APPLIED AND ENVIRONMENTAL MICROBIOLOGY, May 1993, p. 1269-12730099-2240/93/051269-05$02.00/0Copyright X 1993, American Society for Microbiology

Role of Campylobacterjejuni Flagella as Colonization Factorsfor Three-Day-Old Chicks: Analysis with Flagellar Mutants

IRVING NACHAMKIN,1* XIAO-HE YANG,' AND NORMAN J. STERN2

Department ofPathology and Laboratory Medicine, University ofPennsylvania School ofMedicine,Philadelphia, Pennsylvania 19104-4283, 1 and Richard B. Russell Agricultural Research Center,

Agricultural Research Service, U.S. Department ofAgriculture, Athens, Georgia 306132

Received 8 December 1992/Accepted 3 February 1993

Campylobacter jejuni, an important cause of human gastrointestinal infection, is a major food-bornepathogen in the United States and worldwide. Since poultry becomes colonized and/or contaminated during theearly stages of production and is a major food-borne source for this organism, we studied the role of C. jejuniflagella on the ability of the bacterium to colonize the chicken gastrointestinal tract. Three-day-old chicks wereorally challenged with a motile wild-type strain of C. jejuni IN9 or with flagellar mutants created from IN9 bydisrupting the flagellin genes with a kanamycin resistance cassette by using shuttle mutagenesis (A.Labigne-Roussel, P. Courcoux, and L. Tompkins, J. Bacteriol. 170:1704-1708, 1988). One mutant, IN9-N3,lacked flagella and was nonmotile. The other, IN9-N7, produced a truncated flagellum and was partially motile.Three-day-old chicks were orally challenged with different doses of the wild-type strain and the two mutants.At challenge doses ranging from 3.0 x 104 to 6.6 x 10i CFU per chick, only the fully motile, wild-type straincolonized the chick ceca. Our results show that intact and motile flagella are important colonization factors forC. jejuni in chicks.

Campylobacter jejuni is recognized as one of the mostcommon causes of bacterial gastroenteritis in the UnitedStates, other developed countries, and developing nations(26, 33). Humans acquire campylobacter infection by han-dling or ingesting contaminated food, milk, or water (27, 33).Poultry serves as the primary source of contamination (4, 12,27, 33). C. jejuni is a normal inhabitant of the gastrointestinaltract of poultry (27), and the organism is typically acquiredduring the first 3 to 4 weeks of production. In surveys ofbroiler chicken carcasses, 45 to 85% of retail-ready birdswere contaminated with C. jejuni (5, 27). Thus, understand-ing the factors that allow Campylobacter strains to colonizethe poultry gut is important in designing strategies to preventintestinal colonization.Campylobacter species are motile by means of a single

polar, unsheathed flagellum at one or both ends of theorganism. Flagella have been shown to be necessary forcolonization and/or infection in a number of models ofinfection (1-3, 18, 20, 22). The contribution of flagella tocolonization of the chicken gastrointestinal tract has notbeen reported previously. Two genes, flaA andflaB, that are

involved in expression of the flagellar filament, have beenidentified in C. jejuni (7, 21) and C. coli (11). In C. jejuni81116 (21), onlyflaA is expressed, whereas in C. coli VC167(11), some flaB is also expressed. During the past 5 years,molecular methods for creating stable mutations in theCampylobacter genome have been developed (13, 14) andapplied to studies of the flagellar genes in C. jejuni and C.coli (9, 36). By using gene replacement mutagenesis wherebya kanamycin resistance gene is inserted into one or both ofthe genes and inactivating a particular gene, flaA appears tobe involved predominantly in the expression of intact andfully motile organisms (9, 36). Using these methods, we havedisrupted the flagellar genes of C. jejuni IN9 to produceseveral mutants either lacking flagella (nonmotile) or with

* Corresponding author.

altered flagella (partially motile). We tested these mutantsfor the ability to colonize the 3-day-old chick by oralchallenge with different doses and found that only thewild-type organism colonized the gut.

MATERIALS AND METHODS

Bacterial strains. C. jejuni IN9 and IN1 were initiallyobtained from two human patients with gastrointestinalinfection. The environmental source from which these pa-tients acquired infection is not known. C. jejuni IN9 was nottypeable by heat-labile (HL) serotyping (15) and was sero-

type 13,16,22 by heat-stable (0) serotyping (23). C. jejuniIN1 was serotyped as HL7 (7). Escherichia coli S17-1(DT1697) was used as the host for cloning experiments (25).E. coli C600 (leu thr thi lacY supE44 tonA) harboring theconjugative plasmid pRK212.2 (6) was used as the host strainfor conjugation experiments.

Shuttle mutagenesis. The flaA gene from C. jejuni IN1(HL7) was amplified by polymerase chain reaction (PCR) as

previously described (7), BamHI and SalI restriction siteswere added by PCR, and the gene was cloned into thesuicide plasmid pILL533 (34), yielding plasmid pXY102.pILL533 is similar in structure and function to pILL560,which was previously described by Labigne-Roussel et al.(13). Inserts were verified by PCR with oligonucleotideprimers directed against the 5' and 3' flaA region as previ-ously described (7). A 1.4-kb ClaI-HindIII fragment contain-ing a campylobacter kanamycin resistance gene (Kin) was

purified from pUOA13 (35), and the 3' termini were filled inwith Klenow fragment by the method of Sambrook et al.(24). The kanamycin gene was inserted into the uniqueEcoRV site of theflaA gene by blunt-ended ligation, yieldingpXY120, and inserts were selected on agar containing 32 ,ugof kanamycin per ml. Inserts were verified by PCR andrestriction mapping. E. coli harboring the conjugative plas-mid pRK212.2 (6) was transformed with pXY120, and the

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flaA-Km gene was mobilized into C. jejuni IN9 by conjuga-tion as described previously (13).

Motility. Kanamycin-resistant transconjugates were se-lected and tested in 0.4% Muller-Hinton agar for motilitycharacteristics. A suspension of organisms (ca. 109 CFU/ml)was placed in a central well of the soft-agar plate, and plateswere incubated for 48 h at 37°C under microaerophilicconditions. The diameter of swarming from the central wellwas measured.

Immunologic studies. Flagellin epitopes were analyzed inthe bacterial mutants by using Western immunoblot analysisas described previously (1).

Southern hybridization. Mutants were characterized byhybridization with probes prepared by using the PolarPlexChemiluminescent Blotting Kit (Millipore, Bedford, Mass.).Purified flaA and Km genes were biotinylated by randompriming and hybridized to EcoRV-digested chromosomalDNA of the organisms as specified by the manufacturer.Hybridization was detected by adding streptavidin and bio-tinylated alkaline phosphatase and then developing the blotwith Lumigen-PPD substrate. The emitted light from thereaction was captured on X-ray film after exposure for 1 h.

Electron microscopy. Each of the mutants and the wild-type isolate were examined by transmission electron micros-copy for the appearance of flagella. Organisms were grownovernight on Mueller-Hinton agar plus kanamycin for themutants and on plain Mueller-Hinton agar for the wild-typestrain. A light suspension of each was prepared in buffered0.1% glutaraldehyde-0.01% tannic acid and spotted on elec-tron microscopy grids. The grids were stained with 1%phosphotungstic acid and examined.

Experimental animals. Chicks used in all experiments wereobtained from a local hatchery (Athens) on the day of hatch.The birds were transported immediately to the AgricultureResearch Station facilities and placed in raised, wire-floorisolation units, ventilated with positive-pressure filtered air.Fecal material from paper pads on which chicks weretransported was cultured by enrichment methods (30) todemonstrate the absence of Campylobacter spp. beforechallenge experiments were performed. Chicks were placedin each isolation unit, provided with feed and water adlibitum, and maintained at ca. 35°C with constant lightingthroughout the experiments. The birds were allowed 48 h toacclimate to the units before experimental treatments werebegun.

Challenge studies. Each group of chicks was challenged 48h posthatch. All isolates were grown on Brucella-ferroussulfate-sodium bisulfite-sodium pyruvate (Brucella-FBP)agar plates (8) under a microaerobic atmosphere at 42°C for20 to 24 h. Mutant strains were grown on Brucella-FBP agarcontaining 32 ,ug of kanamycin per ml. Organisms weresuspended in phosphate-buffered saline (PBS: pH 7.2), andserial dilutions were made in PBS according to the cultureoptical density. Plate counts were determined to quantify thechallenge dose. Each dose was provided in 0.1 ml of PBSadministered by direct gavage into the crop by using anarrow-gauge hose at the end of a needle and syringe. Oneday after challenge, fecal samples were collected from eachof the isolation units. Approximately 5 g of feces was diluted1:4 in PBS and then plated onto Brucella agar with 5% lysedhorse blood, 32 mg of cefoperazone per liter, and 15 mg ofcycloheximide per liter (Campy-Cefex medium) (32). Initialstudies showed that the wild-type and mutant strains used inthis study grew well on Campy-Cefex medium. Plates wereincubated for 24 h under microaerobic conditions (5% 0210% CO2, 85% N2) and examined for characteristic colonies

of C. jejuni. Colonies were inspected under phase-contrastmicroscopy to confirm the characteristic morphology anddarting motility of the organism. At 5 days after challenge,the birds were sacrificed by cervical disarticulation. Cecawere aseptically removed, and C. jejuni organisms werequantified by serial dilution plating on Campy-Cefex medium(with and without 32 ,ug of kanamycin per ml). We calculatedthe colonization quotient (CQ) for each group. The CQ is themean log1o number of C. jejuni organisms per gram of cecalmatter for the individuals within each group and challengedose (29).

Statistical analysis. One-way analysis of variance wasperformed by using GraphPad Instat statistical software,version 1.1 (GraphPad Software, Inc., San Diego, Calif.).

RESULTS

Two flagellar mutants, IN9-N3 (Fla- Mot-) and IN9-N7(Fla+truncated Mot+'-), were prepared by inserting a kana-mycin resistance gene into the flagellin gene locus and wereselected for further animal studies. We compared the motil-ity characteristics of the two mutants with those of thewild-type strain. On semisolid motility agar, the wild-typestrain, C. jejuni IN9, produced a zone of growth with adiameter of 30 mm after 48 h of incubation. The mutants, incontrast, showed no zone in mutant IN9-N3 and a partialzone (diameter, 11 mm) in mutant IN9-N7. There was somevariability in the zone diameters observed for IN9 andIN9-N7 on repeat experiments; however, IN9-N7 alwaysexhibits motility approximately one-third that of the wildtype. Motility in soft agar has never been observed withIN9-N3. Both IN9 and IN9-N7 exhibited motility by wetmount examination. We examined these mutants by electronmicroscopy (Fig. 1) and observed that IN9-N3 did notproduce a flagellum but that IN9-N7 had a short flagellumcompared with the wild-type strain.Western immunoblot analysis of the wild-type strain, IN9,

and mutants were consistent with our findings by electronmicroscopy. Using monoclonal antibody Fll directedagainst the flagellum, we detected the 62-kDa subunit flagel-lin protein in the wild-type strain (Fig. 2). The nonmotilemutant, IN9-N3, did not exhibit the 62-kDa subunit. Incontrast, the partially motile mutant, IN9-N7, exhibited abarely visible 62-kDa band and also showed a prominent ca.20-kDa band reactive with the antibody.We analyzed chromosomal DNA obtained from the wild-

type and mutant strains by Southern hybridization with flaAand Km probes (Fig. 3). The fla genes in both IN9-N3 andIN9-N7 had been disrupted with the Km gene, as evidencedby the altered hybridization pattern with flaA (Fig. 3A).Further analysis of the digests with the kanamycin resistanceprobe showed the presence of the 1-kb internal Km genefragment in the chromosome (Fig. 3B). Both mutants werestable in vitro; reversion to wild-type motility was notobserved.Using the wild-type and mutant strains, we were able to

assess the role of flagella on the ability of C. jejuni tocolonize the chicken gastrointestinal tract. In preliminaryexperiments to determine the colonization dose (CD50S)chicks were challenged with 2.7 x 105, 2.7 x 103, and 2.7 x101 CFU of C. jejuni IN9, 1.6 x 105, 1.6 x 103, or 1.6 x 101CFU of C. jejuni IN9-N7, and 2.4 x 105, 2.4 x 103, and 2.4x 101 CFU of C. jejuni IN9-N3. None of the strainscolonized any of the 90 chicks challenged at these doses,with a lower limit of detection in our system of 40 CFU/g ofcecal material (CQ < 1.6). An additional challenge experi-

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FIG. 1. Electron micrographs of C. jejuni strains used in thisstudy. The motile wild-type organism (strain IN9) has a normalflagellum; the nonmotile mutant (strain IN9-N3) has no flagellum;and the partially motile mutant (strain IN9-N7) has a truncatedflagellum. Photographs are representative morphotypes observedfor each strain. Several dozen fields were examined, and more than95% of the bacteria exhibited the morphology for the particularstrain. Bacteria were prepared and stained as described in Materialsand Methods.

ment with a dose of 1.0 x 107 CFU of C ;ejuni IN9, 2.0 x107 CFU of C. jejuni IN9-N7, and 6.0 x 10 CFU of C. jejuniIN9-N3 showed that only the wild-type strain colonizedeight of eight chicks with a CQ of 6.99. None of the chickschallenged with either IN9-N7 or IN9-N3 became colonized.

In a final study, three groups of chicks were challengedwith three increasing doses of C. jejuni (Table 1). Again,only the group challenged with the wild-type strain becamecolonized, whereas neither mutant was isolated from chickson day 8. Cultures of fecal material obtained 24 h postchal-lenge contained only the wild-type strain, IN9.

62-

FIG. 2. Western immunoblot analysis of wild-type and mutantstrains with monoclonal antibody Fll. The lanes are labeled withthe strain names: IN9 is the wild-type strain; IN9-N3 (lane N3) is thenonmotile strain; and IN9-N7 (lane N7) is the partially motile strain.

DISCUSSION

Several investigators have successfully used gene ex-change mutagenesis in Campylobacter spp. to study the roleof flagellin genes (9, 36). Labigne-Roussel et al. were the firstto develop shuttle mutagenesis in C. jejuni (13, 14). Theyconstructed a conjugative suicide plasmid that could repli-cate in Escherichia coli and could be mobilized into Campy-lobacter species but could not replicate in the recipient cell.Homologous genes inserted in the suicide plasmid could berescued into the recipient chromosome by homologous re-combination. Guerry et al. (9) and Wassenaar et al. (36) tookadvantage of this system by disrupting cloned flagellin geneswith a kanamycin cassette and exchanging the disruptedgenes in the wild-type strain by conjugation, natural trans-formation, and/or electroporation.Using this approach, we were able to prepare two flagellin

mutants that either lacked intact flagella or produced analtered flagellum. Whether the Km cassette is located inflaA, flaB, or both is uncertain. On the basis of previousstudies, it seems likely that IN9-N3 has aflaA flaB mutation,since insertion of a Km gene into both genes resulted inaflagellate, nonmotile mutants (9, 36). IN9-N7, in contrast,probably contains a flaA mutation, resulting in the produc-tion of short flagella that may confer partial motility (9, 36).Insertions of the Km gene that inactivate flaA result in theproduction of short flagella composed of FlaB protein (9, 10,36). Further studies are under way to localize the exactinsertions in the two mutants. On the basis of Southern andWestern analysis of the strains, we suspect that the weakreaction of monoclonal antibody Fll with the 62-kDa bandin the partially motile mutant, IN9-N7, represents somecross-reaction of this antibody with FlaB. The appearance ofa ca. 20-kDa band may represent reactivity with the portionof flagellin translated upstream to the Km gene insertion inflaA.

In experiments to assess the role of flagella on campylobac-ter colonization, only the fully motile and flagellate wild-typestrain colonized the chicken gastrointestinal tract at challenge

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A.flaAprobe

23.19.4 -

6.6 -

4.4 -

2.3 -

2.0 -

1.353 -

1.078 -

B.Kmprobe

IN N N1 9 3 7

INN N pUOA9 3 7 13

2.3 -

2.0 -

1.353 -*1.078 -

.872 -,

.603 -^i '

FIG. 3. Southern hybridization analysis of EcoRV-digestedchromosomal DNA from the wild type (IN9) and mutant strains(IN9-N3 and IN9-N7) by using biotinylatedflaA (A) and kanamycin(B) probes as described in Materials and Methods. Size markers arelocated in the left-hand lane of each panel. In panel A, lane 1contains chromosomal DNA from C. jejuni IN1 (HL7) from whichtheflaA constructs were derived; lane IN9 contains wild-type strainIN9; lanes N3 and N7 contain, respectively, strains IN9-N3 andIN9-N7, the mutants that result from insertion of the flaA-Km geneinto IN9. In panel B, lane IN9 contains the wild-type strain IN9probed with the 1-kb Km probe; Lanes N3 and N7 show the mutantsIN9-N3 and IN9-N7, respectively, containing the Km gene afterinsertion of the flaA-Km gene; Lane pUOA13 contains the controlplasmid, pUOA13, from which the Km gene was originally derived.

doses ranging from 104 to 108 CFU. Mutants were notdetected in cecal cultures from any of the chicks during theexperiments, suggesting that these mutants were quicklycleared from the gastrointestinal tract after oral inoculation.The CD50% for our wild-type strain is somewhat higher thanfor other C. jejuni isolates (29); this may be related to thehuman origin of the isolate. The doses required for coloniza-tion of chickens may be affected by passage through chickens(28); however, we have not passaged our strains throughchickens to determine whether there is any increase in theability of these strains to colonize the chicken gastrointestinaltract. The variation in colonizing dose may be explained byprevious observations by Stern et al. (29), which showedconsiderable interexperiment variation in establishing theCD50%s but consistent results within a particular experiment.Further, there was considerable variation in the CQ forchickens challenged with different doses of C. jejuni IN9, asshown by the large standard deviation within each group(Table 1). Consequently, there was no statistical differencebetween the CQs for the different challenge doses.

TABLE 1. Colonization of 3-day-old chicks after challenge withflagellar mutants of C. jejuni

Strain Challenge level No. CQ No. positive(CFU/chick) tested (%)

IN9 (Fla' Mot') 3.0 x 104 9 4.73 ± 2.84a 7 (78)3.0 x 106 9 3.52 ± 3.03 6 (67)3.0 x 108 10 5.94 ± 2.21 9 (90)

IN9-N7 6.6 x 104 9 <1.6 0 (0)6.6 x 106 8 <1.6 0 (0)6.6 x 108 10 <1.6 0 (0)

IN9-N3 5.3 x 104 10 <1.6 0 (0)5.3x106 9 <1.6 0(0)5.3x108 9 <1.6 0(0)

No statistical difference between CQ values for each challenge dose withIN9 by one-way analysis of variance (P = 0.2028).

Our results provide strong evidence that campylobacterflagella are important colonization factors for chickens.Whether flagella, per se, are involved in attachment or solelyin motility is an important question not resolved in thisstudy. However, a partially motile mutant, IN9-N7, did notcolonize the chicken gastrointestinal tract. This informationsuggests that, at the very least, a fully intact and motileflagellum is necessary for colonization. Motility is probablya major factor in the ability of Campylobacter organisms tocolonize the gastrointestinal mucus layer (10). Flagella maynot be the only colonization factors produced by C. jejuni.Meinersmann et al. (16, 17) have described variants of C.jejuni that lacked the ability to colonize chickens at an oraldose of 105 CFU and that did not appear to have alteredflagella and/or motility (16). The colonizing strain was asso-ciated with a 69-kDa surface antigen, but the nature of thiscomponent has not been identified.

C. jejuni causes a human illness with important health andeconomic implications (26). Although a number of strategiescould be used to minimize campylobacter infections inhumans, a program directed toward preventing colonizationof poultry with this organism would have significant impli-cations for reducing the incidence of human infections (5,27). Chickens colonized with C. jejuni develop humoral andbile secretory immunoglobulin A antibodies directed againstthe organism (19). Secretory immunoglobulin A antibodiesappear to be lowest at hatch and increase by 4 weeks incolonized chickens. Since chicks become colonized with C.jejuni early in the growth phase, strategies to stimulatesecretory immunoglobulin A might affect the ability of C.jejuni to colonize the chick gut. Although this was notstudied directly, Stern et al. (31) found that C. jejuni prein-cubated with rabbit hyperimmune serum inhibited cecalcolonization. Future studies on factors that promote coloni-zation of C. jejuni in poultry, such as flagella, may lead touseful methods to prevent colonization.

ACKNOWLEDGMENTSWe thank Mike Musgrove, Margaret Myszewski, and Sharilyn

Morris for carrying out the chicken challenge studies. The assis-tance of Melanie Minda in electron microscopy is gratefully ac-knowledged. We thank Lucy Tompkins, Stanford University, forproviding plasmid pILL533; Diane Taylor, University of Edmonton,for providing pUOA13 and E. coli S17-1; and Donald Helinski,University of California, San Diego, Calif., for providing pRK212.2.We also thank Paul Edelstein for review of the manuscript and forsuggestions.

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Some of the work performed in this study was supported in partby grant AI-24122 from the National Institutes of Health (to I.N.).

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