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Serotyping of Campylobacter jejuni isolated from sporadic cases and outbreaks in British Columbia


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Serotyping of Campylobacter


Isolated from Sporadic

Cases and



British Columbia

P. M. S.MCMYNE,1*J. L.PENNER,2R. G. MATHIAS,' W. A. BLACK,' ANDJ. N. HENNESSY2 DivisionofLaboratories, Ministry of Health, Vancouver, BritishColumbia, Canada V6J4M3';and

Department ofMedicalMicrobiology, Faculty of Medicine, University of Toronto, Banting Institute,Toronto,

Ontario, Canada MSG JLS2

Received3February 1982/Accepted12May 1982

Campylobacterjejunifromsporadiccasesandoutbreaks ofgastroenteritiswere

serotyped onthe basisof heat-extracted soluble thermostable antigensidentified

with the use of the passive hemagglutination technique. A total of168 isolates

were separated into45 differenttypes. Thelargestproportion ofthe isolatesfell into three serotypes, each with 11 to 12.5% of the total number. Three less frequentlyoccurring serotypeseachincludedapproximately5%, and the remain-ing 50% oftheisolatesweredistributed among39otherserotypes. Inmostcases,

serotypingdemonstrated that epidemiologically linked isolateswereof the same

serotype, but the outbreak strains could belong either to frequently or to

infrequently isolatedserotypes.Thehighcorrelation between clinical findings and serotyping results confirmed the applicability of the serotyping scheme in epidemiological investigations of C. jejuni infections.

In the last 10 years, it has been well docu-mented thatCampylobacterjejuni bacteriaare a common causeof enteritis in humans


8, 13, 20, 23).Methods have been developed and wide-ly


for routine isolation of these agents (6, 8, 12, 23), but much still remains to be learned about the


of the infec-tions.

Previous studieshave drawn attention to the


transmission of the infectious agents through contaminated water, milk, orfood (10, 11, 18, 19, 22, 25). Some indirect evidence suggests that household pets or domestic ani-mals may be the source ofhumaninfections (2, 3, 24). To confirm these suspected routes of sources andtransmissions, effective discrimina-tion between strains of the species is required. Progress in thisareahas beenlimited. Sincethe bacteria have onlyafew biochemical reactions that may beroutinely demonstrable inthe clini-callaboratory (6, 12, 16), it isnotexpected that biotyping will be adequate for


studies. Interest has therefore focused on the use ofserotyping methods for epidemiological tracing. Several different serotyping schemes havealready been described, but all are still in the developmental stages (1, 14, 15, 17, 21). Adoption by publichealth laboratories and ref-erence centers ofone scheme or another will dependto alarge extentupon the effectiveness ofthe scheme in thepractical situation.

Inthiscommunication,wedescribeastudyin which one such scheme was used to serotype isolates submitted to apublic health laboratory

with the objectives ofassessing the scheme for its performance in


studies and for




into the


of infections caused by C.jejuni.


Isolation and identificationof bacteria. Fecal

speci-mensinCary-Blair transport medium (7) from different

areasin theprovinceweresent totheBritish Columbia

Provincial HealthLaboratory for isolationand

identifi-cation ofdiarrhea-producing pathogens. Forisolation

of C. jejuni, eachspecimen wasinoculated onto

Co-lumbia agar base medium(Oxoid)supplemented with

7%horse blood and antibiotics (SR85; Oxoid). The

plates were incubated at 43°C for 48 h in an atmo-sphere of 5% oxygen, 10% carbon dioxide, and 85%

nitrogen. Colonies that were gram negative with

curvedorS-shaped morphology, positive intests for

oxidase and catalase production, and susceptible to

nalidixic acid (30



Sourcesofstrains to beserotyped. Strains fell into

threecategories: (i) strains fromanoutbreak of

gastro-enteritisatNakusp, BritishColumbia; (ii) strains from

sevenfamilies; and (iii) strains from sporadiccases.

Serotyping. Subcultures ofC.jejuni were sent in

Amiestransport medium (Difco Laboratories) to the

laboratory ofone ofus(J.L.P.) for serotyping. The

swab from the transport medium was first used to

inoculate5ml ofthioglycolatebroth(BBL

Microbiolo-gy Systems) and then ablood agar plate (Columbia

agar with 5% whole horse blood). The inoculated

media were incubated at 37°C in a CO2 incubator

(Forma Scientific, Marietta, Ohio)set tomaintain an

atmosphere of5%CO2. This atmosphere, unsuitable

forprimaryisolation, readily supportedthegrowthof

strainssubmitted forserotyping. After 48 h, each plate

was checked for purity and then transferred to and


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TABLE 1. Communityoutbreak at Nakusp, British Columbia, Canada




specimen Serotype Titer"


1 27 9/7/80 1 1:5,120

2 20 11/7/80 1 1:5,120

3 21 14/7/80 1 1:5,120

4 56 23/7/80 1 1:2,560

5 39 30/7/80 1 1:2,560

6 59 30/7/80 1 1:640

7 25 17/7/80 5 1:5,120

25 29/7/80 5 1:2,560

aTiters areforantisera against the indicated

sero-type. Reactions in antiseraagainst all other serotypes were '40.

spreadoverthe surface of four bloodagarplates. After

incubation for48 h at 37°C, 1.5 ml of saline (0.85%

NaCl) was added to each plate, and bacteria were

scraped from the four plates and pooled in

screw-capped tubes.Thetubeswereheated for1hinboiling

water. After coolingand centrifugation (8,000 x g for 10min), the supernatant fluid wasremoved and used

tosensitize sheep erythrocytes forpassive


Ifpurecultureswere not seen onfirsttransfer, the

swabfrom thetransportmedium(maintainedat4°Cin

theinterval)wasused toinoculateaplate of Columbia

agar base (Oxoid) containing 7% horse blood and

antibiotics according to the formula of Skirrow (23)

and incubated for48hat37°CintheCO2 incubator. If

Gram stains revealed the typical morphology of C.

jejuni, colonieswereselected toinoculateblood agar

platesasabove. Growth fromthioglycolate brothwas

usedtoinoculate Skirrow mediumwhen subculturing


Passive hemagglutination was performed as

de-scribed previously (21). Eachantigenpreparation was

titrated against 55 antisera prepared against stock


microti-trationplates. Antiserawereserially twofold diluted in

11 wells. Another well without antiserum contained

only diluent. Suspensions of antigen-coated sheep

erythrocytes were addedtoand mixed with the

con-tentsofeach well. After1hof incubationat37°Cand

overnightstorageat4°C,thehighest dilution inwhich

agglutination wasobservedwastaken as the titer. No

agglutination ofsensitizederythrocyteswasobserved

in the well that servedas acontrol. Each isolate was

then assigned a serotype according to the antiserum

(orantisera)inwhichagglutinationoccurred. Antisera

were notabsorbed. In instancesofmultipledisplaysof

antigens,thetiters obtainedwerecomparedwith those

of knowncross-reactionsof standard strainsto identi-fytheserotype(s) present. Thisprocedurewas under-takentodetectas many combinationsofantigenson unknownstrainsaspossible.


Retrospective serotyping of isolates from

pa-tientsofacommunityoutbreak ofgastroenteritis.

Ninestrainswereobtained fromeight



Nakusp, British Columbia, whereanoutbreak of gastroenteritis was reported (9). Six isolates reacted only in antiserum againstserotype1 and titers ranged from 1:640to1:5,120(Table 1). No significant reactionswere seenwith theother 54 antisera. Three isolates, twofromoneindividual andathird fromanother, reacted only in

antise-rum against serotype 5. The third isolate was

obtained 1 month after the outbreak and was

therefore considered not tohave been associat-ed withthe outbreak; thus, it is not included in Table 1.

Those strains belonging to serotype 1 were

positive in tests for hippurate hydrolysis and were susceptible to 10 p.g of ampicillin per ml. The two serotype 5 isolates were hippurate negative and were not inhibited by

concentra-tions of the antibiotic less than 40 p.g/ml. Having determined that this typing method discriminated between isolates of the species and identified thecommonstraininvolved in this outbreak,wearrangedaprogramforcontinuous serotyping between the laboratories at

Vancou-verand Toronto.

Serotypes of isolates from family outbreaks. Isolatesfrom seven suspected family outbreaks

are shown in Table 2. It can be seen that in all but oneoutbreak, eachfamily member or animal contact had an isolate of the same serotype as others involved in the particular outbreak. The serotype of the outbreak strains were different fromone family to the next. They included some verycommon serotypes (1, 2, and 4) in families A, B, and C as well as some less frequently

TABLE 2. Serotypes of isolates infamily outbreaks

Family Patient(age



Family and animal contact ofisolate

A Adultmale(65) 1

Adultfemale(61) 1

Chicken 1

B Malechild 2

Mother 2

Father 2

C Mother(32) 4

Femalechild (6) 4

D Femalechild(6) 5

Femalechild (2) 5

E Male child(9) 11

Female child (6) 11

F Mother(29) 31

Male child(10) 31

G Child(2) 20

Child(0.25) 45

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TABLE 3. Serotype distribution of C. jejuni isolates

No.of isolates No.of isolates

Serotype Separate Epidemiologically Serotype Separate Epidemiologically

isolationsa linkedb isolations' linkedb

1 19(11.3) 5 + 2c 19 2(1.2)

1/4 1 (0.6) 20 1 (0.6)

2 20 (11.9) 2 21 2(1.2)

2/4 1 (0.6) 22 1 (0.6)

2/13 1 (0.6) 23 1 (0.6)

3 6 (3.6) 23/26 2(1.2)

4 21 (12.5) 1 24 1 (0.6)

4/13/16 1 (0.6) 28 1 (0.6)

4/13/34 1 (0.6) 31 7(4.2)

4/16 1 (0.6) 33 1 (0.6)

4/19 1 (0.6) 34 2(1.2)

5 9 (5.4) 1 37 1 (0.6)

5/30 1 (0.6) 41 5(3.0)

5/31 2 (1.2) 42 3(1.8)

6/7 2 (1.2) 43d 4(2.4)

8 8 (4.8) 45 6(3.6)

10 2 (1.2) 46d 1 (0.6)

11 3 (1.8) 1 47 3(1.8)

13/16 8 (4.8) 48 1 (0.6)

13/19 1 (0.6) 50 3(1.8)

14 2 (1.2) 51 1 (0.6)

16 1 (0.6) 53d 1 (0.6)

18 6 (3.6)

aSeparate isolations includessporadic isolates plusoneisolate from each outbreak.Percentages areshown

within parentheses.

bIncludes all butoneisolatefrom each outbreak.

cFiveisolateswerefromcommunity outbreak (Table 1), andtwowerefromfamilyBoutbreak(Table 2).

dNew serotypesidentified inthis study.

occurringtypes (11 and31)infamilies EandF. Isolatesfrom siblings in family Gwerefoundto be different in serotype (20 and 45). Repeated resubmissions for serotyping of these two iso-lates (under coded designations) produced the sameresults. Itshouldbe notedthat theisolate of serotype 20 was

hippurate negative

and the other of serotype 45 was hippurate positive. Thus, differentiation byserotypingwas consist-entwithdifferentiation by


The lack of correlation betweenserobiotypingandthe clini-cally expected results for family G remains unexplained.

Prevalence ofserotypes among British Colum-bia isolates. To show prevalence of each sero-type moreaccurately, onlyoneisolate fromeach outbreak was considered ascontributing to the prevalence ofthat particular serotype. Hence, only168ofthe 181serotypedisolates were taken intoaccount in determining the serotype distri-bution (Table 3). Isolates sent for repeat sero-typing were not included. The most common isolateswereserotype 4followed byserotypes 1 and 2. This contrasts with results from a previ-ousstudy inwhich it was foundthat isolates of serotypes 2 and 1 were most common (21). More than one-halfoftheisolates(50.3%)fellintosix

serotypes (1, 2, 4, 5, 8, and


Similar concentrations of isolates withinasmall number ofserotypes were also noted



hospitals (21). Isolatesthatreacted in both anti-sera13and16constituted 5.0% of the totaland, asintheprevious study,werethemost frequent-ly occurring of the multiplyagglutinating types. Only 15 others


reacted in two or more antisera. The number that reacted only in one antiserum therefore formed the largest group


No untypable isolates were recorded in this study. Twelve isolates that were initially classifiedas untypable werefound tobelongto one oranotherof fournewtypes(serotypes43, 45, 46, and 53) identified in thisstudy.


Wereport thesuitability ofaserotyping meth-od for discriminating between strains ofC. je-juni. The passive hemagglutination technique used waspreviously reportedtoseparate strains ofC.jejuni onthe basis ofthermostableantigens (15,21). With thismethod, onlythe heat-extract-ed,solubleantigens having affinityfor the eryth-rocyte surface are measured, thereby circum-ventingtheproblem ofautoagglutinability found

VOL. 16,1982

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with Campylobacter cell suspensions (1, 17).

The large size of the erythrocyte, acting as antigen carrier, increases the sensitivity of de-tecting antigen-antibody reactions compared with direct bacterial agglutination.

Theserotyping results ofstrains from a com-munity outbreak correlated well with clinical data and withbiochemical and antibiotic sensi-tivitytest results. This collaborative study was therefore undertaken to further assess the reli-ability of the serotyping scheme and to extend knowledge of the epidemiology of the species with serotyping results.

In the course of this study, 181 isolates were typed andassigned to 1 or more of 45 serotypes. Insix separateinstances,familyoutbreaks were foundtobe causedbyaserotype common to all members of the particular family or to their animal contact. In another family, serotyping separated isolates of different biotypes. Results ofthese smaller outbreaks confirmedthe effec-tiveness of serotyping for epidemiological pur-poses.

The serotyping scheme is now capable of identifying55 differentantigenic specificities by means of the same number of antisera. Some isolates were found to react in two or more antiseraagainst serotypes notpreviously known tobe related. Thus, some strains possess com-plexes of antigens. Isolates with such multiple specificitieswill be useful in defining new

sero-types in ascheme that isexpectedtobe expand-ed to include cross-absorbed antisera as in the well-knownKauffman-White schemefor Salmo-nella spp.

Itis clearfromthis studyandfromothers that there are considerable geographical differences amongthe serotypes in their


of isola-tion(15, 21). It canbeexpectedthatthenumber of serotypes will continuetoincreaseasisolates from more widely separated sources are

exam-ined. The detection of four new serotypes (43, 45, 46, and53) amongonly 181 isolates ofthis study illustratesthepoint.Humaninfectionsare

notrestricted to particular serotypes. Sporadic casesandoutbreaksmay be causedby frequent-ly isolated types such as those ofserotypes 1, 2, and4orbyinfrequentlyoccurringtypes suchas

11 and31.

Morework is necessarytoclarifythe epidemi-ological significance of Campylobacter infec-tions in human and animal populations. This study has shown the applicabilityof thepassive hemagglutinationtechniquetothis task. Further evaluationsof the schemearetherefore

warrant-ed and should be conducted independently by other laboratories who will undertake the tasks of reconstituting the scheme and testing it in practice. Presently,theDivisionof Laboratories of theMinistry of Health in British Columbia is

one of the laboratories that is participating in thistesting program.


WethankG.Geddes,T. Matheson,and K.Tanner ofthe BritishColumbia Ministryof Health Laboratories and M. M.

Goodbodyatthe Universityof Toronto forskilledtechnical


This research was supported by the British Columbia

Health Care Research Foundation and Health and Welfare Canada.


1. Abbott, J. D., B. Dale, J. Eldridge, D. M. Jones, and E. M. Sutcliffe. 1980.Serotyping of Campylobacterjejunil

coli.J.Clin. Pathol. 33:762-766.

2. Blaser, M., J. Cravens,B. W. Powers,andW.L. Lang.

1978.Campylobacter enteritis associated with canine in-fection. Lancet ii:979-981.

3. Blaser, M., F. M. LaForce, N. A. Wilson, and W. L. L. Wang.1980.Reservoirsforhuman campylobacteriosis.J. Infect. Dis. 141:665-669.

4. Bokkenheuser, V. D., N. J. Richardson, J. H. Bryner, D.J. Roux, A.B. Schutte, H.J.Koornhof, I. Frieman, and E. Hartman. 1979. Detectionof enteric campylobac-teriosisinchildren. J.Clin.Microbiol. 9:227-232.

5. Bruce, D., W. Zochowski, and I. R. Ferguson. 1977.

Campylobacterenteritis. Br. Med. J. 2:1213.

6. Butzler,J. P., and M. B. Skirrow. 1979.Campylobacter

enteritis. Clin.Gastroenterol. 8:737-765.

7. Cary, S. G., and E. B. Blair. 1964. Newtransportmedium

forshipmentofclinical specimens.I.Faecalspecimens.J. Bacteriol. 88:96-98.

8. Dekeyser, P., M. Gossuin-Detrain, J. P. Butzler, and J. Sternon.1972. Acuteenteritis due to related vibrio: first positive stool cultures. J. Infect.Dis. 125:390-392. 9. DiseaseSurveillance,BritishColumbia Ministryof Health.


10. Grant,I. H., N. R. Richardson, and V. D. Bokkenheuser. 1980. Broiler chickens as potential sourceof Campylo-bacter infections in humans. J. Clin.Microbiol. 11:508-510.

11. Haley,C. E., R. A.Gunn,J. M. Hughes, E. C. Lippy, and G. F. Craun. 1980. Outbreaks of waterborne disease in theUnitedStates,1978. J. Infect. Dis. 141:794-797.

12. Kaplan,R. L.1980.Campylobacter,p. 235-241. In E. H.

Lennette,A.Balows,W.J.Hausler, Jr.,andJ. P. Truant

(ed.), Manualof clinicalmicrobiology, 3rded. American

SocietyforMicrobiology, Washington,D.C.

13. Karmali, M. A., and P. C. Fleming. 1979.Campylobacter

enteritis in children. J. Pediatr. 94:527-533.

14. Kosunen,T.U., D. Danielsson, and J. Kjellander. 1980.

Serology ofCampylobacterfetus ss.jejuni ("related" campylobacters). ActaPathol. Microbiol. Scand. Sect. B 88:207-218.

15. Lauwers,S., L.Vlaes,andJ. P.Butzler. 1981.

Campylo-bacterserotypingandepidemiology.Lancet i:158-159. 16. Leapers,S., and R.J.Owen. 1981.Identification of

cata-lase-producing Campylobacterspeciesbased on biochem-ical characteristics and on cellular fatty acid

composi-tions. Curr. Microbiol. 6:31-35.

17. Lior, H., D. L. Woodward, J. A. Edgar, and L.J. La-Roche. 1981.Serotyping byslideagglutinationof Campy-lobacter jejuniandepidemiology.Lancet ii:1103-1104. 18. Mentzing,L.-O. 1981.Waterborne outbreaks of

campylo-bacterenteritis in centralSweden.Lancet ii:352-354. 19. Park, C.E., Z. K. Stankiewicz, J. Lovett, andJ. Hunt.

1981. Incidence ofCampylobacterjejuniin fresh

eviscer-atedwhole market chickens. Can. J. Microbiol. 27:841-842.

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20. Pearson, A. D., W. G. Suckling, I. D. Ricciardi,M.Knill,

andE. Ware. 1977.Campylobacter-associated diarrhoea in Southampton. Br. Med. J. 2:955-956.

21. Penner, J.L., and J. N. Hennessy.1980. Passive

hemag-glutination technique for serotyping Campylobacter fetus subsp.jejunionthebasis of soluble heat-stable antigens. J. Clin.Microbiol. 12:732-737.

22. Robinson,D. A., W. J. Edgar, G. L. Gibson, A. A.

Mat-chett, and L. Robertson. 1979. Campylobacter enteritis

associated withconsumption of unpasteurized milk. Br. Med. J. 1:1171-1173.

23. Sklrrow, M. B. 1977. Campylobacter enteritis:a"new"

disease. Br. Med. J.2:9-11.

24. Svedhem, A., and G. Norkrans. 1980. Campylobacter jejuni enteritistransmitted fromcat toman.Lancet


25. Yanagisawa, S. 1980. Large outbreak of campylobacter enteritisamongschool children. Lancet 11:153.

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