Serogroups of Campylobacter jejuni, Campylobacter coli, and Campylobacter fetus defined by direct immunofluorescence

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Vol.17, No. 3 JOURNALOFCLINICAL MICROBIOLOGY,Mar. 1983,P. 529-538

0095-1137/83/030529-10$02.00/0

Copyright01983, American Society for Microbiology

Serogroups of

Campylobacter jejuni, Campylobacter coli, and

Campylobacter

fetus Defined by Direct Immunofluorescence

G. ANN

HtBERT,l*

D.G. HOLLIS,2R. E.WEAVER,2A. G.STEIGERWALT,2 R. M. McKINNEY,2 ANDD. J. BRENNER2

Hospital Infections Program1 and Division of Bacterial Diseases,2 Center for Infectious Diseases, Centers for DiseaseControl, Atlanta,

Georgia

30333

Received1 September 1982/Accepted 24 November 1982

Rabbits were inoculated with whole, formalinized Campylobacter jejuni, C.

coli, andC.fetus cells;the C.jejuni and C. coli immunogens were identified by

theirDNA relatedness at the species level to the type strains of C. jejuni or C.

coli. The designation C. coliwasnot used among the other C. jejuni strains; they were classified as hippurate-positive or hippurate-negative C. jejuni.

Immuno-globulin Gwasisolated fromtheantisera and labeled with fluorescein isothiocyan-ate.These conjugates defined10serogroups of C. jejuni, 2 serogroups of C. coli,

and2 serogroupsofC.fetus. Ofthe 316 strains of C. jejuni tested, 258 (82%) were

groupable; 173 were single-serogroup strains, and 85 were mutliple-serogroup

strains. Of the 226 strains in C. jejuni serogroups, 223 (98.7%) were hippurate

positive;

of the 27 strains in C. coli serogroups, 26 (96.3%) were hippurate

negative. Five strainswereequally reactive in immunofluorescent staining with a

conjugate

foraC. jejuniserogroupandaconjugate fora C.coliserogroup. A total

of 58 strains of C. jejuniwereungroupable: 33(13%) ofthe 259hippurate-positive

strains and25

(44%)

of the 57hippurate-negative strains.All 121strains of C. fetus

tested were groupable as A, B, or A:B. The 14 conjugates used to define

serogroupsof C.jejuni, C. coli,and C.fetus reacted with the flagella but not the

cells of other Campylobacter species and were negative on 256 other bacteria

from21 genera.

The

organisms

now classified in the genus

Campylobacter have been aproblem for veteri-narians formany years. A"vibrio" was

report-edasthecauseof abortion in sheep in 1909 and

incattle in1913(18).A"spirillum"wasisolated from aborted calves in 1918, and laterthe

orga-nism was named Vibrio fetus by Smith and Taylor (33). The first human infection was

re-ported in France in 1947 (36). Then, in 1957, King reported 11 human blood isolates in the United States; 7 strains were V.

fetus,

and 4

strainswere a

closely

which she

designated

"related vibrios"

(14).

Oneof those

V.

fetus strains (1134)

and the two

surviving

strains of related vibrio (2718 and 2737) are

included in the present immunofluorescence study.

King reported

that the related vibrios

grewat42butnot at

25°C

andwere

antigenically

different in

serological

studies;

she also

identi-fied them as

possible

causes of

gastroenteritis.

In 1963, Sebald and Veron (29)

proposed

the

genus Campylobacter, and in 1973, Vdron and

Chatelain

(35)

reclassified V.

fetus

as

Campylo-bacterfetusand the related vibrios as C.jejuni

and C. coli. C. jejuni is now

recognized

as a

leading cause of

gastroenteritis

throughout the

world(4, 13, 27).

The various

Campylobacter

species

are

bio-chemically

relatively inactive,

andthefew

phe-notypic differencesreported forC.jejuni and C.

coliwereobtained withteststhataredifficultto

duplicate

and that do not

give

reproducible

results(31). Wecannotseparate C. coli from C.

jejuni

biochemically (8).

The biochemical

char-acteristics ofthe type strains of both

species

fit thisdefinition:

gram-negative

vibrio

forms;

posi-tive catalase,

oxidase,

nitrate

reactions,

and

motility;

no fermentation or oxidation of

glu-cose;no

H2S

produced

in

triple-sugar

ironagar;

growthinbrucellabroth

containing

1%

glycine

and 0.16% agar;

growth

at 42 but not at

25°C;

sensitive to nalidixic acid

(30-p.g

disk);

and

re-sistant to

cephalothin (30-,ug

disk).

Recently,

Owen andLeaper

(25)

studiedthe DNA charac-teristics oftype and reference strains of various

species of

Campylobacter

and showed that the C. jejuni, C.

coli,

and C.

fetus

type strains

belong to separate

species.

Their data support

theclassificationsystemofVeronandChatelain (35). They

suggested

the use of

hippurate

hy-529

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530 HEBERT ET AL.

drolysistodifferentiate betweenC. jejuniandC.

coli; however, only three strains of each species

werestudied. Untilasufficient number of strains

have beenexaminedto document thereliability

ofa defined hippurate hydrolysis test for

sepa-rating species and until the several tests for

hippurate hydrolysis have been examined to

defineastandard method,wewill identify these

strainsasC. jejuniandreportthe hippuratetest

result. In this report, the designation C. coli is

used only for C. jejunistrains examined in DNA

homology studiesand showntobehighly related

to C. coli. The other strains are designated

hippurate-positive or hippurate-negative C.

je-juni.

Most of theantigenic studies of

Campylobac-terspecies have been done with animal strains.

In 1953, Marsh and Firehammer (19), at the

Montana Veterinary Research Laboratory in

Bozeman, Mont.,described five serotypesofV.

fetus using a tube agglutination test; these are

usually referred to as the Montana serotypes.

Studies published in 1958 (22) and 1959 (23)

divided V. fetus into two serotypes; one study

used a complement-fixation test, and the other

usedanagglutinationtest. In1971, Bergetal.(3)

reported five serotypes of V. fetus using both

tube and slide agglutination tests. The Berg

serotypeCwasthesameasMontanaserotypeI,

andthese strains are nowknowntobe C.jejuni

(3, 32).

Several techniques have recently been

de-scribed for serotyping clinical isolates of C.

jejuni: Abbottetal.(1)used atubeagglutination

test with heat-labile and heat-stable antigens;

Penner and Hennessy (26) and Lauwers et al.

(15) used a passive hemagglutination test with

heat-stable soluble antigens; and Lioret al. (16)

usedaslideagglutinationtestwithlive bacteria.

We now report thedevelopment of direct

fluo-rescent-antibody (FA) tests for identifying and

serogrouping C. jejuni, C. coli,and C.fetus.

MATERIALS ANDMETHODS

Cultures. All of the strains ofCampylobacter

spe-ciesdescribed inanearlierreport(8) andmany

addi-tionalorganismswereexamined during this study. The

total of 733 isolates included 316strains of C.jejuni, 110 strains of C.fetus subsp. fetus, 11 strains ofC. fetus subsp. venerealis, 2 strains of C. sputorum subsp. sputorum, 2 strains of C. sputorum subsp. bubulus, 3 strains of C.sputorumsubsp.mucosalis, 4 strains of C. fecalis, 29 Campylobacter-like

orga-nisms, and 256 otherorganisms representing 39

spe-ciesfrom21othergenera.Mostof theCampylobacter strains were in the culture collection of the Special Bacteriology Laboratory at the Centers for Disease Control, Atlanta, Ga. J. C. Penner (University of Toronto, Toronto, Ontario, Canada) supplied 71 strains of C. jejuni from Canada. M. B. Skirrow (Worcester Royal Infirmary, Worcester, England)

supplied10strains of the nalidixicacid-resistant ther-mophiliccampylobacters(NARTC)fromGreat Britain (31); theseNARTC strainswereamong the Campylo-bacter-like organisms. Most of the strains of other genera werefrom thecollection in the Enteric Bacteri-ology SectionattheCenters for DiseaseControl.

Biochemicalcharacteristics. All of the

Campylobac-ter strains were examined for sodium hippurate hy-drolysis by the rapidtest ofHwangand Ederer(11). Since several modifications of their procedure are currently being used to examine Campylobacter strains, and since the hippurate hydrolysis test may prove to be a valuable tool for classifying these organisms, we restatebelowtheprocedure used dur-ing this study.A1% solution of sodiumhippuratewas preparedinsterile distilled water,dispensed in 0.4-ml amounts into screw-captubes (13 by 100 mm), and frozenat-20°Cuntilused.A3.5% solutionninhydrin

wasprepared in a 1:1 mixture ofacetoneandbutanol and stored in the darkat roomtemperature. Aloopful of 18-to24-hgrowthfrom bloodagar(heartinfusion agar containing 5% defibrinated rabbit blood) was emulsified in athawed tube of1% sodiumhippurate, andthedensesuspensionwas incubatedat35°C in air for 2 h. After incubation, 0.2 ml of the ninhydrin solutionwasadded. Thescrewcapwastightened,the contents were either mixed well ornot mixed atall, and the tube was reincubated at 35°C. After10min, the tubewasexamined for colordevelopment;adeep purplecolor,usuallyasdarkascrystal violet, indicat-edtheformation ofglycinewhenhippuratewas hydro-lyzed by the organism. If the contents had not been mixed, the color was in a band above the layer of settled cells, but if thecontents had been mixed, the colorwasevenlydispersed.Acolorlesstolight-purple reaction was recorded as negative for hippurate hy-drolysis. Many of the Campylobacter strains were examined by gas-liquid chromatography for cellular fatty acids. The24-hgrowthonbloodagarplateswas removed and processed as described previously (8, 24).

DNA homology. Each ofthe strains used in DNA homology studies was grown on 20 to 25 bloodagar plates for 18 to 24 h at 35°C in approximately 5%

oxygen. Thecellgrowthwasharvested andsuspended in 30ml of buffercontaining0.1 MNaCl,0.05 MTris, 0.05 M EDTA, and 50

p.g

of pronase per ml. The extraction andpurificationofDNA and detailsof the in vitrolabeling and hydroxyapatite procedures used tomeasure DNArelatedness have beendescribed(5). Immunogen strains and history. The strains used as immunogens and their histories are shown in Table 1. Antigen preparation. All cultures were grown on plates of blood agar for 18 to 24 h at 35°C in approxi-mately 5% oxygen as previously described (8). The cell growth was harvested and suspended in 0.01 M sodium phosphate buffer (pH 7.6) containing 0.85%

NaCI and 1.0% Formalin (1% Formalin-phosphate-bufferedsaline[PBS]).Thecellsuspensionswereheld at4°C overnight; thecells were then centrifugedand resuspended in 0.5% Formalin-PBS to a final cell concentration of 40 international units of turbidity (17),orapproximately4 x 109bacterialcells per ml.

Immunization.Young adult New Zealand white rab-bits were prebled and then immunized with 10 ml of antigengiven as five separate doses of 2 ml each as follows:intradermallyonday 1,distributed in 15 to 20

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CAMPYLOBACTER DIRECT FA SEROGROUPS 531

TABLE 1. Immunogen strains

Strain Organism Source Country Date Hippurate

isolated hydrolysis

KC159=' C.jejuni PasteurInstitute CIP702 France +

KC1589a C. coli Pasteur Institute CIP7080 France

KC1495b C. coli M. B. Skirrow GreatBritain

KC1494c C.jejuni M. B. Skirrow Great Britain +

KC1480d C. jejuni K. Watson Scotland 1978 +

2718e Related vibrio Human blood UnitedStates 1956

3695f Related vibrio Chicken UnitedStates 1958 +

A46469 Related vibrio Human blood UnitedStates 1%6 +

A73159 Related vibrio Human blood UnitedStates 1967 +

B2015 C.jejuni Retroperitoneal abscess Australia 1969

B7607h Related vibrio Human feces Belgium 1970 +

B8788 C. jejuni Human blood UnitedStates 1971 +

E8134 C. jejuni Human feces UnitedStates 1980 +

E8765 C. jejuni Human feces UnitedStates 1980 +

F2565 C. jejuni Human feces UnitedStates 1981 +

B8833' C.fetus subsp. fetus Humanblood UnitedStates 1971 1134' C.fetus subsp.fetus Human blood UnitedStates 1952

aType strain(35).

bObtained asC. coli 4620 and also listed as NCTC 11353 (31).

cObtainedasC. jejuni5636 and also listed as NCTC 11168 (31).

dObtainedasstrainCOP (37, 38).

eOne of theoriginal four human isolates reported by King (14).

fDatafrom serology studies reported in1961 by Middlekamp and Wolf (21). gUsedinBelgium by Dekeyseretal. (6) to prepare antisera.

hIsolated by Dekeyseretal. (6) and reported as the first positive stool culture from a patient with acute enteritis.

'AlsolistedasATCC 33249.

iDesignated Vibriofetus(14);listed by Vdron and Chatelain (35) as CIP 5443 and designated C. fetus subsp. venerealis biotype intermedius. In our laboratory, however, this strain grows in1% glycine and gives a small amount of H2S on lead acetate paper over brucella broth with 0.02% cystine-hydrochloride. These two characteristics describe C.fetus subsp. fetus and separate it from the subspecies venerealis.

sitesontheback; intramuscularlyonday 48,1ml into each hind quarter;andintravenouslyondays 56, 63, and 70. The rabbitswerebledonday 77,andtheserum washarvested thefollowing day.

Conjugate preparation. ImmunoglobulnG(IgG)was isolated fromthe rabbit antisera andpreimmune sera

by affinitychromatographyonacolumn ofproteinA

(Staphylococcus aureus) covalentlybound to Sepha-roseCL-4B(PharmaciaFineChemicals,Div. of

Phar-macia, Inc.,Piscataway, N.J.)aspreviouslydescribed (7).TheIgGwaslabeled with fluorescein isothiocyan-ate (FITC) by the direct method to achieve final fluoresceintoproteinratios ofapproximately30tLgof FITCpermgofprotein (9, 10).UnreactedFITCwas

removedbydialysisin PBS(pH9.0 to9.5). The final

conjugateswereadjustedto contain10mgofIgGand 0.1%NaN3perml.

Conjugateevaluation. All 17 of the Campylobacter conjugates were titrated with theirhomologous

anti-gensandwith theheterologous antigensof the other 16

immunogens.Thetestantigenswerefresh cell suspen-sions in 1% Formalin-PBS. Smears of the antigens

were preparedonmultiwelled slides(Cel-Line Asso-ciates, Inc.,Minotola, N.J.),airdried,andgentlyheat fixed. Serialtwofold dilutions oftheconjugates were

madein0.01Mphosphatebuffer(pH 9.0).Thesmears werestainedwith theconjugatedilutionsbystandard direct FAprocedures (12). The stainedsmears were

examinedby incident illumination witha50-Whalogen lamp through a Leitz Dialux microscope equipped withaPloemilluminator,anFITC interferencefilter, and a100x oil objective. The conjugate titers were

expressedasreciprocalsof the dilutions from 10 mg of

IgGper ml thatretaineda3to4+ staining intensityof the cells. The working dilution of a conjugate was definedasonedoubling dilution less thanthatof the titer. The preimmune conjugates were tested at one dilutiononly (1:4or2.5 mgofIgGperml).Each of the final immune conjugates was tested at a1:8 dilution (1.25 mg ofIgG perml) against296 otherantigensthat included all of the C. sputorum, C.fecalis,and Cam-pylobacter-like strains and 256 other bacteria

repre-senting39speciesfrom 21 other genera. The

miscella-neous strains were primarily enteric organisms, including Vibrio,Salmonella,andShigella,but Staph-ylococcus andLegionellastrainswerealsoexamined. Polyvalent conjugates. The polyvalent conjugates were prepared by combining equal volumes of the

appropriateconcentrations of individualconjugatesso

thatthefinalconcentration of each component of the

poolwasusuallytwodoublingdilutionsless than that

of the titer of that component. The two C. fetus

conjugates were combined to make oneofthese

re-agents, butthe otherconjugateswereused invarious combinationsas theywere developed and the study

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532

HtBERT

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Examination of isolates. A loopful of 24-h growth from bloodagarwassuspendedin 1%Formalin-PBS to form a light cell suspension (McFarland no. 1 standard). Smears were prepared and stainedas de-scribed above. Initialtestingwasdone with all conju-gates atthe 1:8dilution,but latertestingwasdonewith the working dilutions. Strains were assigned to a

serogroup onthe basis ofstainingreactions with the working dilution ofaconjugate.

RESULTS

The data onhippurate hydrolysisformanyof

thestrains in thisstudywerereported earlier(8).

Inthatreport, asin this,wedidnotseparate C.

jejuni and C. coli on the basis ofbiochemical

characteristics, but stated that 156(81%)of192

C.jejuni strains testedwere hippurate positive,

and all strains of otherCampylobacter species

andsome similarorganismswerehippurate

neg-ative. We havenowtestedatotalof 316 strains

ofC.jejuni for hippurate hydrolysis and found

259 (82%) positive and 57 negative.

The dataoncellularfatty acid analysis for 89

of these C.jejuni strains were reported earlier

(8). We have now examined 111 strains of C.

jejuniby gas-liquid chromatographyforcellular

fatty acids. A C19 cyclopropane acid identified

as lactobacillic acid was found in all 60 of the

hippurate-positive strains tested, although only trace amounts (0.4 to0.9%) were measured in

oneof the strains; the relative concentration of

this acid in the other 59 hippurate-positive

strainsranged from 3 to 30%. Ofthe 51

hippu-rate-negative C.jejuni strains studied, 38 (74%)

contained greater than 2% concentrations of

lactobacillic acid; this acid was not detected in

13 of thehippurate-negative strains.

Labeled DNA from thetypestrainof C.jejuni

showed 55to 100% relatedness to the other 10

hippurate-positive strains tested and 29to46%

relatedness to the 4 hippurate-negative strains

tested when reactions were done ata stringent

65°C incubation temperature, at which only

highly similar nucleotide sequences can

hybrid-ize (Table 2). Labeled DNAfrom thetypestrain

of C. coli showed 30to52%relatednesstothe 11

hippurate-positive strains and 92 to100%

relat-ednessto theother 3hippurate-negative strains

under the samereactionconditions. These data

indicated that the hippurate-positive isolates

tested were strains of C.jejuni and the

hippu-rate-negative isolates tested were strains of C.

coli.

The homologous andheterologous direct FA

staining titers and theserogroupdesignationsof

the Campylobacter conjugates are shown in

Table 3. The homologous serogroup titers

ranged from 32 to 1,024. Only three of the

conjugates gave heterologous serogroup

stain-TABLE 2. DNArelatedness of thetypestrains of

C.jejuniand C. coli to variousreferencestrains of

Campylobacter

RBRat65°Cwith the

followingsourceof

SourceofunlabeledDNA labeled

DNA'

C.jejuni C. coli CIP 702 CIP7080

C.jejuni

KC1590(CIP 702) 100 35

KC1494(NCTC 11168) 75 45

KC1480(COP) 94 52

2737(related vibrio) 84 35

3695 84 37

A4646 55 30

A7315 82 42

B7607 83 51

B8788 81 44

E8134 84 51

E8765 88 41

C. coli

KC1589(CIP7080) 29 100

KC1495(NCTC 11353) 33 100

2718(related vibrio) 39 100

B2015 46 92

aRelativebindingratio(RBR) = (percent

heterolo-gous DNAboundtohydroxyapatite)/(percent

homolo-gousDNA boundtohydroxyapatite) x 100.

ing,andthose titers were less than theworking

dilutions of the conjugates: (i) the C. jejuni

serogroup 4 conjugate had a specific titer of 64

and gave 3 to 4+ staining of the C. jejuni

serogroup 3 immunogen at a 1:16 dilution, but

only 1 + staining of that strain at the 1:32

work-ing dilution;(ii)the C. jejuniserogroup8

conju-gatehad a specific titer of 1,024 and gave 3 to 4+

stainingofthe C. coli serogroupC-1 immunogen

at a1:16dilution,but wasnegative on that strain

at the working dilution; and (iii) the C. fetus

serogroupAconjugatehad a specific titer of 128

and gave 3 to 4+ staining of the C. coli

sero-groupC-1 immunogenat a 1:8dilutiononly.

Of the 17 hyperimmune conjugates prepared

during this study, 3 are not shown in Table 3.

The C.jejuni conjugate prepared with the type

strainKC1590(CIP702) had a titer of 64, but the

C. jejuni conjugate E8765 had a titer of 128 on

the cells ofE8765 and KC1590, so E8765 was

selectedto represent serogroup 7. The C.

coli

conjugates preparedwith the type strain KC1589

(CIP 7080) and with strain KC1495 had equal

titersof1,024on thecellsofboth strains,so the

type strain conjugate, KC1589, was selected to

represent serogroup C-1. The C. coli conjugate

preparedwiththe related vibrio,strain2718, had

atiterof 128 on the cells of 2718 and B2015, but

C.coliconjugate B2015had a titer of 512 on both

cellsand wasselectedtorepresent serogroup

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TABLE 3. Homologous and heterologous titers ofCampylobacter conjugates Conjugate serogroups and direct FA stainingtitersa

Antigen Serogroup C. jejuni C.coli C. fetus

1 2 3 4 5 6 7 8 9 10 C-1 C-2 A B

C.jejuni

B7607 1 128 _b _ _ _ _ _ _ _ _ _ _ _ _

KC1494 2 - 128 - - -

-3695 3 - - 64 16 - - -

-F2565 4 - - - 64 - - -

-E8134 5 - - - - 64 - - -

-A4646 6 - - - 32 - - -

-E8765 7 - - - 128 - - -

-B8788 8 - - - 1,024 - - -

-A7315 9 - - - 512 - - - -

-KC1480 10 - - - 64 - - -

-C.coli

KC1589 C-1 - - - 16 - - 1,024 - 8

-B2015 C-2 - - - 512 -

-C.fetus

B8833 A - - - 128

-1134 B - - - 64

C. sputorum

subsp.sputorum

(2)c.-subsp.bubulus (2) - - -

-subsp. mucosalis

(3).-C.fecalis (4) - - - _ _ _ _ _ _

Campylobacter-like

organisms(29)d

aReciprocal of

highest

dilution

retaininga3to4+ intensity.

bNegative.

CNumbersinparenthesesrefertothenumbers

of

strainstested.

dIncludesthe 10NARTC

strains

fromM. B.

Skirrow

(31).

2. The conjugates prepared from preimmune

sera of the rabbits used in this study did not

reactwith

Campylobacter

antigens.

Noneof the

Campylobacter

conjugates

react-ed with the cells of C. sputorum, C.

fecalis,

NARTC,

and other

Campylobacter-like

orga-nisms tested. Eachof theseCampylobacter

con-jugatesgave

good

staining

of the

flagella

ofmany

of theC.jejuni, C.

coli,

C.

fetus,

C. sputorum,

NARTC,

and other

Campylobacter-like

strains

tested. No

flagella

were seen on the four C. fecalis strains by immunofluorescent

staining,

but

flagella

maynot havebeenpresent in these

preparations.

The

specificity

of the

hyperim-muneconjugateswasfurtherconfirmedwhen no

significantstainingwasobservedamong the 256

other strainsof

heterologous species.

As this study progressed, more and more

conjugates

were

prepared

andadded tothetest

panel;

therefore,

in the latter partofthe

study,

polyvalent reagents were used to reduce the

amountof timeandmaterials

required

toscreen

newisolates. Positive

specimens

were then

ex-amined with the individual components of the reactivepolyvalent

conjugate

todetermine their

serogroup. The four current

polyvalent

conju-gateswere

prepared

asshown in Table4. Each

of these

conjugates

gave 3to4+

staining

ofthe

cells of eachof its components.

Thedirect FA

staining

reactions of 437

Cam-pylobacter strains from

multiple

human and

non-humansources areshown in Table5.Of the 316

C.jejuni strains

tested,

258

(82%)

were

group-able; 227 reacted with C.jejuni

conjugates,

and 31 reacted with C. coli

conjugates.

Positive

staining reactions with the

conjugates

of C.

jejuniserogroups

1,

2, 3, 4,

and 5 and C.

coli

serogroupC-1werethemost

frequently

encoun-tered andaccounted for227

(72%)

ofthestrains. Amongthe 258

groupable

strains,

therewere173

single-serogroup strainsthat

belonged

to1 of12

serogroups and 85

multiple-serogroup

strains

that gave

equal

reactions with from 2 to 4

different

conjugates.

The C.

jejuni

strains from

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TABLE 4. Preparation ofpolyvalentCampylobacterreagents

Polyvalent Conjugate Specific Working Dilutionsto

conjugate serogroups titers dilutions be pooled'

PolyI 1 1:128 1:32 1:8

2 1:128 1:32 1:8

3 1:64 1:16 1:4

4 1:64 1:16 1:4

Poly II 5 1:64 1:16 1:4

6 1:32 1:16 1:4

7 1:128 1:32 1:8

8 1:1,024 1:256 1:64

PolyIII 9 1:512 1:128 1:32

10 1:64 1:16 1:4

C-i 1:1,024 1:256 1:64

C-2 1:512 1:128 1:32

PolyIV A 1:128 1:32 1:16

B 1:64 1:16 1:8

aCombinedequal volumes ofeach componentatthelisted dilutiontoachievetheworkingdilution chosen for the polyvalent.

Canada belongedto most ofthe same C.jejuni

and C. coli serogroups found in our culture

collection and were predominately

single-sero-groupstrains. Therewasnocorrelation between

thisdirect FA data and thebiotypedataobtained

earlier (8) with many of these same strains,

except for the hippurate test; 224 (86%) of the

259

hippurate-positive

strainswere placedin C.

jejuni serogroups,and 29

(51%)

ofthe 57

hippur-ate-negative strainswereplaced in C. coli

sero-groups. All ofthe C.fetus strains tested were

groupable. Asshown inTable6, 77(70%)of the

110 C.fetus subsp.fetus strainsand 10 (91%)of

the 11 C.fetus subsp. venerealis strains tested

belongedtoserogroup A;this includedthe type

strains of both subspecies, CIP 5396 and CIP

6829. The remaining strain of C. fetus subsp.

venerealis and 21 (19%) of the C. fetus subsp.

fetus strains belonged to serogroupB. The other 12 (11%) strains of C.fetus subsp. fetus were

equally reactive with both ofthe C.fetus

conju-gates, and theytherefore constituted serogroup

A:B.

Only 9 of the 258groupable C. jejuni strains

had divergent serogroup and hippurate

reac-tions; 3 of the 227 strains in the C. jejuni

serogroups were hippurate negative, 1 of the 31

strains in the C. coli serogroups was hippurate

positive,and5strainswereequally reactive with aconjugateof each species (Table 7). These nine strains have been tested many times with the

same results.

Only 58 (18%) of the 316 C. jejuni strains

tested were ungroupable; 33 of these strains

werehippurate positive, and 25 were hippurate

negative (Table5). Veryfewof the58

ungroupa-ble strains were totally nonreactive, however

(Table 8). Mixed staining reactions were seen

with 18 of the hippurate-positive and 11 of the

hippurate-negative strains; the cells of these

strains gave a mixture of 1+, 2+, 3+, and 4+

staining intensities with the working dilution of one or more

conjugates.

Repeated testing with

fresh growth did not alter the results, so these

strains could notbe assignedtoserogroups. No

cellularstainingwas seen with theremaining 29

strains, but many of these negative strains had

flagella which reacted with one or more

conju-gates.

Of the 13 hippurate-negative strains that did

notcontain lactobacillicacid,11 wereamongthe

ungroupable strains; 4 had mixed staining

reac-tions and 7 were negative. The remaining two

strains without lactobacillic acid, related vibrio

2718 andKC1593,belongedtoC. coli serogroup

C-2.

DISCUSSION

One oftheadvantages of the direct FA test is

the opportunity to observe the morphology of

the reactiveantigen, and among these

Campylo-bacter strains there was much variety. Most of

the cells seen by immunofluorescence were

curved and S-shaped rods with tapered ends,

many with a single, polar flagellum at one or

bothends of the cell. A few strains had mostly

thin, straight rods with no apparent curvature.

Many strains seen with the C.jejuniconjugates

also hadacoccoidform,sometimes with a single

flagellum like that seen by electron microscopy

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TABLE 5. Direct FA staining reactions of 437 Campylobacter strains

No. ofstrains from various sources Direct FA Total Special BacteriologyCulture Collection

serogroup strains Human isolates Nonhuman isolates cultures

Feces Blood Other Cow Fowl Other (Canada)

49 23 13

26 17 4

2 1

1 1

8 3 3

1 1 6 4 2 1 1 1 35 17 1 2 2 18 8 13 7 9 6 4 4 2 12 1 10 1 1 7 6 4 3 2 33

1(spinal fluid) 1 1 1 (water) 1 1 1 5 1 3a ₂ 6 2 1 1 1 1 5 2 1 3 1 1 1 (dog) 1 (dog) 1 1 1 1 2 3 1 3 1 1 1 1 10 5

18 4 6

4 2

9 1 3

25 4 9

87 12 22

1 1

2 1 1 (dog)

1(peritoneal abscess) 1(gall bladder)

4 64 gd

11 1 (joint fluid) 20 2(pleural fluid)

6(pigs) 1 (monkey) 6 2(1 dog, 1 water) 4

aSpinal fluid, uterus,gall bladder.

bUG, Ungroupable; HP, hippurate positive. 'HN,hippuratenegative.

dBone, placenta,threespinalfluids, four joint fluids. IBaboon,twoguinea pigs, three sheep.

(28)and lessfrequentlywith twoflagella arising

from opposite sides of the cell. These coccal

forms fluoresced much brighterthan the other,

moretypical,curved rodsin thesamefield,asif

the antigens were more concentrated per unit

area. The coccal forms were dominant in

anti-gens prepared from overnight growth at 42°C,

but not uncommon from growth at 35°C. The

antigensofsome strainswereverypleomorphic

and included curvedrods,cocci,andlong,wavy filaments in the same preparation.

Although we did not attempt a systematic

study offlagella, we recorded some interesting

observations. For example, with the same C.

jejuni antigen,wecouldseemanyhighly

fluores-cent flagella, but no cells, with the C. jejuni

serogroup 3 conjugate; very faint 1+ cells with

4+ flagella usingtheserogroup1 conjugate;and

10 4 1 1 1 C.jejuni 1 1:2 1:2:5 1:3:4 1:4 1:4:5 1:5 1:9 1;C-1 2 2:4 2:5 3 3:4 3:4:5 3:4:5:6 3:6 4 4:6 S 5:6 5:7 6 7 8 9 10 UG,HPb C.coli C-i C-1;8 C-2 UG,HNc C.fetus A A:B B 8 2 3 2 1 4 4 3 1 1 1 14 2 2 3 3 VOL. 17, 1983

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TABLE 6. Direct FAstaining reactions of 121 strains of C.fetus

C. fetus No. of No. ofstrainsin each C.fetus serogroup

strains

subsp. tested A B A:B Nonreactive

fetus 110 77" 21" 12 0

venerealis 11 10' 1 0 0

aIncludes thetypestrain CIP5396and ATCC strains 33246,33247, and 33249. bIncludes CIP5443 andATCC 33248.

CIncludes thetypestrain CIP 6829.

4+ cells without flagella using the serogroup 8

conjugate. Most of our conjugates gave

heter-ologous staining oftheflagella, butnotthe cells,

of other Campylobacter species. These same

reagentsdidnotreact with the flagella of strains of Salmonella, Vibrio, and many other enteric

bacteria. Thepresence orabsence of fluorescent

flagella did not interfere with interpretations of

cellular fluorescence; it was evident, however,

that the flagellar antigens of Campylobacter

species could be studied by direct

immunofluo-rescence.

Many of the Formalin-PBS antigens in our

collection are now 2 years old, and very few

exhibit any deterioration. All of the antigens

have been stored at4°C in screw-capped tubes.

Autoagglutination was very seldom seen; when

it occurred itwas weak, leaving plenty ofloose

cellstoexamine.

Ofhistorical interest isthefact thatoneof the first reported fecal isolates of C. jejuni, strain

B7607from Belgium, isnowthe reference strain

for C.jejunidirect FA serogroup 1; one of the

original related vibrios, strain 2737, is now a C.

jejuni by DNA homology and is also in

sero-group1. Theotherrelated vibrio, strain2718,is now aC.coli by DNA homologyandisin C.coli

direct FA serogroupC-2.

Our DNA homology data add support to the

use ofthe hippurate hydrolysis test to separate

C.coli from C. jejuni. Amongthe 15 strains ofC.

jejuni tested,thehippurate-positivestrainswere

highlyrelatedtothe typestrain of C.jejuni, and

the hippurate-negative strains were practically

identicaltothetype strain of C. coli. Thedirect

FA data also correlate with the hippurate data;

amongthe 316 strains of C.jejuni tested, only 1

ofthe hippurate-positive strainsof C.jejuniwas

ina C. coliserogroup, and only 3 of the

hippu-rate-negative strains of C. jejuni were in C.

jejuni serogroups. These strains and the five

strains thatwereequallyreactive with bothaC.

jejuniand a C. coli conjugate will be examined

by DNA homology to determine whether the

serological orbiochemical dataare aberrant.

The direct FAtestforC.fetus has been used

by veterinarians for many years. In 1965,

Mel-lick et al. (20) reported the use of a V. fetus

conjugatetoexaminesmearsofbovine preputial

fluid,andno crossreactionswereseen among17

other species of bacteria. Also in 1965, Belden andRobertstad (2) reported theuseof direct FA reagents to Montana serotypes I, II, and V to

place V. fetus isolates of human, bovine, and

ovineoriginin four differentserogroups defined asI, II,V,and II:V.SinceMontanaserotypeIis

C.jejuni, their serotypes II, V, and II:V for V.

fetusareprobably identical toour serogroupsA,

B, and A:Bfor C. fetus. Taul and Kleckner(34)

used direct FA reagents to V. fetus and V.

TABLE 7. Nine strains withdivergentserogroup andhippuratereactions

Hippurate Immunofluorescence

StrainSourcehydrolysis Serogroup Species

E9454 Feces 2 C.jejuni

F589 Feces 4 C.jejuni

F1753 Blood 4 C.jejuni

E8343 Feces + C-i C.coli

E6162 Feces + 1; C-1 C.jejuni; C. coli

KC1481a Feces + C-1;8 C. coli;C.jejuni

F1789 Feces C-1;8 C. coli;C. jejuni

KC1570b Pig C-1;8 C. coli;C.jejuni

KC1572C Pig C-1;8 C. coli;C.jejuni

a StrainMELfrom K. Watson in Scotland.

bStrain PC 66 from J. L. Penner in Canada.

' Strain PC67fromJ. L. Penner inCanada.

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TABLE 8. Data on the 58ungroupable strains of C. jejuni

Total No. of strainsfrom varioussources

no.of Hippurate Direct FA Human Nonhuman

strains hydrolysis reaction'~

Feces Blood Cow Chicken Dog Other

18 + Mixed 13 2 1 1 1 (goat)

15 + Negative 9 3 1 1 1 (gazelle)

11 - Mixed 6 2 1 1 1 (turkey)

14 - Negative 8b 1 5 (1 turkey, 1 water,

3 teals)

aMixed, afew cells were fluorescent at each of several intensities with one or more conjugates at their working dilution; negative, no cellular staining but frequent flagellar reactions.

bIncludesonestrainfrom a gall bladder.

bubulusto examinesmears of bovine semen and

preputial scrapings and saw no cross staining

between the two species. Shires and Kramer

(30) used the direct FA test to detect C. fetus

subsp. venerealis in bovine cervicovaginal

mu-cus smears. Allofthese studiesreport no

differ-entiation between the two subspecies of C.

fetus; however, the subspecies fetus can cause

abortionincattle,and the subspeciesvenerealis is amajorcauseof bovine infertilitythroughout the world. Thus, the detection of either subspe-cies ofC. fetus can be significant in infertility

studies ofdairy and beefherds, because

infec-tion with C.fetus is amultimillion dollar prob-lemforthecattle industry. The subspeciesfetus

is also, of course, a human pathogen, but the

subspecies venerealis has not been reported to

be(4, 32). Itwouldseem, therefore, thatexcept

for

epidemiological

purposes,neitherphysicians

nor veterinarians need a diagnostic reagent

ca-pable ofseparatingthe subspecies ofC.fetus.

All of the C. fetus strains examined were

groupable, but 18% ofthe C.jejuni strainscould

not be serogrouped with our present panel of direct FA reagents. More conjugates are

need-ed, and the new immunogens must be selected

from the set ofungroupable strains. Since 13%

ofthe

hippurate-positive

butnearlyhalf

(44%)

of

the

hippurate-negative

strains of C.jejuniwere

ungroupable, the greatest need isforadditional

reagents prepared with antisera

against

hippu-rate-negative

strains. At least two more

sero-groups must be

defined,

therefore,

before our

panel ofFA reagents is sufficient to cover the

diversityofclinically

significant

Campylobacter

spp.

Slideand tube

agglutination,

complement

fix-ation,passive

hemagglutination,

and indirectFA

testsarevaluable

epidemiological

toolsonce an

organismhas beenisolated andidentified.

They

can also be useful in

serological

studies for

retrospective

diagnosis

ofdisease

by using

refer-encestrainsasstandard

antigens.

ThedirectFA

testcan alsogiveserogroup

identity

to

isolates,

and theantigensthatdefine its serogroups could

be used as the reference antigens with an

indi-rect FA test to conduct serological surveys.

However, the direct FA test described in this

study has still another parameter: it should

per-mit the direct detection and identification of

Campylobacter species in clinical and

environ-mental specimens. Ourreagents have been

test-ed for specificity with pure cultures of many

species of bacteria, but theymustnow be tested

ontissuesections, fecal samples,waterand milk

filters,etc.,before the fullpotentialof direct FA

tests for C.jejuni and C. coli can be realized.

Once this is done, clinical diagnostic

labora-tories could begin using the reagents, and per-haps commercial production ofCampylobacter

conjugates forC.jejuni, C. coli,and C.fetuswill

follow.

ACKNOWLEDGMENTS

We thank M. A. Lambert for the additional gas-liquid chromatography studies andM.Veron forarrangingtohave theCIPtypestrainssent to us.

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