicken products. Antimicrob Agents Chemother. 47: 2161-2168.
2003.
5. Kaper, J. B., J.P. Nataro, and H.L.T. Mobley. Pathogenic Escherichia
coli. Nature Rev. Microbiol. 2: 123-140. 2004.
6. Nakamura, K., M Maecla, Y. Imada, T. Imada, and K. Sato. Pathology of
spontaneous colibacillosis in a broiler flock. Vet. Pathol. 22:592-597. 1985.
7. Nolan, L.K., R.E. Wooley, J. Brown, K.R. Spears, H.W. Dickerson, and
M Dekich. Comparison of a complement resistance test, a chicken embryo
lethality test, and the chicken lethality assay for determining virulence of
avian Escherichia coli. Avian Dis. 36:395-397. 1992.
8. Quinn, P.J., ME. Carter, B.K. Markey, and G.R Carter. Clinical
veterinary microbiology. Mosby-Year Book Limited, London, England,
pp. 209-236. 1994.
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Johnson, and L.K. Nolan. Characterizing an APEC pathotype. Vet Res. 36:
1-16. 2005.
10. Rodriguez-Siek, K.E., C.W. Giddings, T.J. Johnson, M Fakhr, C.
Doetkott, and L.K. Nolan. Comparison of Escherichia coli implicated in
human urinary tract infection and avian colibacillosis. Microbiol. 151:

2097-2110. 2005.
11. Skyberg, J.A, S.M Home, C.W. Giddings, R.E. Wooley, P.S. Gibbs, and
L.K. Nolan. Characterizing avian Escherichia coli isolates with multiplex
PCR Avian Dis. 47:1441-1447,2003.
12. Vaillancourt, J.-P., and H.J. Bames. Coliform Cellulitis. In: Diseases
of poultry, 11th ed. Y.M Saif, H. J. Bames, J.R Glisson, A.M Fadly, L.R.
McDougald, and D.E. Swayne, eds. Iowa State University Press, Ames,
Iowa. pp. 652-656. 2003.
13. Zhao, S., J.J. Maurer, S. Hubert, J.F. De Villena, P.F. McDermott, J.
Meng, S. Ayers, L. English, and D.G. White. Antimicrobial susceptibility
and molecular characterization of avian pathogenic Escherichia coli isolates.
Vet. Microbiol. 107:215-24,2005.

Figure 3.1.
Escherichia coli identification scheme .Diagnosis of
colibacillosis is dependent on distinguishing pathogenic isolates from
nonpathogenic, normal intestinal flora isolates of E. coli.

Serogrouping of Isolates
Escherichia coli serogroups are based on O antigens; but serotypes
of E. coli are based on O antigens, as well as flagellar and/or
capsular antigens (5). There is great diversity of serogroups/types
among APEC, although some occur more commonly than others
(e.g., 078 and 02), and many isolates are not typable (9). Routine
serotyping of APEC is usually impractical, but for epidemiologic
studies, isolates can be serotyped for a fee by the Gastroenteric
Disease Center (Pennsylvania State University, University Park,
Penn.).

11

4
PASTEURELLOSIS, AVIBACTERIOSIS, GALLIBACTERIOSIS, RIEMERELLOSIS, AND PSEUDOTUBERCULOSIS
John R. Glisson, Tirath S. Sandhu, and Charles L. Hofacre

SUMMARY. In avian hosts, certain members of the genera Pasteurella, Avibacterium, Gallibacterium and the species Riemerella
anatipestifer and Yersenia pseudotuberculosis cause septicemic and respiratory disease. Acute diseases caused by these Gram-negative
bacteria are often characterized by high flock mortality and morbidity. Clinical signs of acute disease include ruffled feathers, depression,
increased respiratory rate, cyanosis, emaciation, and diarrhea. Lesions may consist of hemorrhages, swollen liver and spleen, focal necrotic
areas in the liver, airsacculitis, and increased pericardial and peritoneal fluids. With R. anatipestifer infection, common gross lesions in ducks
are fibrinous pericarditis, hepatitis and airsacculitis. Chronic disease signs produced by Pasteurella sp., R. anatipestifer and Y.
pseudotuberculosis can include ocular and nasal discharge, swelling of tissues such as joints and wattles, and stunted growth.
Pasteurella
This genus includes P. multocida, the cause of fowl cholera. Two similar organisms, Avibacterium gallinarum (formerly Pasteurella
gallinarum) and Gallibacterium anatis biovar haemolytica (formerly Pasteurella haemolytica) are include in this chapter because they can be
isolated from the respiratory tract and have been associated with respiratory disease in poultry (1,4). Five serogroups of P. multocida (A, B,
D, E and F) have been isolated from avian species, but serogroup A strains are the major cause of fowl cholera. Sixteen somatic serotypes
may occur among strains of P. multocida. However, somatic serotypes 1, 3 and 4 are most commonly isolated.
Agent Identification. Pasteurellas are identified by cell and colony morphology, Gram stain, and reactions in biochemical and other tests. A
mucopolysaccharidase test can be used for presumptive identification of P. multocida serogroups A, D and F. Gel diffusion precipitin tests
are used to determine somatic serotype.
Serologic Detection in the Host. Serologic tests are not commonly used to detect infections by Pasteurella sp. in poultry.

Riemerella anatipestifer
Riemerella anatipestifer infection occurs in ducklings usually at 1-8 wk of age. Primary isolations of R. anatipestifer are made on blood or
trypticase soy agar incubated at 37C in a candle jar or CO2 incubator. At least twenty one serotypes have been identified using agglutination
tests.
Agent Identification. Riemerella anatipestifer is identified by cell and colony morphology, Gram stain, and biochemical characteristics.
Fluorescent-antibody technique can be used to detect and identify R. anatipestifer in tissues or exudates from infected birds.

Serologic Detection in the Host. Serum antibodies against R. anatipestifer can be detected in poultry by ELISA.
Yersenia pseudotuberculosis
Pseudotuberculosis, caused by infection with Y pseudo tuberculosis, is infrequently reported in poultry. Selective media are used for
isolation of the bacterium from feces. There are six serotypes (I-VI) based upon heat-stable antigens using agglutination and agglutination­
adsorption tests. Among strains isolated from birds, serotype I is the most common. Serotypes V and VI have not been reported in birds.
Agent Identification. Yersenia pseudotuberculosis is identified by cell and colony morphology, Gram stain, and reactions in biochemical
and other tests. The bacterium is motile at 25 C.
Serologic Detection in the Host. Serologic tests are not used to detect Y. pseudotuberculosis infections in poultry.

Pasteurella multocida
INTRODUCTION

stage, particularly when the infection is caused by organisms of low
virulence. Finding dead birds may be the first sign of fowl cholera.
Other typical signs are depression, diarrhea, ruffled feathers,
increased respiratory rate, and cyanosis. Commonly observed
lesions in birds dying of acute fowl cholera include passive
hyperemia, hemorrhages, swollen liver, focal necrotic areas in the
liver and spleen, and increased pericardial and peritoneal fluids. In
general, the signs of chronic fowl cholera include swelling of
affected tissues, such as joints and sternal bursae, and exudate from
conjunctivae and turbinates. The focal lesions are generally
characterized by fibrinosuppurative exudate and various degrees of
necrosis and fibroplasia.

The disease caused by infection with Pasteurella multocida is
usually called fowl cholera; however, the term avian cholera is
frequently used when the disease occurs in wild birds. The name
Pasteurella septica infection was sometimes used in older European
literature. Fowl cholera is a common, widely distributed disease of

major economic importance in the United States. The disease
affects all species of birds. Among commercially raised birds,
turkeys and Japanese quail are particularly susceptible. Outbreaks
in wild waterfowl are common and frequently cause high mortality.
Fowl cholera occurs as a primary disease that does not require
predisposing factors, although predisposing factors may increase
severity of outbreaks. Subclinical infections apparently do not exist
in normal flocks, but normal-appearing birds that have survived
outbreaks of the disease frequently remain infected and may serve
as carriers.

SAMPLE COLLECTION

Pasteurella multocida can be isolated readily from the liver, bone
marrow, and heart blood of birds that die of acute fowl cholera and
usually from localized lesions of chronic cholera (5). Bone marrow
and brain are recommended when specimens are not fresh or when
contamination of tissues seems likely (25). To obtain specimens for
microbiologic examination, the surface of the tissue is seared with a
heated spatula, and a sterile cotton swab or wire loop is inserted
through the seared surface. The specimen is transferred to an agar

CLINICAL DISEASE
Fowl cholera may affect birds of any age, but it rarely occurs in
commercially raised poultry of less than 8 wk of age. The infection
often occurs as an acute septicemic disease with high morbidity and
mortality (5). Chronic fowl cholera may follow the septicemic

12

Chapter 4

Pasteurellosis, Avibacteriosis, Gallibacteriosis, Riemerelloisis, And Pseudotuberculosis

medium and incubated at 37 C. Pasteurella multocida grows
aerobically and anaerobically.
Cultures of P. multocida are moderately stable, generally surviving
storage or transportation if maintained in a humid environment,
such as on agar slants in screw-capped tubes. Stab cultures in agar
medium in screw-capped tubes generally survive for weeks. Long­
term storage is best accomplished using lyophilization.

strains of P. multocida. A gel-diffusion precipitin test (GDPT) is
used for serotyping based on differences in somatic antigens
(somatic serotyping) (9). Sixteen serotypes (1-16) have been
reported (2); strains representing each of these 16 serotypes have
been isolated from avian hosts. Frequently, antigens from a single
strain react with more than one type of serum, resulting in serotypes
such as 3,4 and 3, 4, 12. An indirect (passive) hemagglutination
test is used for serogrouping based on differences in capsular
antigens (capsular serogrouping) (3). Five capsular serogroups (A,
B, D, E, and F) have been reported (20). Serogroup A, D, and F
strains produce capsules containing mucopolysaccharides, and
presumptive identification of these serogroups can be made using
specific mucopolysaccharidases in a disk-diffusion test (16). Except
for serogroup E, strains representing all serogroups have been
isolated from avian hosts.
Antisera used in determining somatic serotypes are prepared in
chickens (9, 17). Such typing antisera are available from the

National Veterinary Services Laboratories, Animal and Plant Health
Inspection Service (APHIS), United States Department of
Agriculture (USDA), Ames, Iowa. Laboratories wishing to obtain
such sera should contact the APHIS-USDA Veterinarian-in-Charge
in their state.
Antigens for the GDPT are prepared from 18-to-24-hr growth of
heavily seeded DSA in petri dishes. The cells from one dish are
suspended in 1.0 ml of 0.85% NaCl, 0.02M phosphate, and 0.3%
formalin solution, pH 7.0. The suspension is heated in a water bath
at 100 C for 1 hr and centrifuged at 4000 x g for 30 min. The
supernatant fluid is used for antigen.
The agar gel consists of 0.9% Noble hgar (Difco, Detroit, Mich.)
in 8.5% NaCl solution. Five milliliters of warm (46 C) melted agar
is flooded onto a 25 x 75-mm microscope slide; wells, 4 mm in
diameter and 6 mm from center to center, are cut. Antigen is placed
in a well and antisera are placed in opposing wells. The slide is
placed in a petri dish to prevent drying, and the results are recorded
after 24 hr at 37 C.
Antisera used in the indirect hemagglutination test for capsule
serogrouping are prepared in Pasteurellα-free rabbits (18, 20).
Preparation of high-titered sera requires repeated intravenous
inoculations with formalin-killed capsulated organisms. Currently,
antisera for capsule serogrouping are not available from commercial
or government laboratories.

PREFERRED CULTURE MEDIA AND SUBSTRATES
Dextrose starch agar (DSA), blood agar, or trypticase soy agar
(Becton Dickinson Microbiology Systems, Sparks, Md.) are
recommended for primary isolation of P. multocida. The likelihood
of isolation may be improved by supplementing these media with

5% heat-inactivated serum. The organisms grow readily in tryptose
or trypticase soy broth.

AGENT IDENTIFICATION
Colony Morphology
On DSA, 24-hr colonies are circular, 1-3 mm in diameter, smooth,
transparent, glistening, and butyrous. Colonies on blood agar are
similar to those on DSA but are grayish and less translucent.
Observation of 24-hr colonies on DSA or other translucent agar
using a dissection microscope and obliquely transmitted lighting (5)
provides information on whether or not cells are capsulated.
Colonies that are iridescent contain capsulated cells, whereas
colonies that are noniridescent (blue or blue-gray) contain
uncapsulated cells. Pasteurella multocida produces a distinctive
odor when grown on agar media.

Cell Morphology
Pasteurella multocida cells are typically rods of 0.2-0.4 χ 0.6-2.5
pm occurring singly or occasionally in pairs or short chains. When
grown under unfavorable conditions or after repeated subculture,
cells tend to become pleomorphic. Cells in tissues or exudate
usually show bipolar staining with Giemsa or Wright’s stain.
Capsules can be demonstrated using an indirect india-ink method of
staining. A loopful of dilute bacterial specimen is mixed with a
loopful of india ink on a microscope slide, a coverslip is applied,
and the preparation is examined microscopically at a high
magnification. Capsules appear as clear halos around the bacterial
cells when examined in this manner.

SEROLOGIC DETECTION IN THE HOST

Commercially available enzyme-linked immunosorbent assay
(ELISA) kits may be used to detect a serological response to P.
multocida infection in poultry (IDEXX, Westbrook, Maine and
Synbiotics, Gaithersbury, MD). ELISAs are used primarily to
measure the serologic response following the use of inactivated P.
multocida vaccines in poultry. Serologic tests are rarely used for
diagnosis of fowl cholera.

Biochemical and Other Tests
Evaluation of reactions in differential media is generally made
after 2 days of incubation at 37 C and again after 5 days of
incubation at room temperature (21 C), in case of delayed reactions.
The carbohydrate broth media used for identification (Table 4.1) is
phenol red broth base containing 1% of the carbohydrate substrate.
For detection of hydrogen sulfide (H2S), a filter paper strip
impregnated with lead acetate is suspended above modified H2S
broth (10) during incubation. The presence of indole is indicated by
development of a dark red color when a small amount of modified
Kovac’s indole test reagent is added to a 24-hr culture consisting of
2% tryptose in 0.85% NaCl solution (7). Oxidase production is
determined using Kovac’s oxidase-test reagent and indirect filter
paper procedure (12).
Fructose, galactose, glucose, and sucrose are fermented without
gas production. Inositol, inulin, maltose, salicin, and rhamnose are
not fermented. Indole and oxidase are almost always produced.
Neither hemolysis of blood nor growth on MacConkey’s agar occur.
Differential characteristics are listed in Table 4.1.

DIFFERENTIATION FROM CLOSELY RELATED AGENTS

Differentiation of fowl cholera from other diseases caused by
Pasteurella,
Avibacterium,
Gallibacterium,
Riemerella
anatipestifer, and Yersinia pseudotuberculosis depends primarily
upon isolation and differentiation of causative organisms.
Differential physiologic reactions of these organisms are indicated
in Table 4.1. Several other diseases that are not caused by closely
related agents, such as infectious coryza, fowl typhoid, fowl plague,
duck plague, and infectious synovitis may have histories, signs, or
lesions similar to those of fowl cholera. Differentiation of fowl
cholera from these diseases also depends upon isolation and
identification of causative organisms.

Serologic Identification
Serologic tests are rarely used for identification of P. multocida.
However, these are commonly used for antigenic characterization of

13