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

Sổ tay Phòng thí nghiệm về Phân lập, Nhận dạng và Đặc tính của Mầm bệnh Gia cầm, Phiên bản thứ 5, bắt nguồn từ nhu cầu tiêu chuẩn hóa các phương pháp thử nghiệm và đánh giá vắcxin gia cầm. Trong ấn bản thứ 5, AAAP đã bổ nhiệm Tiến sĩ Louise DufourZavala làm Tổng biên tập, với David Swayne làm cố vấn và biên tập phần. John Glisson, Willie M. Reed, Mark W. Jackwood và James E. Pearson tiếp tục là người biên tập chuyên mục, đóng góp kiến thức chuyên môn của họ về vi khuẩn học, chẩn đoán thú y, sinh học phân tử và virus học.

A LABORATORY MANUAL FOR THE

ISOLATION,

IDENTIFICATION, AND CHARACTERIZATION

A LABORATORY MANUAL FOR THE

Copies Available from:

American Association of Avian Pathologists

953 College Station Road Athens, GA 30602-4875

Frederic J. Hoerr

9

1 2

Cover:

1 Direct fluorescent antibody test on primary chicken embryo kidney cells showing

syncytia formed by ILT virus inprimary 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)andrestriction fragment lengthpolymorphism (RFLP)analysis if the spike (SI) glycoprotein gene of the Arkansas strain of infectious bronchitis virus

(IBV).Lanes1 and 2 are molecular weightmarkers.Lane3is 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 virusRNArun with the USDA type A influenza M gene

real-time RT-PCR teston the Applied Biosystems 7500 FAST system (Erica Spackman, SEPRL, Athens, GA) 6 Microphotograph ofMycoplasma gallisepticum colonies on agar

(35x) (Stan Kleven, PDRC, Athens, GA) 7. Bio Merieux API 20 system for bacteria

identification usinga series of biochemical tests.(Doug Waltman, GPLN, Oakwood, GA).

8 Embryonatedeggcandlingto 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 isolatewas 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 of Avian Pathogens

Library of Congress Catalog Card Number: 2008924452

A Laboratory Manual for the Isolation, Identification and Characterization

of Avian Pathogens

1 DiagnosticPrinciples. Frederic J Hoerr 1

2 Salmonellosis W Douglas Waltman andRichardK.Gast 3

3 Colibacillosis Margie D LeeandLisa K Nolan 10

4 Pasteurellosis, Avibacteriosis, Gallibacteriosis,Riemerellosis, andPseudotuberculosis John R. Glisson, Tirath S Sandhu, and CharlesL. Hofacre 12

5 Bordetellosis Mark W Jackwood 20

6 InfectiousCoryza Pat J Blackall 22

7 Campylobacter in Poultry Jaap A Wagenaar andWilmaF Jacobs- Reitsma 27

8 Spirochetosis.Stephen R Collett 31

9 Erysipelas GeorgeL.CooperandArthur A Bickford 36

10 Listerosis. George L CooperandArthur A Bickford 39

11 Staphylococcosis Stephan G ThayerandW.DouglasWaltman 42

12 StreptococcosisandEnterococcosis StephanG ThayerandW.Douglas Waltman 44

13 ClostridialDiseases StephanG Thayer and David A. Miller 47

14 Tuberculosis SusanSanchezandRichard M. Fulton 53

15 Mycoplasmosis StanleyH.Kleven 59

16 Chlamydiosis. Arthur A AndersenandDaisy Vanrompay 65

17 Omithobacteriosis RichardP Chinand Bruce R Charlton 75

18 Mycoses and Mycotoxicoses R.D. Wyatt 77

19 Adenovirus BrianM.AdairandJ BrianMcFerran 84

20 HemorrhagicEnteritis of Turkeys Marble Spleen Disease of Pheasants F.WilliamPierson and Sctott D Fitzgerald 90

21 InfectiousLaryngotracheitis Deoki N. TripathyandMaricarmenGarcia 94

22 Marek’s Disease PatriciaS.WakenellandJagdev M Sharma 99

23 Duck VirusEnteritis Peter R Woolcock 106

24 Herpesviruses of Free-Living and Pet Birds ErhardF Kaleta 110

25 Pox Deoki N TripathyandWillie M Reed 116

26 BudgerigarFledgling Disease and OtherAvianPolyomavirusInfections Branson W. RitchieandPhil D Lukert 120

27 PsittacineBeak and FeatherDisease BransonW Ritchie and Phil D Lukert 122

28 ChickenAnemiaVirus. M. StewartMcNultyandDanielTodd 124

29 Avian Influenza David E Swayne,Dennis A Senne and David L Suarez 128

30 NewcastleDisease Virus and OtherAvianParamyxoviruses.DennisJ.AlexanderandDennis A. Senne 135

31 AvianMetapneumovirus Richard E. GoughandJanice C. Pedersen 142

32 InfectiousBronchitis JackGelb,Jr.andMark W. Jackwood 146

33 TurkeyCoronavirus MarkW JackwoodandJames S. Guy 150

34 Enteric Viruses Don Reynolds andChing Ching Wu 153

35 Oncornaviruses,Leukosis/Sarcomas and Reticuloendotheliosis Aly M. Fadly,RichardL Witter, and Henry D Hunt 164

36 AvianEncephalomyelitis.Louis van derHeide 173

37 DuckHepatitis. Peter R Woolcock 175

38 Turkey Viral Hepatitis WillieM Reed 179

39 ViralArthritis/Tenosynovitis and OtherReovirusInfections JohnK Rosenberger and Erica Spackman 181

40 ArbovirusInfection. Eileen N. Ostlund and JamesE Pearson 184

41 InfectiousBursalDisease JohnK.Rosenberger,Y.M Saif, and DaralJ Jackwood 188

42 Parvovirus of Waterfowl. Richard E. Gough 191

43 Cell-CultureMethods Karel A Schat and Holly S. Sellers 195

44 VirusPropagationinEmbryonating Eggs Dennis A. Senne 204

45 VirusIdentification and Classification PedroVillegas and IvanAlvarado 209

46 Titration of BiologicalSuspensions PedroVillegas 217

47 SerologicProcedures StephanG ThayerandCharlesW Beard 222

48 Molecular IdentificationProcedures. Daral J JackwoodandMark W Jackwood 230

49 Antigen Detection Systems. MaryJ Pantin-JackwoodandSandraS Rosenberger 233

Appendixof Abbreviations and Acronyms used intheText 241

QuickReferenceDiagnosticChart 244

Index 247

iii

CONTRIBUTING AUTHORS Brian Adair

Veterinary Sciences Division

Agri-Food and Biosciences Institute

Central Veterinaiy Laboratory (Weybridge)

New Haw, Addlestone

Animal Research Institute

Locked Mail Bag No 4

School of Veterinary Medicine

University of California - Davis

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: colletts@uga.edu FAX: (706) 542-5630

George L Cooper

School of Veterinary Medicine University of California-Davis Calif Vet Diagnostic Lab System Turlock Branch

P.O Box PTurlock, California 95381E-mail: gcooper@cvdls.ucdavis.edu FAX: (209) 667-4261

Richard M Fulton

Diagnostic Center for Population and Animal Health Michigan State University

PO Box 30076Lansing, Michigan 48909E-mail: fulton@dcpah.msu.eduFAX: (517)355-2152

Maricarmen Garcia

Department of Population Health College of Veterinary Medicine The University of Georgia

953 College Station Rd

Athens, Georgia 30602-4875 E-mail: mcgarcia@uga.edu FAX: (706) 542-5630

Richard K Gast

USDA, ARS

Russell Research Center

950 College Station Road

Athens, GA 30605

E-mail: rgast@seprl.usda.gov

FAX: (706) 546-3035

Jack Gelb, Jr.

Department of Animal and Food Sciences

College of Agricultural Sciences

531 South College Avenue

Department of Population Health

College of Veterinary Medicine

The University of Georgia

Central Veterinary Laboratory (Weybridge)

New Haw, Addlestone

North Carolina State University

College of Veterinary Medicine

Department of Population Health & Pathobiology

4700 Hillsborough Street

Raleigh, North Carolina 27606

E-mail: Jim Guy@NCSU.edu

FAX: (919)513-6464

Frederic J Hoerr

Thomas Bishop Sparks

State Diagnostic Laboratory

Department of Population Health

College of Veterinary Medicine

The University of Georgia

953 College Station Road

Athens, Georgia 30602-4875

E-mail: chofacre@uga.edu

FAX: (706) 542-5630

Henry Hunt

USDA- Agricultural Research Service

Avian Disease and Oncology Laboratory (ADOL)

3606 East Mount Hope Road

East Lansing, Michigan 48823

1680 Madison AveWooster, Ohio 44691E-mail: jackwood.2@osu.eduFAX: (330) 263-3760

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: mjackwoo@uga.edu FAX: (706) 542-5630

Wilma F Jacobs-Reitsma

RIKILT Institute of Food SafetyBomsesteeg 45

6708 PD WageningenThe NetherlandsE-mail: Wilma.jacobs@wur.nlFAX: +31-317-417717

Stanley H Kleven

Department of Population Health College of Veterinary Medicine University of Georgia

953 College Station Rd Athens, Georgia 30602-4875 E-mail: skleven@uga.edu FAX: (706) 542-5630

Margie D Lee

Department of Population Health Poultry Diagnostic and Research Center The University of Georgia

953 College Station RoadAthens, Georgia 30602-4875 E-mail: mdlee@uga.edu FAX: (706) 542-5771

Phil D Lukert

Department of Medical MicrobiologyCollege of Veterinary MedicineUniversity of Georgia

Box 207Colbert, Georgia 30628 E-mail: plukert@uga.edu FAX: (706) 542-5771

J Brian McFerran

19 Knocktem GardensBelfast BT4 3LZNorthern IrelandFAX: +44-1232-658040

v

Dept, of Vet Microbiology and Preventive Medicine

College of Veterinary Medicine

Southeast Poultry Research Laboratory USDA-ARS

934 College Station Road

Center for Molecular Medicine and Infectious Diseases

Virginia-Maryland Reg College of Vet Medicine

Virginia Polytechnic Institute and State University

West Lafayette, Indiana 47907-2026E-mail: wreed@purdue.eduFAX: (765) 496-1261

Donald L Reynolds

2520 Veterinary AdministrationIowa State University

1802 Elwood DriveAmes, Iowa 50011E-mail: DLR@iastate.eduFAX: (515) 294-8956

Branson W Ritchie

Department of Medical MicrobiologyCollege of Veterinary MedicineUniversity of Georgia

Athens, Georgia 30602FAX: (706) 542-6460Email: britchie@uga.edu

John K Rosenberger

Aviserve, LLCDelaware Technology Park

1 Innovation Way, Suite 100Newark, DE 19711

E-mail: aviserve@aol.comFAX: (302) 368-2975

Sandra S Rosenberger

Aviserve, LLCDelaware Technology Park

1 Innovation Way, Suite 100Newark, DE 19711

E-mail: aviserve@aol.comFAX: (302) 368-2975

Y M Saif

Food Animal Health Research ProgramOhio Agricultural Research and Development Center The Ohio State University

1680 Madison AvenueWooster, Ohio 44691E-mail: saif.l@osu.eduFAX: (330) 263-3677

Susan Sanchez

Athens Diagnostic Laboratory

0103 Athens V.M Diag Lab

501 D.E Brooks DrAthens, GA 30602E-mail: ssanchez@uga.eduFAX: (706)542-5568

Tirath S Sandhu

Cornell University Duck Research Laboratory

37 Howell PlaceP.O Box 427Speonk, New York 11972E-mail: Sandhu37@optonline.net

Karel A Schat

Unit of Avian Health

College of Veterinary Medicine

Department of Population Health

Poultry Diagnostic and Research Center

The University of Georgia

953 College Station Road

Animal and Plant Health Inspection Service

United States Department of Agriculture

Department of Veterinary PathoBiology

258 Veterinary Science Building

College of Veterinary Medicine

USD A-Agriculture Research Service

Southeast Poultry Research Laboratory

934 College Station Road

Athens, GA 30605

E-mail: erica.spackman@ars.usda.gov

FAX: (706)546-3161

David L Suarez

USD A-Agriculture Research Service

Southeast Poultry Research Laboratory

934 College Station Road

Athens, GA 30605

E-mail: david.suarez@ars.usda.gov

FAX: (706)546-3161

David E Swayne

USD A-Agricultural Research Service

Southeast Poultry Research Laboratory

934 College Station Road

953 College Station RoadAthens, Georgia 30602-4875E-mail: sthayer@uga.eduFAX: (706) 542-0252

Daniel Todd

Dept of Agriculture and Rural Development from N Ireland Veterinary Sciences Division

Stormont, Belfast BT4 3SDUK

E-mail: Daniel.Todd@dardni.gov.ukTel: +44 2890 525773

Deoki N Tripathy

Department of Veterinary PathobiologyCollege of Veterinary MedicineUniversity of Illinois

104 West McHenryUrbana, Illinois 61801E-mail: tripathy@uiuc.eduFAX: (217) 244-7421

Daisy Vanrompay

Ghent UniversityFaculty of Bioscience engineeringDepartment of molecular biotechnologyCoupure Links 653

9000 Ghent, BelgiumE-mail: daisy.vanrompay@UGent.beFAX: +32 09 2646219

Louis Van Der Heide

Dept Of Pathobiology U-89

PO Box 37

12 Yeomans RoadColumbia, Connecticut 06237E-mail: louisandingrid@yahoo.comFAX: (860) 486-2794

Pedro Villegas

Department of Population HealthCollege of Veterinary MedicineUniversity of Georgia

953 College Station RoadAthens, Georgia 30602-4875E-mail: pedrov@uga.eduFAX: (706) 542-5630

Jaap A Wagenaar

Department of Infectious Diseases and ImmunologyOIE Reference Laboratory for Campylobacteriosis and WHO Collaboration Centre for Campylobacter Faculty of Veterinary Medicine

Utrecht UniversityP.O Box 80.165

3508 TD UtrechtThe NetherlandsE-mail: i wagenaar@uu.nlFAX: +31 (0)30-2533199

vii

Patricia S Wakenell

Department of Veterinary Medicine

Population Health & Reproduction

USDA- Agricultural Research Service

Avian Disease and Oncology Laboratory

3606 East Mount Hope Road

East Lansing, Michigan 48823

Ching-Ching Wu

Purdue UniversityAnimal Disease Diagnostic Laboratory

406 South University StreetWest Lafayette, Indiana 47907-2065E-mail: Wuc@purdue.edu

FAX: (765) 497-1405

Roger Wyatt

195 Edgewood Dr SWAthens, GA 30606(706) 548-2297E-mail: mycotec@charter.net

viii

This manual has its origins in the need for a book to codify standardized method for testing and evaluating poultry vaccines The

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

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 Editor- in-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

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

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 Enzyme-

linked 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

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 post­administration 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 so- called 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 wild­type 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

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,

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

1

FredericJ. Hoerr

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

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

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

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

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

http://web.memberclicks.com/mc/page.do?sitePageId=27915&orgl 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 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

blood plate test, are used for verification of the Salmonella status of flocks participating in the National Poultry Improvement Plan

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

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 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

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

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

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

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

3

W Douglas Waltman and Richard K.Gast

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)

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 3-

or 4-square inch gauze pads moistened with DSSM are drawn by 3- 4-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

Chapter 2 Salmonellosis

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 10-

15 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

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

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

selective media

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 41-

42 C

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)

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

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

XLT4 (BD Diagnostic Systems, Sparks, MD) This medium inhibits

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 lactose-

fermenting 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 non-

host-adapted salmonellae Consequently, all plates should be

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 AO AC 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

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 AO AC 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

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

salmonellae are present, screening many more colonies may be necessary to ensure the absence of S enteritidis, S pullorum, or S

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

Chapter 2 Salmonellosis

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

and Morganella) produces a reddish or port-wine colored slant,

indicating lysine deamination, and a yellow (acid) butt on LI agar

(acid) butt, with some H2S production Rarely, both slant and butt

are yellow

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

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

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

degrees of success due to sensitivity and specificity problems and thus have not found widespread commercial application Because S

typhoid 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

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

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.

Chapter 2 Salmonellosis Table 2.1 Typical biochemical reactions of Salmonella K

Media S pullorum S gallinarum Paratyphoid

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 orpiecesoftissuemay be inoculated into a non-selective broth, such

asbrain-heart infusion or nutrient broth

c

Brilliant green agar supplemented withnovobiocin (BGN) incombination

withxylose-lysine-tergitol(niaproof) 4 (XLT4) agarare preferred (refer to

text). S pullorum is suspected, MacConkey (MAC) agar may be

substituted for XLT4 agar).

D Colony lift immunoassays mayincrease the reliability of detecting Group

DSalmonella (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 ismotile

F Turnsdeep 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. AO AC 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.

4 Fanelli, M J., W. W Sadler, C E Franti, and 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. Russek-

Cohen Monitoring poultryfarms forSalmonella bydrag swabs sampling and antigen captureimmunoassay. Avian Dis. 33:684—690 1989.

8 Miller, R G.,C R Tate, E T. Mallinson,andJ A. Scherrer Xylose

lysine tergitol 4: an improved selectiveagar 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 ofpoultry, 9th ed B W Calnek, H J Bames, C. W Beard, W.M

Reid, andH.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 forSalmonelladetection

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 ofSalmonellainpoultryand

poultry environments Avian Dis 35:88-92.1991

17 Waltman, W D., and A M Home Isolation ofSalmonella 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 ofSalmonella. Avian Dis 37:884—887 1993.

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.

INTRODUCTION

Colibacillosis of poultry is a common systemic infection caused by

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

in human beings (4,10)

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).

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

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

PREFERRED CULTURE MEDIA AND SUBSTRATES

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

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

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

enteric organisms On MacConkey's agar, most E coli and some

(lactose positive) whereas Klebsiella and Enterobacter aerogenes form large mucoid pink colonies (8) However slow lactose-

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 non­pink 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,

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

The site of sample collection, condition of the carcass, and nature

of the lesion are important when deciding whether isolation of E

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-wk-

old) 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

associated 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)

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.)

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, andH.L.T Mobley. PathogenicEscherichia coli. Nature Rev.Microbiol 2: 123-140. 2004

6. Nakamura, K., M Maecla, Y Imada, T Imada, and K. Sato.Pathology of spontaneous colibacillosis ina 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 complementresistance test, a chicken embryo lethality test, andthe chicken lethality assay for determining virulence of

avianEscherichia 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 BookLimited, London, England,

pp 209-236 1994.

9 Rodriguez-Siek, K.E., C.W Giddings, M Fakhr, C Doetkott, T.J Johnson, and L.K Nolan. Characterizingan 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 Characterizingavian Escherichia coliisolates with multiplex PCR Avian Dis 47:1441-1447,2003.

12 Vaillancourt, J.-P., and H.J. Bames Coliform Cellulitis In: Diseases

ofpoultry, 11th ed Y.M Saif, H. J. Bames, J.RGlisson,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 pathogenicEscherichia coliisolates

Vet Microbiol 107:215-24,2005.

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

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

Pasteurella

This genus includes P multocida, the cause of fowl cholera Two similar organisms, Avibacterium gallinarum (formerly Pasteurella

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

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 agglutination­adsorption 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

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

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

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

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

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 agar12

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

PREFERRED CULTURE MEDIA AND SUBSTRATES

Dextrose starch agar (DSA), blood agar, or trypticase soy agar

(Becton Dickinson Microbiology Systems, Sparks, Md.) are

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

odor when grown on agar media

Cell Morphology

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

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

Serologic Identification

Serologic tests are rarely used for identification of P multocida

However, these are commonly used for antigenic characterization of

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-USD A 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

SEROLOGIC DETECTION IN THE HOST

Commercially available enzyme-linked immunosorbent assay (ELISA) kits may be used to detect a serological response to P

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

DIFFERENTIATION FROM CLOSELY RELATED AGENTS

Differentiation of fowl cholera from other diseases caused by

Pasteurella, Avibacterium, Gallibacterium, Riemerella

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

13

John R. Glisson, TirathS.Sandhu,and Charles L. Hofacre

Table 4.1 Differential characteristics ofPasteurella, Avibacterium, Gallibacterium, Riemerella, and Yersinia.

Test P multocida A gallinarum G.anatis biovar R anatipestifer

Infection with A gallinarum often occurs in the respiratoiy tracts

of chickens and turkeys (6, 13), but disease is generally manifested

only when this infection occurs with other respiratoiy tract

infections Attempts to isolate A.gallinarum from normal flocks

have not been successful The infection is widely distributed It has

been reported in the United States, Australia, Japan, Nigeria, Iran,

and Israel Generally it is not considered to be of economic

significance

CLINICAL DISEASE

Disease manifestations in which A gallinarum infection occurs

usually affect the respiratory tract and have a chronic course

Swollen and inflamed wattles resulting from infection with only A gallinarum have been reported in chickens (13)

SAMPLE COLLECTION

nasal cleft, trachea, air sacs, and lungs of birds exhibiting respiratory disease The organism is less frequently isolated from livers, heart blood, and joints Avibacterium gallinarum grows aerobically and anaerobically Specimens are collected using the same methods as described for P multocida

PREFERRED CULTURE MEDIA AND SUBSTRATES

Chapter 4 Pasteurella,Avibacteriosis, Gallibacteriosis,Riemerellosis, and Pseudotuberculosis

AGENT IDENTIFICATION

Colony and Cell Morphology and Biochemistry

On DSA or blood agar at 37 C, 24-hr colonies are 1-2 mm in

diameter, smooth, entire, low convex, and transparent Colonies on

DSA are iridescent when observed with obliquely transmitted light,

often with concentric rings

Gram-negative short rods that occur as single organisms and short

chains, stain bipolarly, and are capsulated They become

pleomorphic with repeated subculturing and growth under less than

optimum conditions

Glucose, sucrose, and maltose are fermented without gas

production Lactose is not fermented, and indole is not produced

Neither hemolysis of blood nor growth on MacConkey’s agar occur

Differential characteristics are listed in Table 4.1

Serologic Identification

Serologic tests are rarely used for identification of A gallinarum

Antigenic differences have been demonstrated among strains of A

been defined Serologic testing has indicated that minor antigens of some strains of A. gallinarum are shared with P multocida.

SEROLOGIC DETECTION IN THE HOST

poultry

DIFFERENTIATION FROM CLOSELY RELATED AGENTS

Differentiation of A gallinarum infections from those caused by

determination and comparison of selected physiologic characteristics (Table 4.1) Other infections that may cause similar signs or lesions include infectious coryza, mycoplasmosis, and bordetellosis Differentiation depends upon isolation and identification or differentiation of the agents

Gallibacterium anatisbiovarhaemolytica

INTRODUCTION

to be a secondary pathogen that requires some predisposing factor

before producing disease However, too little information is

available to clearly establish the role of this organism in avian

disease production In normal-appearing flocks, the reported

incidence of infection is quite high in chickens but low in turkeys

and geese Infections in avian hosts are widely distributed, as

indicated by isolations in the United States, England, Germany,

France, Israel, Syria, Nigeria, Taiwan, and Australia This disease

is not considered economically important

CLINICAL DISEASE

Disease manifestations related to G anatis biovar haemolytica

infections most frequently involve the respiratory tract Septicemic

manifestations with petechial hemorrhages in the viscera and areas

of liver necrosis have been reported Salpingitis and peritonitis may

occur in laying hens

SAMPLE COLLECTION

G anatis biovar haemolytica usually can be isolated from the

trachea, lungs, liver, or oviduct of an infected bird

AGENT IDENTIFICATION Colony Morphology

On blood agar at 37 C, 24-hr colonies are generally 0.5 mm in diameter, smooth, entire, low convex, and translucent A wide zone

of β-hemolysis is produced on blood agar On DSA, the colonies are iridescent when observed with obliquely transmitted light and often exhibit concentric rings

Cell Morphology

non-spore-forming, pleomorphic rods (0.5 x 1.6 pm) that often exhibit bipolar staining They generally occur singly but occasionally form short chains

Biochemical and Other Tests

Glucose, sucrose, mannose, glycerol, and fructose are fermented without gas production within 24 hr of incubation at 37 C Lactose

is usually fermented after 3 days Arabinose, dulcitol, rhamnose, inulin, and salicin are not fermented Indole is not produced Differential characteristics are indicated in Table 4.1

Serologic Identification

Serologic tests are not used for identification of G anatis biovar

from avian hosts, and no avian serotypes have been established

SEROLOGIC DETECTION IN THE HOST

Serologic tests are not used to detect G anatis biovar haemolytica infection in poultry

Riemerella anatipestifer

INTRODUCTION

serositis, duck septicemia, new duck disease, or anatipestifer

syndrome is a septicemic disease of ducks, geese, turkeys, and

prevalent worldwide and causes significant economic loss due to

high mortality, weight loss, and condemnations The acute form of

the disease can cause mortality as high as 75% in ducks, especially

at farms where infection persists because hatches are frequently moved from one pen to another to create space for the next hatch Adverse environmental conditions and concomitant disease often

disease is not of public health importance In the United States, federal or state notification is not required

15

John R. Glisson, Tirath S Sandhu,and Charles L Hofacre

CLINICAL DISEASE

The acute form of the disease usually occurs in ducklings 1-8 wk

of age Chronic infections may occur in older birds High mortality

has been reported in turkeys 6-15 wk of age (23) Riemerella

guinea fowl, partridges, quail, and chickens Clinical signs of the

disease include ocular and nasal discharge, sneezing, greenish

diarrhea, tremors of the head, neck and legs, ataxia, and coma The

common gross lesions are fibrinous pericarditis, perihepatitis,

airsacculitis, and meningitis In females, the oviduct is filled with

caseous yellowish white exudate Chronic and localized infections

result in synovitis/arthritis and dermatitis Infections originate from

exposure via the respiratory tract or through abrasions or cuts in the

skin

SAMPLE COLLECTION

Riemerella anatipestifer can readily be isolated from heart blood,

brain, pericardial exudate, air sacs, lungs, oviduct, and liver

Isolations should be attempted from infraorbital sinuses and trachea

to detect carriers or inapparent infections Specimens should be

obtained by a wire loop or sterile swab through a seared surface In

chronic or localized infections, R anatipestifer can be isolated from

the exudate The specimen is streaked directly onto the surface of

agar media, which are then incubated at 37 C in a candle jar for 24-

72 hr Incubation under increased CO2 and humidity in a candle jar

or CO2 incubator enhances the growth for primary isolation If the

cultures cannot be made within a reasonable time, the specimens or

tissues should be kept at 4 C or frozen for shipping or later

processing

PREFERRED CULTURE MEDIA AND SUBSTRATES

Although R anatipestifer grows readily on routine media, growth is

enhanced by using enriched media such as blood agar or trypticase

soy agar containing 0.05% yeast extract Tryptose broth or

trypticase soy broth is used for broth cultures Addition of

gentamicin (5 mg/liter) to enriched solid medium has proven

contaminated specimens from the processing plant, even though

growth of other organisms is not completely inhibited

AGENT IDENTIFICATION

Colony and Cell Morphology and Biochemistry

morphologic and biochemical characteristics After 24 hr of growth

on blood agar, colonies are 1-2 mm in diameter, convex, entire,

transparent, glistening, and butyrous Some strains may produce

mucoid growth on solid media On trypticase soy agar, colonies

appear bluish and are iridescent when observed with obliquely

transmitted light Some strains have been observed to produce off-

white growth, which may change to grayish brown after 3-5 days

Growth in broth produces slight turbidity

Most R anatipestifer strains become nonviable after 3^4 days on a

solid medium at room temperature or 37 C In broth the organisms

may survive for 2-3 wk under refrigeration Cultures can be stored

as freeze-dried for longer periods Riemerella anatipestifer cells are

Gram-negative, nonmotile, non-spore-forming rods that occur

singly, in pairs, or occasionally in chains The cells are 1-5 pm

long and 0.1-0.4 pm wide and may show bipolar staining A capsule has been shown with the india ink method of staining

(Table 4.1), but has been reported to produce acid in glucose, mannose, maltose and dextrin when grown in buffered single substrate medium (11) The organism liquefies gelatin and produces a slight alkaline reaction in litmus milk Some strains produce urease and arginine dihydrolase No growth occurs on MacConkey’s agar and no hemolysis takes place on blood agar The organism produces oxidase, catalase, and phosphatase, but is indole negative

Riemerella anatipestifer is identified by enzymatic activity

(Apizyme - API laboratory Products Ltd., St-Laurent, Quebec, Canada) It is positive for leucine-, valine-, and cystine-arylmidases, phosphoamidase, α-glucosidase, and negative for a- and β- galactosidases, β-glucuronidase, β-glucosidase, α-mannosidase, β- glucosaminidase, ornithine and lysine decorboxylases (22)

Serologic Identification

Using agglutination tests, 21 serotypes have been reported (15, 21) Antisera are made in rabbits Antigen for rabbit inoculation is made by harvesting 24 to 48-hr growth from an agar plate in 0.85% NaCl solution containing 0.3% formalin The inactivated cells are washed twice by centrifugation in the above solution and adjusted

to an optical density of 0.2 at 525 nm in a spectrophotometer Young rabbits are immunized by inoculating through the marginal ear vein successive doses of 0.05, 0.1, 0.2, 0.5, 1.0, 1.0, 1.5, and 2.0

ml of standardized cell suspension, at 3- to 4-day intervals Rabbits are bled for serum collection when maximum titers are obtained Agglutination titers are low, usually on the order of 1:50 to 1:400.The plate agglutination test is done by mixing a drop of undiluted antiserum with one or two colonies from a 24-hr growth Agglutination within a few seconds indicates a homologous serotype reaction The tube agglutination test is performed by mixing an equal amount of formalinized cell suspension (adjusted

to 0.2 optical density at 525 nm) with each dilution of serum and incubating at 37 C Agglutination is recorded after 24 and 48 hr.The fluorescent antibody technique may be used to detect and identify R. anatipestifer cells directly in tissues or exudate from

infected birds

SEROLOGIC DETECTION IN THE HOST

Enzyme-linked immunosorbent assay (ELISA) is commonly used for early detection of R anatipestifer infections (8) Cell lysate is used as an antigen in ELISA It is not serotype specific and will show positive reactions with antisera against heterologous serotypes, but ELISA is more sensitive than agglutination test The GDPT is unreliable because of an apparent discrepancy in duck immunoglobulins, which are almost nonfunctional in precipitin reactions (24)

DIFFERENTIATION FROM CLOSELY RELATED AGENTS

Diagnosis should be based on isolation and identification of the causative organism, because similar gross lesions are produced by

Ducklings infected with Salmonella may sometime show nervous signs similar to those of R anatipestifer infection Chlamydiosis should also be considered in differential diagnosis

Chapter 4 Pasteurella, Avibacteriosis, Gallibacteriosis, Riemerellosis,and Pseudotuberculosis

Yersinia pseudotuberculosis

INTRODUCTION

The disease caused by infection with Y.pseudotuberculosis is

pseudotuberculosis (19) It can affect domesticated, feral, or caged

birds and a variety of mammals, including humans Although this

disease is infrequently reported in the United States and is not

considered economically important, it has caused death losses as

high as 80% in turkey flocks

CLINICAL DISEASE

Pseudotuberculosis usually occurs as a septicemia or bacteremia of

short duration, followed by chronic focal infections In very acute

cases, birds may die before other signs of disease are observed,

although signs usually are present for 2 wk or more Affected birds

exhibit weakness, ruffled feathers, diarrhea, and breathing

difficulties Emaciation and paralysis occur occasionally Swollen

livers and spleens and enteritis are common lesions in acute cases

Necrotic foci in visceral organs and muscles and enteritis are

common lesions in chronic cases Osteomyelitis occurs in some

affected turkey flocks

SAMPLE COLLECTION

liver, spleen, or lung using blood or trypticase soy agar and

incubation at 37 C (19) The method of Paterson and Cook (14) is

recommended for isolation of Y pseudotuberculosis from feces To

use this method, inoculate the surface of Paterson and Cook agar

medium with a 10% suspension of feces in phosphate buffer (pH

7.6) Recovery of Y pseudotuberculosis is enhanced by storing the

agar The organism grows both aerobically and anaerobically

PREFERRED CULTURE MEDIA AND SUBSTRATES

Trypticase soy agar and blood agar are effective for primary

isolation Differential media are as described for P. multocida For

isolation from feces, Paterson and Cook medium is recommended

(14) (Table 4.2)

AGENT IDENTIFICATION

Colony Morphology

After incubation at 37 C for 24 hr on trypticase soy agar, colonies

are smooth, round, entire, grayish yellow, butyrous, and 0.5-1 mm

in diameter Older colonies are 2-3 mm in diameter, raised, flat,

dry, and irregular with rough edges

Table 4.2 Medium of Paterson and Cook

Trypsinized meat agar

200.0Peptic digest of sheep blood

10.0Novobiocin (0.2%)

0.5Erythromycin (0.2%)

0.5Mycostatin (50,000 units/ml)

0.8Crystal violet (0.1%)

0.5

Cell Morphology

0.8-5.0 pm Coccoid forms usually stain bipolar Neither spores nor capsules are formed Peritrichous flagella usually develop between 20 C and 30 C Motility is best shown in semisolid media

at 25 C

Biochemical and Other Tests

Maltose, trehalose, and usually sucrose are fermented, and acid but not gas is produced Production of H2S is variable Urease and catalase are produced, but not indole and oxidase No hemolysis occurs on blood agar Differential characteristics are listed in Table 4.1

Serologic Identification

Serologic testing is not used for identifying strains of

are six serotypes (I-VI), four of which have two subtypes each (A and B) These serotypes and subtypes are based on 15 heat-stable somatic antigens (1-15), which are demonstrated by agglutination and agglutination-adsorption tests Additional serotypes VH and Vin and a subtype C of serotype Π have been proposed Among strains from birds, serotype I is most common, followed by serotypes Π and IV Serotypes V and VI have not been reported in birds, and type ΠΙ is rare Five heat-labile flagellar antigens (a, b, c,

d, e) are recognized but are rarely used for serologic characterization

SEROLOGIC DETECTION IN THE HOST

Serologic techniques are not used to detect Y pseudotuberculosis infection in poultry

DIFFERENTIATION FROM CLOSELY RELATED AGENTS

Differentiation of Y pseudotuberculosis from avian Pasteurella is

based on differences in biochemical characteristics, as indicated in Table 4.1 Diseases with similar lesions or signs, such as

tuberculosis, E coli infections, and salmonellosis, are differentiated

from pseudotuberculosis through isolation and identification or biochemical differentiation of causative agents

REFERENCES

1 Blackall, P.J H Christensen, T. Beckenham, L.L. Blackall, and M Bisgaard Reclassification of Pasteurella gallinarum, [Haemophilus] paragallinarum, Pasteurella avium and Pasteurella volantium as Avibacterium gallinarum gen nov., comb, nov., Avibacterium

paragallinarum comb, nov., Avibacterium avium comb, nov and

Avibacterium volantium comb. nov Int.J. Evol Microbiol 55: 353-362, 2005.

2 Brogden, K A., K R Rhoades, and K L Heddleston Anewserotype

of Pasteurella multocida associated with fowl cholera Avian Dis 22:185-

190 1978.

3 Carter, G.R. Studies onPasteurella multocida. I Ahemagglutination

test for the identification ofserological types. Am.J. Vet. Res 16:481^484

1955.

4 Christensen, Η.,M.Bisgaard,A.MBojesen, R Mutters, andJ.E Olsen Genetic relationships among avian isolates classified as Pasteurella

haemolytica, Actinobacillus salpingitidis or Pasteurellaanatis with proposal

of Gallibacterium anatisgen nov., comb nov. anddescription of additional

genomospecies with Gallibacterium gen nov Int J.Syst. Evol Microbiol 53: 275-287,2003.

5 Glisson, J R., C L Hofacre and J. P Christensen Fowl cholera 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, A Blackwell Publishing Company, Ames, Iowa pp.658-676 2003.

17

RGlisson, Tirath S Sandhu, and Charles L Hofacre

6. Hall,W. J., K L Heddleston, D. H Legenhausen, and R W Hughes

Studies on pasteurellosis. I. new species ofPasteurella encountered in

chronic fowl cholera Am J Vet Res 16:598-604 1955.

7 Hans, G H., and S Gabriel Modified stable Kovac’s reagent for

detection of indole Am.J. Clin Pathol 26:1373-1375 1956

8 Hatfield, R Μ., B A Morris, and R R Henry Development of an

enzyme-linked immunosorbent assay for the detection of humoral antibody

to Pasteurellaanatipestifer. Avian Pathol. 16:123-140 1987

9 Heddleston, K L., J.E Gallagher,andP. A. Rebers Fowl cholera: gel

difiusion precipitin test for serotyping Pasteurella multocida from avian

species Avian Dis. 16:925-936 1972

10 Heddleston, K L., T. Goodson, L Liebovitz, and C I Angstrom.

Serological and biochemical characteristics of Pasteurella multocidafrom

free-flying birds and poultry Avian Dis. 16:925-936 1972.

11 Hinz, K Η., M Ryll, and B Kohler Detection of acid productionfrom

carbohydrates by Rimerella anatipestifer and related organisms usingthe

buffer^ single substrate test Vet Microbiol 60:277-284. 1998

12. MacFaddin, J F Oxidase test In:Biochemical tests for identification

of medical bacteria, 2nd ed Williams and Wilkins,Baltimore, Md pp

249-253. 1980

13 Mushin, R., R Bock, and M Abrams. Studies on Pasteurella

gallinarum Avian Pathol. 6:415-423 1977.

14 Paterson,J S. and RCook method for the recovery ofPasteurella

pseudotuberculosis from feces. J Pathol. Bacteriol 85:241-242 1963

15 Pathanasophon, P., P Phuektes, T Tanticharoenyos, W Narongsak, and

T. Sawada A potential new serotype of Riemerella anatipestifer isolated

from ducks in Thailand Avian Pathol 31:267-270. 2002

16. Rimler, R B Presumptive identification ofPasteurella multocida

serogroups A, D, and F by capsule depolymerisation with

mucopolysaccharidases Vet. Rec 134:191-192. 1994.

17 Rimler, R B., R. D Angus, and M Phillips Evaluation of the

specificity of Pasteurella multocida somatic antigen-typing antisera

prepared in chickens using ribosome-lipopolysaccharide complexes

inocula Am J Vet Res 50:29-31 1989.

18 Rimler, R B., and K A. Brogden Pasteurellamultocida isolated from

rabbits and swine: serologictypesand toxin production. AmJ. Vet Res

47:730-737 1986.

19 Rimler, R B., andJ R Glisson Pseudotuberculosis In: Diseases of

poultry, 10th ed B W. Calnek, H J. Bames, C. W Beard, L. R

McDougald, and Y. M Saif,eds Iowa State University Press, Ames,Iowa,

McDougald, and D E. Swayne, eds Iowa State University Press, A

Blackwell Publishing Company Ames, Iowa pp 676-682.2003

22 Segers, P.,W Mannheim, M Vancanneyt, K DeBrandt, K H Hinz,

K.Kersters, andP. Vandamme Riemerella anatipestifer gen nov., comb, nov., the causative agent of septicemia anserum exudativa, and its

phylogenetic affiliation within the Flavobacterium-Cytophaga rRNA

homology group Int J Syst Bacteriol 43:768-776 1993.

23 Smith, J M, D D Frame, G. Cooper, A. A Bickford, G Y

Ghazikhanian, and B J Kelly Pasteurella anatipestifer infection in

commercial meat-typeturkeys in California Avian Dis. 31:913-917 1987

24 Toth, T E., and N L Norcross Precipitating and agglutinating activity in duck anti-soluble protein immune sera. Avian Dis 25:338-352.

1981

25 Waltman, W.D and A.M Home Characteristics of fowl cholera

diagnosed in Georgia 1989-1991. Avian Dis. 37:616-621, 1993

5 BORDETELLOSIS

Mark W Jackwood

SUMMARY Bordetellosis is an acute highly contagious disease of the upper respiratory tract of young turkeys (4-8 wk of age) The disease is caused by a gram-negative nonfermentative bacteria, Bordetella avium Members of the genus Bordetella are well known for their ability to colonize and damage ciliated epithelial surfaces in the respiratory tract and B. avium is no exception. Bordetellosis is most severe when it occurs in conjunction with other respiratory infections, especially Newcastle disease, or when turkeys are immunosuppressed Mortality can be high when poor management (inadequate ventilation, dust, chilling, or filthy conditions) is a complicating factor and there is

an associated colibacillosis Bordetella avium is an opportunistic pathogen in chickens

Agent Identification Bordetellosis can be diagnosed using a combination of clinical signs (snick or cough with a catarrhal nasal discharge) and bacterial culture

Serologic Detection in the Host Enzyme-linked immunosorbent assays and microagglutination tests are accurate and sensitive for detecting

antibodies in turkeys Test results can be an aid in diagnosis as well as monitoring vaccination

INTRODUCTION

In the 1970s an acute upper respiratory disease emerged as a major

disease problem in young turkeys The disease was more severe the

earlier posthatch turkeys become infected Initially some confusion

occurred as to the identification of the causative agent but a

definitive study established the agent as a new species in the genus

early years following its recognition, the disease and associated

colibacillosis produced high mortality but in recent years the

disease or the susceptibility of turkeys to the disease has changed

and the number and severity of reported outbreaks has lessened

Some of the possible explanations for the change in the character of

the disease include the presence of maternal antibody, as it is well

established that passive immunity is highly protective (9), or that

significance of bordetellosis is not as great now as when it was first

recognized but it still is considered to be a major cause of

respiratory disease in young turkeys

disease can only be reproduced following initial infection with an

upper respiratory disease virus vaccine such as infectious bronchitis

virus or Newcastle disease virus (4) Recent studies have reported

antibodies against B avium have been detected in peafowl (2).

CLINICAL DISEASE

Bordetellosis is characterized by an abrupt onset of a snick

(sneezing) in young turkeys Other signs include watery eyes,

submaxillary edema, and a clear nasal discharge that can usually be

expressed by placing gentle pressure on the nares Mouth breathing,

altered vocalization, dyspnea, huddling, and decreased consumption

of feed and water are also common Morbidity is high 80-100% in

young turkeys, and mortality is low (1% to 10%) except when other

infections occur simultaneously (colibacillosis) or when

management is poor, then mortality can be high (>50%) The

infection also adversely affects growth rate and toxins associated

with the bacteria damage tracheal cartilage beneath colonized

ciliated respiratory epithelium Mortality has been reported from

suffocation due to excessive mucus production in the trachea and

when damaged tracheas collapse (12)

SAMPLE COLLECTION

The bacterium is best cultured by swabbing the anterior trachea

through a midcervical aseptic opening Bacterial culture of sinus

material is usually not productive because of overgrowth by Proteus

sp Cultures should be taken early during the infection while cilia are heavily colonized with B avium If cultures are taken late in the

infection the most predominant bacteria isolated will likely be

Escherichia coli.

PREFERRED CULTURE MEDIA AND SUBSTRATES

but the bacteria is usually isolated from clinical material using MacConkey agar because this agar quickly demonstrates the bacteria’s nonfermenting characteristic and differentiates it from E

respiratory disease Initial isolation of B. avium is best done on MacConkey agar increased to 2.5% agar content to slow the spread

of faster growing organisms (6) Growth of B avium in liquid media requires that the cultures be aerated by agitation or other means because it is a strict aerobe

AGENT IDENTIFICATION

After 24 hr of incubation on MacConkey agar, colonies of

secured early they are often pure but when cultures are taken late there is often contamination with other bacteria especially E. coli

When such contamination occurs it is important to look in the more diluted part of the streak for typical colonies

Three colony types of B avium have been identified The most typical type is that described above with colonies being small, compact, translucent, and pearl-like with entire edges and glistening surfaces These colonies will be 0.2-1 mm in diameter after 24 hr and 1-2 mm after 48 hr Many isolates develop a raised brown- tinged center when grown to >48 hr on MacConkey’s agar A smaller percentage of isolates dissociate into a rough colony type having a dry appearance and a serrated irregular edge Rough colonies represent a phase-shift to a nonpathogenic form of the bacterium (3)

Biochemical tests that are used in distinguishing B avium from other nonfermenting bacteria include the oxidase test (+), catalase test (+), urease test (-), nitrate reduction test (-), and the ability to alkalinize certain amides and organic salts A microcupule test kit called the Rapid API 20 NE (Non-Fermenter Test, bioMerieux-

A very useful correlation has been made between the ability of

and pathogenicity (6) This association with pathogenicity is thought to be related to the ability of pathogenic strains to adhere to cilia The hemagglutination assay is performed by heavily inoculating solid media (blood agar, brain-heart infusion agar, or MacConkey’s agar) with suspected B avium isolates and incubating19

Mark W.Jackwood

36-48 hr at 37 C Bacterial growths are washed from the plate with

phosphate-buffered saline (PBS) and diluted to a concentration of

approximately 5 χ 109 cells/ml (0.5 optical density at 600 nm)

Equal volumes of bacterial suspension and erythrocyte suspension

(2% packed-cell volume in PBS) are mixed on a glass slide or plate

and observed for agglutination after gentle rocking Alternatively,

50:1 mixture of bacterial suspension and erythrocyte suspension

(0.5% packed-cell volume in PBS) are mixed in U-bottom shaped

wells of microtitration plates and allow to stand 1 hr at 25 C A

included with each test run

Comparable results can be obtained using erythrocytes fixed in

formaldehyde (F W Pierson, Virginia Polytechnic Institute and

State University, pers comm.) Fixed guinea pig erythrocytes are

prepared as follows:

and mix well

2 Centrifuge at 500 x g (2000 rpm) for 10 min Resuspend

erythrocytes in fixative containing 40 ml of PBS and 10 ml of 40%

formaldehyde The final pH of the solution should be

approximately 7.4

3 Mix gently on a rotary shaker at room temperature overnight

4 Centrifuge at 500 x g (2000 rpm) for 10 min and wash pellet 10

times with PBS

2% and store at 4 C lite fixed cells are stable for at least 6 mos

SEROLOGIC DETECTION IN THE HOST

An in-house enzyme-linked immunosorbent assay (ELISA) can be

commercially available ELISA has been developed and marketed

The commercially available ELISA kit is excellent and the results

are very compatible with the in-house ELISA test (9)

Prior to the development of the ELISA a microagglutination

procedure was performed (5) as outlined below

Microagglutination antigen is produced as follows:

broth with an isolated colony of B avium and incubate aerobically

at 37 C for 48 hr in a shaking-water bath or shaking incubator

2 Inoculate 500 ml of sterile broth in a 1000-ml flask with 10 ml of

broth culture from step 1 Incubate in a shaking-water bath or

incubator at 37 C for 48 hr When given ideal growth conditions, B

which interferes with the microagglutination test (1) This problem

can be circumvented by growing the bacterium on agar and

harvesting the cells in PBS Then, continue with step 3

neotetrazolium chloride stain Incubate 4 hr at 37 C following the

last addition of neotetrazolium chloride

overnight at 37 C

5 Centrifuge for 30 min at 10,960 x g Wash and centrifuge

antigen three times in PBS with 0.01% merthiolate

6 Centrifuge antigen in a graduated tube at 10,960 x g for 20 min,

discard the supernatant, and resuspend to make a 1:20 dilution of

packed cells in PBS with 0.01% merthiolate This is the stock

antigen Pass the stock antigen through a 22-gauge needle with a

syringe, run a block titration, aliquot the remaining antigen, and

freeze the aliquots at -30 C or below

Microagglutination antigen standardization (block titration) is as

follows:

1 Use separate microtitration plates for both B. avzwm-positive and

negative sera First make serial twofold dilutions of sera in test

tubes beginning with 1:10 and ending with 1:640 About 0.5 ml of

each dilution is needed for one microtitration plate

2 Drop 0.1 ml of stock antigen into row A, columns 1-8 of each plate

3 Drop 0.05 ml of PBS with 0.01% merthiolate into rows B through H, columns 1-8

well-defined, visible button in the negative control serum and detects antibody (no button) in the positive control serum It is necessary to do a block titration only once for each lot of B avium antigen produced Mark the appropriate dilution on the stock antigen for later reference

The actual microagglutination test is run as follows:

1 Make a 1:5 dilution of each serum sample, including both positive and negative controls

2 Drop 0.05 ml PBS with 0.01% merthiolate into each well of a microtitration plate

3 Drop 0.05 ml of diluted serum to the first row of wells Be sure

to include positive and negative control sera with each plate

4 Make twofold dilutions of the sera from 1:10 to 1:1280 down the plate

using the dilution factor from the block titration The antigen should be passed through a syringe with a 22 gauge needle to break

up all antigen clumps

6 Drop 0.05 ml of diluted B avium antigen into all wells

7 Incubate the test plate at room temperature for 24 hr and read Any titer 1:20 or greater is considered to be positive

The microagglutination test is not as user friendly as the ELISA but the test is sensitive and accurate It detects immunoglobulin M (IgM), which is the first antibody produced following infection The microagglutination test will not detect maternal antibody, which is immunoglobulin G (IgG) High levels of maternal antibody protect poults during the first 2 wk of life when the birds are most susceptible to the disease (12) Antibody titers (IgG) to B avium can be detected by ELISA 2 wk postinfection, with peak titers occurring between 3 and 4 wk postinfection (5,8,9)

DIFFERENTIATION FROM CLOSELY RELATED AGENTS

Bordetellosis in poultry should be differentiated from mycoplasmosis, chlamydiosis, cryptosporidiosis, Omithobacterium

Newcastle disease, influenza, and turkey rhinotracheitis (pneumovirus infection) The most closely related agents to be

(also referred to as B avium-tike bacteria in the literature) Both can

be isolated from the upper respiratory tract, but neither have shown

to cause disease in turkeys B avium causes agglutination of guinea

pig erythrocytes, is urease-negative, and does not grow on minimal essential medium agar or in 6.5% NaCl broth B bronchiseptica may cause hemagglutination, but it produces a positive reaction for

essential medium agar and in 6.5% NaCl broth (6) Molecular tests

Ribotyping using PvuH or restriction endonuclease analysis using

organisms but the REA test was found to be best as a routine epidemiologic tool (11)

Chapter 5 Bordetellosis

REFERENCES

1. Domingo, D D., M W Jackwood, and T P Brown. Filamentous forms

ofBordetella avium', culture conditions and pathogenicity Avian Dis.

36:706-713 1992

2 Hollamby, S., J G. Sikarskie, andJ Stuht. Survey of peafowl(Pavo

cristatus) for potential pathogens at three Michigan zoos J Zoo Wild. Med.

34:375-379. 2003

3 Jackwood, M W., D A Hilt, S. M Mendes, andP D.Cox Bordetella

aviumphase-shift markers: characterization of whole cell, cell envelope,and

outer membrane proteins J. Vet Diag. Invest 7:402-404 1995.

4 Jackwood, M. W., S. M McCarter, and T. P.Brown Bordetella avium'

an opportunistic pathogen in leghorn chickens Avian Dis 39:360-367.

1995.

5 Jackwood, D. J., and Y M Saif Development and use of a

microagglutination test to detect antibodies to Alcaligenes faecalis in

turkeys Avian Dis 24:685-701 1980.

6 Jackwood, M W.,Y. M Saif, P D Moorhead, and R. N Dearth.

Further characterization of the agent causing coryzainturkeys Avian Dis

29:690-705 1985

7 Kersters, K., K.-H Hinz, A Hertle, Segers, A Lievens, O

Siegmann, andJ DeLey Bordetella avium sp nov isolated from the

respiratory tracts of turkeys and other birds. Int J Syst Bacteriol

34:56-70 1984

8 Lindsey, D G., P D. Andrews, G. S. Yarborough, J K Skeeles, B Glidewell-Erickson, G. Campbell, and Μ B. Blankford Evaluation of a commercial ELISA kitfor detection and quantification of antibody against

Bordetella avium. In: Proc 75th Annual Meeting of the Conference of

Research Workers in Animal Diseases,Chicago, Ill. Abstract #31 Nov

14 15,1994.

9 Neighbor, N. K., J.K Skeeles, J. N. Beasley,and D L Kreider Useof

an enzyme-linked immunosorbent assay to measure antibody levels in turkey breeder hens, eggs, and progeny following natural infection or immunization with acommercial Bordetella avium bacterin. Avian Dis.

35:315-320 1991.

10 Raffel, T R., K B. Register, S A Marks, and L. Tempel Prevalence

of Bordetella avium infection in selected wild and domesticated birds in the eastern USA J.Wildl. Dis 38:40-46 2002.

11 Register,K B., R E Sacco, and G E. Nordholm Comparison of ribotyping and restriction enzyme analysis for inter- and intraspecies

discriminationofBordetella aviumandBordetella hinzii.J Clin Microbiol 41:1512-1519 2003

12. Skeeles, J K., and L. H Arp Bordetellosis (turkey coryza) In:

Diseases of Poultry, 10th ed B. W Calnek, H.J.Barnes, C W Beard, L R McDougald, and Y M Saif Iowa State University Press, Ames,Iowa pp 275-287.1997

21

6 INFECTIOUS CORYZA

Pat J Blackall

SUMMARY Infectious coryza, an acute upper respiratory tract disease of chickens, is caused by the bacterium Avibacterium

head-like syndrome, arthritis, and septicemia are either unusual or probably due to complications associated with other infectious agents

Agent Identification Diagnosis of infectious coryza is preferably made by the isolation and identification, by biochemical properties, of the bacterium A polymerase chain reaction (PCR) test, which can be applied either to suspect colonies or directly to samples from chickens, is now available The PCR test is particularly suited for those laboratories that lack suitable expertise and experience in the growth and

phenotypic identification of A paragallinarum or where samples are transported for long periods to the laboratory Although most isolates

of A paragallinarum are dependent upon V factor for growth in artificial media (meaning they show the traditional satellitic growth), some

isolates are V factor-independent This variation in growth factor requirements, along with the existence of nonpathogenic V factor­dependent organisms, increases the need for biochemical identification or the use of the PCR test Serotyping of isolates is important to guide the use of vaccines

Serologic Detection in the Host A range of serologic tests to detect antibodies has been described with hemagglutination-inhibition tests now being the most widely used

INTRODUCTION

Infectious coryza is an acute respiratory disease of chickens The

clinical syndrome has been recognized since the 1930s and has been

described in the early literature as roup, contagious or infectious

catarrh, and uncomplicated coryza (6) Early workers identified the

causative agent as Haemophilus gallinarum, an organism that

required both X (hemin) and V (nicotinamide adenine dinucleotide;

NAD) factors for growth in vitro However, from the 1960s to the

1980s, all isolates of the disease-producing agent have been shown

to require only V factor and have been termed Haemophilus

paragallinarum (6) V factor-independent H paragallinarum

isolates have been encountered in the Republic of South Africa (21)

and Mexico (15) Most recently, a polyphasic taxonomic study has

shown that the Haemophilus paragallinarum is not a member of the

genus Haemophilus (2) Thus, H paragallinarum was allocated to

a new genus - Avibacterium - along with several other chicken

associated members of the family Pasteurellaceae (2) Hence, the

causal agent of infectious coryza is now called Avibacterium

or -independent This text will use the new terminology of A

the older terminologies

The disease occurs worldwide and causes economic losses due to

an increased number of culls and a marked reduction from 10% to

more than 40% in egg production, particularly on multiage farms

The disease is essentially limited to chickens and does not threaten

public health

CLINICAL DISEASE

Infectious coryza may occur in growing chickens and layers The

most common clinical signs are nasal discharge, facial swelling,

lacrimation, anorexia, and diarrhea Decreased feed and water

consumption retards growth in young stock and reduces egg

production in laying flocks (6) In layers, the disease can have a

modi greater impact than the relatively simple scenario described

above As an example, an outbreak of the disease in older layer

birds in California, which was not associated with any other

pathogen, caused a total mortality of 48% and a drop in egg

production from 75 to 15.7% over a 3 wk period (8) The disease

can have significant impact in meat chickens In California, two

cases of infectious coryza, one complicated by the presence of

increased condemnations, mainly due to airsacculitis, that varied

of other pathogens, have been reported in broiler and layer flocks in South America (27) Infectious coryza can also be a problem in the village production system e.g there are reports of outbreaks in such chickens in Thailand (32) and Indonesia (24) Overall, there is considerable evidence that infectious coryza outbreaks can have a much greater impact in developing countries than in developed countries

SAMPLE COLLECTION

Two to three acutely diseased chickens should be killed and the skin over their sinuses seared with a heated spatula The skin is then incised with a sterile scalpel blade, and a sterile cotton swab is inserted into the sinus cavity Typically, in the early phase of the

Swabs of the trachea and air sacs may be taken, although the organism is less frequently isolated from these areas Avibacterium

than 5 hr outside of birds Sinus swabs held in Ames Transport medium (without charcoal) can yield positive cultures for up to 8 days at transport temperatures of either 25 C or room temperature (9)

Live bird sampling is also possible In this technique, gentle milking pressure is exerted on the sinus area and mucus forced from the nostril The expressed mucus should be sampled by a bacteriologic loop, with care being taken to avoid touching the surface of beak or nostril A swab of the expressed mucus is also the optimal sample for the A paragallinarum PCR Swabs collected by this method will still yield a PCR positive reaction after storage in glycerol-enriched phosphate buffered saline for 180 days at either 4 C or -20 C (12)

PREFERRED CULTURE MEDIA AND SUBSTRATES Artificial Media

Blood agar is commonly used for the isolation of A

22

Chapter 6 Infectious Coryza

such as Bacto-tryptose-blood-agar base (Difco, Detroit, Mich.) and

is enriched with 5% erythrocytes, which may be from any animal

The inoculum is streaked onto the blood agar plate in the

conventional maimer, after which the plate is cross-streaked with a

nurse culture Blood agar is deficient in V factor, and the role of the

nurse culture is to excrete excess V factor to support the growth of

nurse cultures, it is recommended that Staphylococcus hyicus, a

normal inhabitant of the skin of chickens, be used Avibacterium

C A convenient procedure is to use candle jars if CO2 incubators

are not available

An alternative isolation (and maintenance) medium that is

particularly suited to laboratories in developing countries has been

described by Terzolo et al (31) This medium consists of Columbia

blood agar base (Becton Dickinson Microbiology Systems, Sparks,

Md.) with 7% lysed equine blood The lysed equine blood is

prepared by holding fresh equine blood at 56 C for 40 min with

occasional stirring The lysed blood can be stored at -20 C A

selective version of the medium, which should only be used in

parallel with the nonselective medium, contains bacitracin (5 U/ml),

cloxacillin (5 pg/ml), and vancomycin (25 pg/ml) The plates are

incubated under a microaerophilic atmosphere

Several complex media have been described that support growth

of avian hemophili (a term used generically to refer to bacteria

within this group) Such media, although not suitable for isolation

due to problems with overgrowth by contaminants, are particularly

useful for characterization tests following initial isolation Two

media that have proven very useful are Haemophilus maintenance

medium (HMM) and supplemented test medium agar (TM/SN),

originally described by Rimler et al (25, 26) HMM base consists

of 1% polypeptone (BBL), 1% biosate peptone (BBL), 0.24% beef

extract, 0.005% para-aminobenzoic acid, 0.005% nicotinamide,

0.1% starch, 0.05% glucose, 0.9% NaCl, 0.23% leptospira base

Ellinghausen-McCullaugh-Johnson-Harris (EMJH) (Difco), and

2% Noble agar (Difco) Immediately before being poured, this

medium is supplemented with 0.0025% reduced NAD and 1%

chicken serum TM/SN base consists of 1% biosate peptone (BBL),

1% NaCl, 0.1% starch, 0.05% glucose, and 1.5% Noble agar

(Difco) and is supplemented with 5% oleic albumin complex, 1%

chicken serum, 0.0005% thiamine, and 0.0025% reduced NAD A

modified version of TM/SN which consists of brain heart infusion

agar that is supplemented with 5% oleic albumin complex, 1%

chicken serum, 0.0005% thiamine, and 0.0025% reduced NAD has

proven to be as suitable as the original formula given above Broth

versions of HMM and TM/SN are prepared by omission of the agar

Chicken Embryos

Avian hemophili can be propagated in 5 to 7-day-old chicken

embryos, commonly by inoculation via the yolk sac route After

overnight incubation, large numbers of hemophili are present in the

yolk, which then can be harvested and preserved by freezing at -70

C (or lower) or by lyophilization

Chicken Inoculation

Another efficient diagnostic procedure is to inoculate suspect

exudate into the infraorbital sinus of two or three susceptible

chickens (preferably 4 wk old or more) The appearance of the

typical clinical signs of infectious coryza in 24-48 hr is diagnostic

On occasion, if the number of viable organisms is low, particularly

in chronic cases, the incubation period may be delayed for up to 1

wk In such cases, a second passage may be required to produce the

typical rapid onset of clinical signs If the exudate is heavily

contaminated with extraneous bacteria, the chicken inoculation test

can be more reliable than culture In such instances, most of the

extraneous bacteria will be cleared by the host defense mechanisms,

whereas A paragallinarum will colonize and produce typical disease

AGENT IDENTIFICATION Background

dependent organism that can be isolated from chickens Nonpathogenic avian hemophili have been recognized since the 1930s The organisms have been the subject of several taxonomic changes - being initially named as Haemophilus avium (16) and

species A (22) In a recent polyphasic study, these non-pathogenic organisms have been transferred to the genus Avibacterium as A

member of the new genus is A. (Pasteurella) gallinarum.

A range of other hemophili, none of which are yet assigned to named species, have been isolated from birds other than chickens The identification of these other hemophili will not be discussed further here

Morphology

Examination of a direct smear of the exudate by Gram stain can be useful for initial assessment of the microbial flora The finding of gram-negative rods in direct smears, however, is not sufficient grounds for definitive diagnosis and must be followed by culture.After incubation of blood agar plates for 24—^48 hr, V factor­dependent isolates of A paragallinarum produce tiny dewdrop colonies up to 0.3 mm in diameter adjacent to the nurse culture The colonies become smaller with increasing distance from the nurse culture For the satellitic growth to be obvious, cultures must

be examined within 24-48 hr The V factor-independent A

not show satellitic growth Colonies of A avium, A volantium,

much bigger than V factor-dependent A paragallinarum Some

isolates of A volantium may produce a yellowish pigment.

Growth Requirements

Most avian hemophili have a requirement for V factor but not for

X factor (6) The determination of the growth factor requirements

of these organisms is not an easy process The use of some brands

of commercial growth factor discs on media such as brain-heart infusion agar or susceptibility test agar can result in a high percentage of cultures that falsely appear to be both X and V factor­dependent The combination of Oxoid growth factor discs (Unipath, Ltd., distr Unipath, Ogdensburg, N.Y.) and TM/S (TM/SN without added NAD) has been shown to be suitable for growth factor testing (5) Some isolates of avian hemophili may have such lowered V factor requirements that the serum must be omitted from TM/S

Alternative tests such as the porphyrin test (18) for X factor testing

or the use of purified hemin (X factor) and NAD (V factor) as supplements to otherwise complete media are possible but are generally too complex for diagnostic laboratories

V factor-independent isolates of A paragallinarum have been recognized to date in the Republic of South Africa (21) and Mexico (15) Hence, diagnostic bacteriologists need to be aware of the possibility of such variants emerging in other areas

Typically, A paragallinarum isolates fail to grow in air, although

some strains will develop this ability on subculturing A avium,

Physicochemical Properties

The ability to reduce nitrate to nitrite and ferment glucose without the formation of gas is common to all of the avian hemophili Oxidase activity, the presence of the enzyme alkaline phosphatase, 23

Pat J.Blackall

and a failure to produce indole or hydrolyse urea or gelatin are also

uniform characteristics A paragallinarum is catalase-negative,

whereas all other members of the genus Avibacterium including the

hemophilic species of A avium, A volantium and Avibacterium

species A are catalase-positive (2)

Carbohydrate fermentation patterns of the avian hemophili are

generally determined using a phenol red broth (Difco) containing

1% NaCl, 0.0025% NADH, 1% chicken serum, and 1%

carbohydrate For routine identification, the use of this broth and a

dense inoculation (i.e., the modified reference technique of Blackall

(1)), is a most suitable approach for determining fermentation

patterns For larger studies, a replica plating technique is more

suitable (1)

Table 6.1 presents those properties that allow full identification of

the avian hemophili The properties of all members of the genus

ferment either galactose or trehalose clearly separates it from all

other members of the genus, both hemophilic and non-hemophilic

Serotyping Tests

Two different, but interrelated, serotyping schemes for A

(19) schemes The most widely recognized and applied scheme is

the Page scheme, which recognizes three serovars (A, B, and C) of

A paragallinarum Although the original scheme was based on an

agglutination test, the Page serovars are best recognized by a

hemagglutination-inhibition (HI) test The antigen for this HI test

can be produced by either of two methods In the first method,

whole bacterial cells are harvested from an overnight broth and

stored at 4 C in 1/100 of the initial broth volume for at least 3 days

(3) Alternatively, the antigen can be produced by the technique

originally used in the Kume scheme (19) In this method, cells of A

(PBS) (pH 7.0), resuspended in 0.5 M KSCN/0.425 M NaCl to a

density equivalent to a MacFarland nephelometer tube number 5

(Difco), held at 4 C for 2 hr with agitation, and then sonicated The

antigen is washed three times in PBS and resuspended in PBS with

0.01% merthiolate to a density equivalent to a MacFarland

nephelometer tube number 5 With either antigen type, the HI test

should be performed with glutaraldehyde-fixed chicken

erythrocytes The erythrocytes are prepared by collecting fresh

chicken blood into an equal volume of Alsever’s solution The

suspension is centrifuged and the erythrocytes are washed three

times in 0.15 M NaCl The erythrocytes are then suspended to 1%

(v/v) in a glutaraldehyde-salts solution and held at 4 C for 30 min

The glutaraldehyde-salts solution is prepared by diluting 25%

glutaraldehyde to 1% in a solution containing one volume of 0.15 M

Na2HPO4 (pH 8.2), nine volumes of 0.15 M NaCl, and five volumes

of distilled water The fixed erythrocytes are collected by

centrifugation, washed five times in 0.15 M NaCl and five times in

distilled water and finally resuspended to 30% (v/v) in distilled

water containing 0.01% merthiolate This stock of fixed

erythrocytes is held at 4 C, and a working dilution of 1% is prepared

in PBS (pH 7.0) containing bovine serum albumin (0.1%) and

gelatin (0.001%) (PBS-B-G) Reports that Page serovar B is not a

true serovar (28) have been discounted, and Page serovar B has

been conclusively shown to be serologically distinct (34)

The Kume scheme is a hemagglutination-inhibition serotyping

scheme using bacterial cells that have been treated with potassium

thiocyanate and then sonicated, and glutaraldehyde-fixed chicken

erythrocytes The preparation of the antigen and the chicken

erythrocytes for the Kume scheme has already been described in the

section on the Page serotyping scheme The nomenclature of the

Kume scheme has been changed since the original publication

Under die altered terminology the three Kume serogroups are

termed A, B, and C (4) This terminology emphasizes that the

Kume serogroups correspond to the Page serovars Within the

Kume serogroups, the use of absorbed antisera allows the recognition of serovars, with the nine currently recognized Kume serovars being A-l, A-2, A-3, A-4, B-l, C-l, C-2, C-3, and C-4 (4) The antisera, with the exception of the antisera for B-l and C-3, need to be absorbed to allow recognition of the Kume serogroups The antisera to be absorbed are diluted 1 in 40 in PBS-B-G The absorption is performed using antigens adjusted to 64 HA units and

at five times die volume of the diluted serum The adjusted antigen

is centrifuged and the supernatant discarded The pelleted antigen

is resuspended in the diluted antiserum The suspension is left at room temperature for 2 hr and then overnight at 4 C The suspension is then centrifuged and the supernatant retained as the absorbed antiserum (still at 1 in 40 dilution) Depending on the antiserum, a succession of absorptions may need to be performed Once the antigen absorption has been completed, the sera need to be absorbed with fixed erythrocytes, using 5 times the volume of fixed erythrocytes compared with the serum The relevant volume of erythrocytes is centrifuged and the supernatant discarded The pelleted erythrocytes are resuspended in the diluted antiserum The suspension is left at room temperature for 2 hr and then overnight at 4C The suspension is then centrifuged and the supernatant retained

as the absorbed antiserum (still at 1 in 40 dilution) The Kume scheme has not been widely applied as it is technically demanding

to perform

Molecular Identification

A polymerase chain reaction (PCR) test that is specific for A

evaluated with a wide range of A paragallinarum isolates and is specific and sensitive The test, termed the HP-2 PCR, has been shown to be suitable for use on purified DNA extracts as well as on crude colony preparations obtained from isolation plates The HP-2 PCR can be used directly on swabs of nasal mucus obtained from the squeezing of the nostril The test can be inhibited in the presence of blood and swabs obtained via by slicing open the infra­orbital sinus during necropsy are not optimal for use in the HP-2 PCR Direct PCR examination of swabs has been shown to outperform culture in China (10, 12) The HP-2 PCR is particularly useful in regions where both NAD-independent A paragallinarum and Omithobacterium rhinotracheale are present Molecular typing methods such as restriction endonuclease analysis (7) and ribotyping (20) have proved useful in epidemiologic studies A PCR-based typing method - ERIC-PCR - has also shown a capacity

to sub-type isolates of A paragallinarum (30).

Maintenance

Although the initial isolation of A paragallinarum from acute

infectious coryza is not difficult, it is a fragile organism requiring special care for propagation and maintenance in the laboratory Cultures can be maintained on blood agar plates by weekly passages Cultures incubated for 24-48 hr at 37 C and then stored

at 4 C in a candle jar will remain viable for up to 2 wk Cultures can be preserved by the lyophilization or freezing (at -70 C or lower) of infected yolk (see the section on chicken embryos) Storage at -70 C of a heavy suspension in the commercial bead systems now commonly available is also possible

SEROLOGIC DETECTION IN THE HOST

Although a range of serologic tests for the detection of antibodies

hemagglutination-inhibition (HI) tests are in widespread use The various HI tests differ in the methods used to prepare the antigen and the type of red blood cells used For the purposes of this overview, the three main HI tests have been termed the simple, extracted, and treated HI tests Although most of these HI tests were originally described as tube or macroplate tests, all can also be

Chapter 6 Infectious Coryza

performed in microtiter trays using appropriate volumes The

simple HI test is based on whole bacterial cells of a Page serovar A

organism (17) Cells are harvested and suspended in PBS

containing 0.01% merthiolate (pH 7.0) Pretreatment of the sera

may be necessary to eliminate nonspecific agglutinins A 5%

suspension of chicken erythrocytes is added to the sera (1:5

proportion) and the mixture is incubated for 2 hr at room

temperature and 12 hr at 4 C (29) Two-tenths milliliter of antigen

(containing 20 HA units/ml) is added to 0.2 ml of serially diluted

serum (initial dilution of 1:5) After incubation at room temperature

for 10 min, 0.4 ml of 0.5% chicken erythrocyte suspension

containing 0.02% (v/v) gelatin is added The HI titers are

determined after incubation for 30-40 min at room temperature

This test has been performed mainly using antigen prepared from A

The extracted HI test is based on potassium thiocyanate-extracted

and sonicated cells of A paragallinarum and glutaraldehyde-fixed

chicken erythrocytes (29) The preparation of the antigen and the

chicken erythrocytes has already been described in the section on

the Page serotyping scheme Although the methodology of the

extracted HI test is as described for the simple HI test, the sera are

pretreated with 10% glutaraldehyde-fixed erythrocytes to eliminate

nonspecific hemagglutinins, and PBS containing 0.1% bovine

serum albumin and 0.001% gelatin is used as the diluent

The extracted HI test has been used to detect antibodies in

chickens receiving inactivated vaccines based on Page serovar C

organisms (29) In chickens infected with a serovar C organism, the

majority of the birds remain negative in this test (36) Note that, in

this report, the erythrocytes were fixed in formalin instead of

glutaraldehyde (36)

The treated HI test is based on hyaluronidase-treated whole

bacterial cells of A paragallinarum and formaldehyde-fixed

chicken erythrocytes (37) Whole bacterial cells that have been

adjusted to 10 times the optical density of 0.4 at 540 nm are treated

with hyaluronidase (50 units/ml) in phosphate buffer (pH 6.0) in a

waterbath at 37 C for 2 hr The treated antigen is then washed twice

in PBS and then resuspended in the original volume of PBS

The test uses the same methodology as that described for the

extracted HI test except that the erythrocytes are 1.0%

formaldehyde-fixed chicken erythrocytes The diluent used in this

HI test is the same as that used in the extracted HI test All sera are

pretreated with 50% (v/v) formaldehyde-fixed chicken erythrocytes

(one volume of antigen with eight volumes of erythrocytes for 2 hr

at 37 C with serum recovered by centrifugation and regarded as being a one in five dilution)

The extracted HI test has been used to detect antibodies to Page serovars A, B, and C in vaccinated chickens (35) Vaccinated chickens with HI titers of 1:5 or greater in the HI tests based on the simple and extracted antigens have been found to be protected against subsequent challenge (29)

DIFFERENTIATION FROM CLOSELY RELATED AGENTS

Infection with A paragallinarum must be differentiated from other diseases, such as chronic respiratory disease, chronic fowl cholera, fowl pox, omithobacteriosis (due to O rhinotr acheale), swollen head syndrome (associated with avian pneumovirus) and hypovitaminosis A, which can produce similar clinical signs

should consider the possibility of other bacteria or viruses as complicating agents, particularly if mortality is high and the disease takes a prolonged course

The simple isolation of a satellitic, gram-negative organism is not sufficient to identify an isolate as A paragallinarum For laboratories with limited facilities, sufficient evidence for a diagnosis of infectious coryza would be the isolation of a catalase­negative, gram-negative organism that exhibits satellitic growth and fails to grow in air, together with a history of a rapidly spreading acute coryza in a flock

The use of the PCR is recommended as the definitive test Laboratories without access to PCR technology should determine growth factor requirements and the ability to ferment glucose,

differentiated from the other members of the genus Avibacterium For laboratories with extensive resources, a complete phenotypic identification based on the properties shown in Table 6.1 is

isolated, diagnostic microbiologists must be aware of the fact that non-satellitic bacteria should be considered as suspect

of the species and have been obtained from chickens showing upper respiratory disease

Table 6.1. Distinguishing properties of the species within the generaA vibacteriumK

Property Avibacterium

gallinarum

Avibacterium paragallinarum

Avibacterium volantium

Avibacterium avium Avibacterium sp A.

PatJ. Blackall

ACKNOWLEDGEMENT

The authors would like to acknowledge the contributionof Dick Yamamoto

who has been the senior author or co-author for previous editions of this

text

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Campylobacter INFECTIONS IN POULTRY

Jaap A Wagenaar and Wilma F Jacobs-Reitsma

SUMMARY Poultry may easily become colonized with Campylobacter jejuni and/or Campylobacter coli These bacterial species do not

cause clinical disease in poultry but contamination of the meat during laughter and processing is a well recognized source of foodbome illness in humans

Agent Identification Campylobacter is isolated from poultry feces and cecal contents using selective agar media under microaerobic conditions Subsequently, suspect isolates are confirmed to be Campylobacter and identified to the species level by using a limited number of biochemical tests or appropriate PCR tests

Serologic Detection in the Host Campylobacter infections are not routinely diagnosed by serologic assays.

INTRODUCTION

The gut of poultry easily becomes colonized with the thermophilic

species are very rarely (< 1%) isolated from poultry Also wild birds

frequently are found to carry Campylobacter spp., with a strong

association of C lari present in seagulls Campylobacters are

commensal organisms without causing any clinical disease in

poultry, though there is an immune response and the presence of the

organism is detected by the host The significance of

not for reasons of clinical poultry diseases Vertical transmission of

the infections does not occur, and eggs are not contaminated

Contamination of meat products is the main issue and consequently

meat producing avian species

Many broiler flocks are colonized at the age of slaughter because

poultry can be infected with low numbers of Campylobacter and

reservoirs in both domesticated and wild warm-blooded animals

(18, 32) The fact that the organism colonizes in high concentrations

results in shedding of high levels of Campylobacter and intense

contamination of the environment Cleaning and disinfection of

poultry houses and the surroundings of the houses between

production cycles is difficult When the houses are stocked with a

new flock of day-old chickens, the infection cycle can easily start

again When Campylobacter-^QsitiNQ birds are processed in the

slaughterhouse, the meat may easily become contaminated with

bacteria from the gut contents Campylobacter will only multiply

under specific atmospheric conditions with reduced oxygen tension

and within a temperature range of 30 - 45 C These conditions are

not present during processing, transport and storage of the meat In

contrast with e.g Salmonella, the Campylobacter concentration will

not increase along the food chain in case of troubles with

maintaining chilled conditions At the consumer level,

but the risk for humans is mainly in cross contamination from the

raw meat to ready-to-eat products like salads Campylobacter is a

highly infectious organism so even ingesting low doses is

associated with a relatively high probability of human illness

In addition to foodbome infections, humans may become ill from

direct contact with infected animals like poultry, but also pets like

(young) cats and dogs Petting zoos are identified as a risk factor

especially when less attention is paid to personal hygiene

gastro-enteritis in humans (30) Campylobacteriosis in humans is

characterized by diarrhea and abdominal cramps Complications

that may occur are reactive arthritis and the Guillain-Barre

syndrome (10, 25)

CLINICAL DISEASE

Sporadic cases of vibrionic hepatitis in poultry have been described, supposedly caused by Campylobacter However, there is only the suggestion of a causative role of Campylobacter without any proof (7) C jejuni may cause illness in ostriches but the economic loss due to Campylobacter infections are assumed to be very limited (28)

Both C jejuni and C. coli have a high prevalence in poultry, but

both species generally are considered as normal gut inhabitants Co­infections with multiple strains frequently occur There is a strong seasonality in the prevalence in broiler flocks with higher isolation rates in summer (18)

Control programs in poultry to reduce the number of infected flocks are implemented to prevent human illness from contaminated poultry meat Epidemiology of Campylobacter infections in broiler flocks is still not completely elucidated Recently a systematic review based on UK data and comprising 159 research papers was published on risk factors for introduction of Campylobacter into broiler flocks (1) Partial depopulation (thinning) and multiple poultry houses on a farm were identified as contributing factors associated with increased risk, and hygiene barrier, parent company and certain seasons of rearing were associated with decreased risk

Diagnostics

Testing poultry for presence of Campylobacter is performed for particular research objectives, for more general monitoring reasons (observing trends), or to identify the Campylobacter status of the individual flock The latter may be used to decide on scheduled slaughter of negative flocks followed by positive flocks

Monitoring programs

Monitoring programs are implemented to identify trends in

programs It offers the possibility to link the poultry data to the

human Campylobacter data in order to assess the contribution of

poultry to the human burden of illness A good example is the obligatory monitoring of Campylobacter in broilers in the EU as prescribed by Zoonoses Directive 2003/99/EC According to this directive the monitoring of broiler flocks started on January 1st,

2005 with the European Food Safety Authority (EFSA) as the responsible agency for compilation and reporting of data collected

by the EU member states At this moment (February 2006) the EU

is unique in the world for this monitoring program

(http ://www.efsa eu.int/science/monitoring zoonoses/reports/catind

ex en.html)

Individual flocks

In some European countries (Denmark, Iceland, Norway) poultry flocks are tested for Campylobacter prior to slaughter, and negative and positive flocks are slaughtered separately The meat from positive flocks is treated (freezing or heat treatment) to reduce the

27

Jnp AWagenaarand Wilma F Jacobs-Reitsma

flocks is sold as fresh meat As consumers are exposed to reduced

levels of Campylobacter compared to the situation without

separation, this approach aims to reduce the burden of illness The

effect of this approach is strongly dependent on the reliability (in

particular sensitivity) of the diagnostic procedure, including

collection and transport of samples, and performance of the

detection assay

SAMPLE COLLECTION

Isolation of Campylobacter from feces and contents of the ceca is

described here as these samples are commonly used for the

diagnosis of a Campylobacter infection in poultry Carcass samples

can be analyzed but this needs a specific approach including

selective enrichment Isolation of Campylobacter from carcass and

meat samples is described in detail elsewhere (12,13)

Collection of samples

A Campylobacter infection rapidly spreads within a flock (31),

and close to 100% of the animals become colonized, shedding >106

cfu Campylobacter per gram feces for a prolonged period of time

(at least till slaughter age of broilers) At a within-flock prevalence

of >40%, 10 samples will be sufficient to detect the Campylobacter

infection at a 95% confidence level In case of scheduled slaughter

it is most important to know the actual Campylobacter status of the

flock just prior to slaughter Consequently, the samples should be

taken as close to slaughter as possible: at the farm or even at the

slaughterhouse In the latter case, reliable results can be obtained by

taking intact ceca for examination Living birds can be sampled by

taking fecal/cecal droppings or cloacal swabs For reliable detection

of Campylobacter by culture, freshly voided feces should be

collected All samples must be prevented from drying out before

culture At the slaughterhouse, ceca can be cut with sterile scissors

from the remaining part of the intestines and submitted intact to the

laboratory in a plastic bag or Petri-dish

Transport of samples

precautions have to be taken to prevent the samples from

dehydration, atmospheric oxygen, sunlight and elevated

temperature When swabs are used, a transport medium (like Amies,

Cary Blair or Stuart) must be used These transport swabs are

available commercially

When only small amounts of fecal/cecal samples can be collected

and transport swabs are not available, shipment of the specimen in

transport media is recommended Several transport media have been

described: Cary-Blair, modified Cary-Blair, modified Stuart

medium, Campythioglycolate medium, alkaline peptone water and

semisolid motility test medium Good recovery results have been

reported using Cary-Blair (15, 23)

No specific recommendation on the temperature for transportation

can be made, but it is clear that freezing and high temperatures

reduce viability In general high temperatures (>20 C), low

temperatures (<0 C) and especially fluctuations in temperature must

room temperature for short periods to avoid unnecessary

temperature shocks When the time between sampling and

processing is longer, storage at 4 (±2) C is advised Transport to the

laboratory and subsequent processing should therefore be as rapid

as possible (preferably the same day, but within at least 3 days)

PREFERRED CULTURE MEDIA AND SUBSTRATES

Isolation of Campylobacter from fecal/cecal or intestinal samples

is usually performed by direct plating onto selective medium or by

using the filtration method on nonselective agar As enrichment of

fecal samples is not performed routinely, enrichment media will not

be discussed in this chapter

Several commercial enzyme immunoassays are available for the

are not validated for routine use in poultry fecal samples

Selective media for isolation

Many media currently are in use for the bacteriological culture of

available A recommendation for a specific medium cannot be given Experience in laboratories is an important factor in the choice of the medium There are differences in medium preferences

in different places in the world Many European countries use mCCDA whereas in the US Campy-cefex and CVA are more commonly used (see list below) A detailed description of the

developments in Campylobacter detection and the variety of

existing media is given by Corry et al (8, 9) Campylobacter- selective media can be divided into two main groups: blood­containing media and charcoal-containing media Both blood components and charcoal remove toxic oxygen derivatives The selectivity of the media is determined by the antimicrobials used Cefalosporins (generally cefoperazone) are used, sometimes in combination with other antibiotics (e.g vancomycin, trimethoprim) Cycloheximide (actidione) or amphotericin B are used to inhibit yeasts and molds (17) All media allow the growth of C jejuni and

C coli but the main difference between the media is the degree of

inhibition of contaminating flora depending on the combination of antimicrobials used There is no medium available that allows

extent, other Campylobacter species (e.g C lari, C upsaliensis,

grow on some of these media, especially at the less selective temperature of 37 C Examples of selective blood-containing solid media are: Preston agar (4), Skirrow agar (24), Campy-cefex (29), CVA (22)

Examples of selective charcoal-based solid media are:mCCDA (modified Charcoal Cefoperazone Deoxycholate Agar)(ll), slightly modified version of the originally described CCDA (5), Karmali agar or CSM (Charcoal-Selective Medium) (14), CAT agar (Cefoperazone, Amphotericin, Teicoplanin agar), facilitating growth of C. upsaliensis (2).

Inoculation of selective media

Samples are sown on to the solid selective agar medium by using a loop or a swab Plating for single colonies needs special attention

Passive filtration

Passive filtration avoids the use of selective media and is useful for the isolation of the more antimicrobial-sensitive Campylobacter species (26) In resource-poor countries this method can be used as

an alternative for the use of expensive selective media For passive filtration, a suspension of the feces is made in PBS (approximately 1/10 dilution) Approximately 100 μΐ of this suspension are then carefully layered on to a 0.45 or 0.65 pm filter, which has been previously placed on top of a nonselective blood agar plate Care must be taken not to allow the inoculum to spill over the edge of the filter The bacteria are allowed to migrate through the filter for 30-

45 min at 37 C or room temperature The filter is then removed, the fluid that has passed through the filter is spread with a loop or spreader to facilitate the isolation of single colonies The plate is incubated microaerobically at 41.5 C (or at 37 C for isolation of

non-thermophilic Campylobacter species).

Incubation Atmosphere

A microaerobic atmosphere of 5-10% oxygen, 5-10% carbon dioxide (and preferably 5-9% hydrogen) is required for optimal growth (9) Appropriate microaerobic atmospheric conditions may