Veterinary Microbiology 101 (2004) 75–82

Correlation between invasion of Caco-2 eukaryotic cells andcolonization ability in the chick gut inCampylobacter jejuni

I. Hänela,∗, J. Müllerb, W. Müllerb, F. Schulzeb

a Federal Research Centre for Virus Diseases of Animals, Institute of Molecular Pathogenesis,Jena, Naumburger Str. 96a, 07743 Jena, Germany

b Institute of Bacterial Infections and Zoonoses, Jena Naumburger Str. 96a, 07743 Jena, Germany

Received 19 September 2003; received in revised form 23 March 2004; accepted 1 April 2004

Abstract

In an in vitro cell culture model using Caco-2 cells the adhesion and invasion properties of 11Campylobacter (C.) jejuniisolates of different origin were studied. Additionally, we investigated the colonization ability of the strains in a chick model.Virtually, all C. jejuni showed cell adherence in the in vitro assay, but there were large differences in the invasion frequenciesamong theCampylobacter isolates. The colonization ability in the chick gut also differed markedly and enabled the formation ofthree groups: non-colonizing, weak or delayed colonization and strong colonization ability. On this occasion, we found a putativecorrelation between invasion of Caco-2 cells and colonization in the chick gut. Non-colonizers are not invasive or only havesmall invasion indexes. Strains which colonize weakly or exhibit delayed colonization have a medium invasion index and strongcolonizers show markedly higher values of this parameter. The characterization of the flagellin gene of the usedC. jejuni strainsresulted in eightflaA types. There was no association betweenflaA type and invasion or colonization ability in the chick gut.© 2004 Elsevier B.V. All rights reserved.

Keywords: Campylobacter jejuni; Caco-2 cells; Invasion; Colonization;flaA

1. Introduction

Although Campylobacter (C.) jejuni is now rec-ognized as a major human enteropathogen, specificvirulence mechanisms in campylobacteriosis are notyet well defined. Numerous attempts have been madeto clarify the mechanisms by which this pathogenexpresses virulence. It has been shown that potentialvirulence factors include chemotaxis and motility,

∗ Corresponding author. Tel.:+49-3641-804426;fax: +49-3641-804228.

E-mail address: i.haenel@jena.bfav.de (I. Hänel).

adhesion, ability of colonization, invasion, epithelialtranslocation, intracellular survival, iron acquisitionand formation of toxins (Ketley, 1997; Wassenaarand Blaser, 1999). Generally,C. jejuni establishes anon-pathologic, commensal relationship in the gas-trointestinal tract of different animals. Since manyhumanCampylobacter infections arise from ingestionof contaminated animal products (poultry, raw milk),we know that the organisms can cause human illness.However, this phenomenon does not exclude the pos-sibility that variation in virulence exists between bac-terial populations or strains (Wassenaar and Blaser,1999). The identification and distinction of strains

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76 I. Hänel et al. / Veterinary Microbiology 101 (2004) 75–82

with different virulence for humans requires meth-ods to distinguish pathogenic from non-pathogenicorganisms. It is possible that the pathotypes ofC.jejuni are as diverse as those ofEscherichia (E.)coli, but the model systems and genetic tools todifferentiate strains according to pathogenic poten-tial are not yet available. The precise means bywhich C. jejuni overcomes host defence factorsare not known. More information is necessary tounderstand the relationship between pathogen andhost.

In the past decade, in vitro assays have permittedthe identification and characterization of a number ofC. jejuni virulence determinants. In certain cases, theidentification of putative virulence determinants fromin vitro assays has been substantiated by in vivo mod-els, thus demonstrating the usefulness of in vitro mod-els (Konkel et al., 2000).

In our investigations, we used 11C. jejuni strainsof different origin. In vitro assays for determination ofadhesion and invasion ability of theseC. jejuni strainswere conducted with Caco-2 cells, a human coloniccarcinoma cell line (Pinto et al., 1983; Zweibaumet al., 1991). Additionally, we investigated the colo-nization ability of these strains in a chick model. Onthis occasion, we found a putative correlation betweeninvasion of Caco-2 cells and colonization in the chickgut. On the other hand, the discrimination between theisolates by a PCR-restriction fragment length poly-

Table 1Adherence to and invasion of Caco-2-cells and colonization ability in the chick gut of 11C. jejuni strains

Strain Origin of isolate Percentage ofinoculum adhereda

Percentage of inoculuminternalizeda,b

Invasion indexa,c Colonizationabilityd

51/89 Human enteritis 5.37± 2.09 0.0073± 0.003 0.17± 0.13 –984djLn Calf intestinal lymph node 0.4± 0 0.0015± 0 0.35± 0 –81116 Human faeces 1.23± 0.68 0.66± 0.66 4.13± 3.19 –C130 Calf abomasum 0.84± 0.42 0.0003± 0.0002 0.04± 0.03 –P1 Reference strain 3.85± 3.09 0.23± 0.18 6.45± 2.07 ++Z2 Unknown 0.53± 0.08 0.029± 0.01 5.9± 2.99 ++T313/W Milk-borne infection 4.69± 0.96 1.79± 0.44 39.6± 14.9 +++340/W Milk-borne infection 12.7± 3.41 4.1± 1.12 32.4± 3.2 +++158/96 Human enteritis 1.36± 0.6 0.51± 0.18 39.2± 13.2 +++73/96 Milk 4.26± 1.58 2.14± 0.77 52.5± 13.6 +++157/96 Human enteritis 2.35± 0.07 0.63± 0.1 26.95± 4.03 +++

a Data are expressed as the mean± standard deviation of at least three independent experiments performed in duplicate.b Calculated as the percentage of bacteria that survived gentamicin treatment.c Invaded bacteria expressed as a percentage of the number of adhered organisms.d Colonization ability: (–) isolates could not be reisolated; (++) weak or delayed colonization; (+++) strong, long lasting colonization.

morphism assay of the flagellin gene (flaA) did notcorrelate with colonization or invasion.

2. Materials and methods

2.1. Bacterial isolates

ElevenC. jejuni strains of different origin (Table 1)were investigated. The non-invasiveE. coli K12 strain(determined in other studies;Dinjus et al., 1998)was included as a negative control for invasion. Thisstrain shows a percentage entry within the scope of0.01–0.001% although 21% of the inoculum werefound to adhere to Caco-2 cells.

2.2. Epithelial cells

Caco-2 cells (ACC 169) were obtained from theGerman Collection of Microorganisms and Cell Cul-tures (DSMZ, Braunschweig). The cells were grownin Dulbecco’s Modified Eagle’s Medium supple-mented with 10% fetal bovine serum (FBS) and1% non-essential amino acids without the use ofantibiotics at 37◦C in a 5% CO2, humidified atmo-sphere. For the experimental assays, Caco-2 cellswere grown in 24-well plastic plates. The cellswere seeded at 4.5 × 104 cells per well and incu-bated for 7 days at 37◦C in a 5% CO2, humidified

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atmosphere. Prior the assay, the cell monolayers wereonce washed with phosphate-buffered saline (PBS),pH 7.2.

2.3. Adherence and invasion assay

Campylobacter strains were grown microaerobi-cally on Mueller-Hinton (MH) agar plates for 48 hat 37◦C. Bacteria were harvested from plates withPBS containing 1% FBS and adjusted spectropho-tometrically to approximately 1× 108 bacteria permilliliter. 0.5 ml of this suspension was inoculatedinto duplicate wells containing confluent monolay-ers of Caco-2 cells. The actual numbers of bacteriain the inoculum added to monolayers were deter-mined retrospectively by serial dilution and platecounting. Eukaryotic cells were incubated for 3 hat 37◦C and 5% CO2 to allow bacterial adher-ence and internalization. For determination of ad-herence, the cells were washed three times withPBS and the cell monolayer was lysed with 1%Triton X-100 and total bacteria associated withthe cells (intracellular and extracellular bacteria)were enumerated by plating serial dilutions of thelysates on MH agar plates and counting the resul-tant colonies. In order to measure bacterial invasion,the infected cells were washed two times with PBSand incubated in fresh PBS containing 1% FBS and150�g/ml gentamicin for 2 h to kill remaining vi-able extracellular bacteria. Quantification of viableintracellular bacteria was performed by washing theinfected eukaryotic cells with PBS twice and sub-sequent lysing with 1% Triton X-100. Followingserial dilution in PBS, released intracellular bacte-ria were enumerated as described for the adherenceassay.

Adherent bacteria were expressed as the percent-age of bacteria counted without antibiotic treatmentreferred to the infection dose. Because the bacterialinvasion rate is very low it can be neglected withoutmarked influence on the result. Invasion ability wasexpressed as the percentage of the bacterial inoculumsurviving the gentamicin treatment. Another measureof invasiveness is the invasion index, which is thenumber of invaded bacteria expressed as a percent-age of the number of adhered organisms. The resultswere the mean of at least three separate determina-tions.

2.4. Experiments on the colonization of C. jejuni inthe chick gut

The design of experiments on colonization of thechick gut by C. jejuni was described in detail bySchulze and Erler (2002). In brief, White Leghornchicks used in this study were hatched from specificpathogen-free eggs, obtained from the companiesLohmann and Charles River. The colonization abilityof theC. jejuni strains was investigated in six experi-ments. In each experiment, day of hatch chicks wererandomly separated into three groups of 24 birds each:two groups were colonized experimentally and oneserved as a control group. Chicks were reared on thefloor in three separated, adjacent rooms with steril-ized wood shavings as litter. They were fed ad libitumwith a commercial starter ration devoid of antibioticor coccidiostat and were provided with chlorinateddrinking water. At 2 or 8 days of age, respectively,the chicks in the experimentally colonized groupsreceived between 3.3 × 107 and 2.0 × 108 colonyforming units (cfu) ofC. jejuni via oesophageal gav-age. Frozen stock cultures ofC. jejuni were thawed,inoculated on MH agar, and incubated microaero-bically for 48 h at 37◦C. Cells were harvested andresuspended in sterile PBS, pH 7.4. The suspensionwas adjusted spectrophotometrically and the meannumbers of cfu per milliliter were determined byperforming standard plate counts.

Seven, 14, 21, 28, 42 and 56 days after inocula-tion, four chicks of each group were sacrificed bycervical dislocation, at which time blood, liver andfaeces were collected for processing. Faecal sampleswere obtained from intact ceca, weighed, homoge-nized with sterile buffer (stomacher), diluted and in-oculated onto CCDA medium (Oxoid) and modifiedSkirrow medium (Oxoid with 30�g/ml Cefoperazone,Sigma; 10�g/ml Amphotericin B, ICN Biomedicalsand 10% calf blood). The liver samples were processedin the same manner. The plates were incubated mi-croaerobically at 37◦C for 48 h, after which time theCampylobacter colonies were enumerated. In the sec-ond part of experiments, a liquid enrichment based onCCDA medium without agar was also used.

Serum was collected after centrifugation andstored immediately at−20◦C. Glycine extractedproteins (strain Penner 1) were prepared as pre-viously described (Blaser and Duncan, 1984) and

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Campylobacter-specific IgG, IgA and IgM antibodieswere measured in serum by an indirect enzyme-linkedimmunosorbent assay (ELISA).

2.5. Flagellin gene typing and RFLP analysis

The chromosomal DNA from the isolates was ex-tracted using the High Pure PCR Template PreparationKit of Roche Diagnostics. Flagellin gene typing wasperformed according to the method ofNachamkinet al. (1993)with minor modifications namely, theuse of a consensus primer set with forward primer5′-ATGGGATTTCGTATTAACAC-3′ and reverseprimer 5′-CTGTAGTAATCTTAAAACATTTTG-3′(Wassenaar and Newell, 2000) and a lower anneal-ing temperature of 45◦C (according toCAMPYNET,2004). PCR was performed with a DNA ThermalCycler (Eppendorf) using the following cycling pa-rameters: 94◦C for 1 min and then 94◦C for 15 s,45◦C for 45 s, 72◦C for 1 min and 45 s for 35 cycles.After the last cycle, the sample was held at 72◦Cfor 5 min. The expected 1.7 kb amplicon representingthe Campylobacter flagellin gene was checked byelectrophoresis on a 1% agarose gel.

The PCR product was digested with 0.067 U/�l ofthe restriction enzymes DdeI or AluI (New EnglandBiolabs) and analyzed by electrophoresis on a 2%agarose gel. The gels were examined using a Bio Imag-ing System (Syngene).

3. Results

3.1. Adhesion and invasion ability of C. jejuni strains

Caco-2 cells were inoculated withC. jejuni and thebacteria assessed for their ability to attach to and sub-sequently enter the cells. All 11 strains investigatedadhered to the Caco-2 cells (Table 1). We observedvariation in the bacterial attachment to Caco-2 cellsbetween 0.4% (C. jejuni 984dJLn) and 12.7% (C. je-juni 340W) of the original bacterial inoculum. Theadherence ability was independent of the coloniza-tion groups in the chick model. However, large differ-ences of invasion frequencies between the 11C. jejunistrains were noted. The percentage entry of the inva-siveCampylobacter isolates should be greater than thepercentage entry of the non-invasiveE. coli strain K12

(0.01–0.001%). According to this criterion, three ofthe tested strains (C. jejuni 51/89,C. jejuni 984dJLnandC. jejuni 130) were not invasive for Caco-2 cells.For strainC. jejuni 81116 the results of seven inde-pendently performed experiments differed in a widerange. In most cases of the experiments, the strain wasnot invasive for Caco-2 cells and therefore the strainC. jejuni 81116 was considered to be not invasive.Two strains (C. jejuni P1 andC. jejuni Z2) showed amedium invasion and five strains (C. jejuni T313W,C.jejuni 340W,C. jejuni 158/96,C. jejuni 73/96 andC.jejuni 157/96) were considered to be highly invasive,having at least 0.5% intracellular bacteria of the inocu-lum which survived gentamicin treatment (Table 1).

3.2. Experiments on colonization in the chick gut

The colonization ability in 8-day-old chicks differedin a wide range. Altogether, 11C. jejuni strains wereinvestigated. The colonization ability was assessed bymeans of the following criteria: first detection afterinoculation, number ofC. jejuni per gram of sample,duration of excretion (up to the end of experiment at 8weeks after inoculation), number of positive animalsout of the group at different investigations, tendencyto invasion of the liver. According to these criteria,we could divide the isolates into the following threegroups:

• No colonization—the isolates could not be reiso-lated. According to this criterion four strains (51/89,984 dJLn, 81116, C130) were arranged in thisgroup.

• Weak or delayed colonization—the strains onlyachieved mediate counts per gram caecal contentand were intermittently excreted (not all animalspositive at different investigations). These strainscould not be reisolated out of the liver. Two strains(P1, Z2) corresponded to these criteria.

• Strong colonization—these strains caused a strong,long lasting colonization (up to the end of experi-ment at 8 weeks after inoculation). Seven days afterinoculation, these strains could be reisolated fromall animals. The highest counts of the pathogenachieved more than 9.0 cfu per gram of caecal con-tent (final viable counts were adjusted to cfu pergram of material in log10 format). Parallel to thehigh density of the bacteria in caeca, the strains

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could also be isolated from the liver (highest amount4.0 cfu). According to these characteristic features,five strains (T 313W, 157/96, 340W, 158Di, 73Di)were arranged in this group.

Additionally, theC. jejuni colonization resulted in amarked IgG (but not IgM and IgA) increase in chicks(Schulze and Erler, 2002). Although someC. jejunicaused a strong, long lasting colonization and couldalso be reisolated from the liver, we observed no im-pairment of behavior of chicks or other clinical signs,such as diarrhea.

3.3. Correlation between invasion of Caco-2 cellsand colonization ability in the chick gut

In general,Campylobacter strains with strong abil-ity to colonize the gastrointestinal tract of chicks werefound to be more invasive in the in vitro cell culturemodel (Table 1). Invasion is the combined process ofbacterial attachment to and penetration into cells. Theratio of number of bacteria internalized to number ofadherent bacteria (invasion index) is a measure of anorganism’s ability to adhere to a eukaryotic cell andhow likely this interaction will lead to internalization(defined byElsinghorst, 1994). The invasion index inconnection to the colonization ability in the chick gutof the examinedCampylobacter strains is shown in

Fig. 1. FlaA types of usedC. jejuni strains. M—molecular weight marker, Contr.—type strainC. jejuni DSMZ 4688.

Fig. 2. Results of the in vivo as well as the results of thein vitro experiments were the basis for differentiationof the Campylobacter strains into three groups. Fourof the 11 isolates (C. jejuni 51/89,C. jejuni 984dJLn,C. jejuni 81116 andC. jejuni 130) which could not bereisolated in vivo were characterized by an invasionindex below 1%. For the two isolates causing a weakor delayed colonization (C. jejuni P1,C. jejuni Z2) theinvasion indexes were 6.45± 2.07 and 5.9 ± 2.99%,respectively. The five Campylobacter strains produc-ing a strong colonization (C. jejuni T313W,C. jejuni340W,C. jejuni 158/96,C. jejuni 73/96 andC. jejuni157/96) were characterized by significant higher inva-sion indexes of over 25% (Fig. 2).

3.4. Characterization of the flagellin gene

The characterization of the flagellin gene of theC.jejuni strains used resulted in eightflaA types. Each ofthe six non or weak colonizing strains had a differentflaA type. ThreeflaA types were detected from analysisof the five isolates producing a strong colonization(Figs. 1 and 2). FlaA type 6 was found in isolateC.jejuni 81116 (non-colonizing) as well as in isolateC.jejuni 340W (strong colonizing). These results do notsupport an association offlaA types with colonizationand invasion.

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0

10

20

30

40

50

60

70

51/89 984djLn 81116 C130 P1 Z2 T313W 340W 158/96 73/96 157/96

inva

sion

inde

x

FlaA type: 7 8 6 2 1 5 3 6 4 4 3

Colonization ability: non colonizing weak or delayed strong

Fig. 2. Correlation between the invasion index: (Invaded bacteria expressed as a percentage of the number of adhered organisms. Data areexpressed as the mean± standard deviation of at least three independent experiments performed in duplicate.), colonization ability: (Thestrains were divided into three colonization types according to criteria described inSection 3.2) in the chick gut andflaA type.

4. Discussion

The cultured eukaryotic cell assay technique has be-come a standard experimental procedure in the studyof bacterial adhesion and internalization.Everest et al.(1992) proposed the use of Caco-2 cells as a modelfor studying adhesion on and invasion of the intestinalepithelia byC. jejuni andC. coli. Adherence to targethost cells is a critical early step in the pathogenesis ofmany bacterial infections, since adherent bacteria canrelease enzymes and toxins, or trigger changes in thereceptor-bearing target cell such as effacing lesionsof microvilli, cytokine production, or invasion into orthrough epithelial cells (Svanborg, 1994). Konkel et al.(2000)proposed a model ofC. jejuni binding to hostcells. The process of adherence is reversible, multifac-torial, motility-dependent and adhesins such as CadF(Konkel et al., 1997) and PEB1/CBF1 (Fauchere et al.,1989; Pei et al., 1998) belong to fundamentally syn-thesized products. Results from our studies show thatall 11Campylobacter strains investigated were able toadhere to Caco-2 cells (Table 1). We observed strainvariations in the extent ofCampylobacter adhesion tothe cell line Caco-2 from 0.4 to 12.7% of the initialinoculum. Analysis of these results for the strains with

non-colonizing and weak or delayed colonizing abil-ities in comparison with the strong colonizing strainsdid not reveal any statistically confirmed differencesfor the adhesion behavior of both groups.

The invasion of host mucosal surfaces byC. jejunialso is an essential early step in pathogenesis. Theresults of intestinal biopsies of patients, infected pri-mates, and several other experimental model animalshave demonstrated that gut cell invasion byCampy-lobacter species also occurs in vivo and support theimportance of bacterial invasiveness as a virulencefactor (Hu and Kopecko, 2000). In our study, thepresence ofC. jejuni within Caco-2 cells was clearlydemonstrated by the gentamicin assay, which providesa quantitative method for comparison of invasion fre-quencies among differentCampylobacter strains. Al-though we found that all investigated strains were ableto adhere to the Caco-2 cells some strains were defi-cient in invasion (Table 1). Adherence is most likelya prerequisite for invasion of epithelial cells by anybacterial pathogen. However, the pathogen may bindto an epithelial cell membrane without subsequentinternalization and the number of adherent bacteriadoes not correlate with the number of intracellularbacteria. Therefore, the invasion index is useful when

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comparing invasiveness between similar pathogens.The difference in internalization between the strainssuggests thatCampylobacter entry into eukaryoticcells may be a phenomenon clearly distinct from at-tachment. Others have also reported that the abilityof C. jejuni to invade cultured human epithelial cellsis strain dependent and quite variable in efficiency(Everest et al., 1992; Konkel and Joens, 1989; Newellet al., 1985; Ketley, 1997). Newell et al. (1985)foundthat environmental isolates were much less invasivefor HeLa cells than were clinical isolates.Everestet al. (1992)observed a statistically significant dif-ference in the level of invasion betweenC. jejunistrains from individuals with colitis and those fromindividuals with non-inflammatory diarrhea.

In addition to the investigation of adhesion and in-vasion properties ofC. jejuni in an in vitro assay,we investigated the colonization ability of the 11 se-lected strains in vivo in the chick model. There werelarge differences between theC. jejuni isolates.C. je-juni strains could be grouped into three colonizationtypes (Schulze and Erler, 2002). Four out of 11 iso-lates could not be reisolated (non-colonization type),two isolates caused a weak or delayed colonizationand fiveC. jejuni strains produced a strong, long sus-tained colonization.Korolik et al. (1998)reported sim-ilar findings and also defined three types ofC. jejunistrains. The first type contains the strains that failedto colonize chicks and were classified as transient, thesecond type could colonize but was soon displacedand the third type which is an efficient colonizer andis able to displace other strains present in the chickintestine. Subsequently,Ringoir and Korolik (2003)showed in the chick model five colonization pheno-types forC. jejuni strains isolated from chicken fae-ces and from humans suffering from gastroenteritis.The results of our in vitro and in vivo investigationssuggest that there is a putative correlation of the de-fined three types of colonization in the chick gut withthe invasion ability in Caco-2 cells. Non-colonizersin the chick model were not invasive in the in vitromodel. Strains which colonized weakly or delayed,had a medium invasion index (below 10) and strongcolonizers showed markedly higher values. At present,we cannot give a conclusive explanation for this ob-servation. A correlation between invasion of cell cul-tures and colonization ability was also found in otherinvestigations.Ziprin et al. (2001)studied virulence

genes, includingciaB, which encodes a protein neces-sary for maximum invasion of cultured epithelial cells,andpldA, which is associated with phospholipase ac-tivity. The corresponding defect mutants were unableto colonize the chick gut. A reduction of adherenceand invasion ofC. jejuni mutants in vitro has beenshown to correlate with reduced virulence in vivo. Amotile, non-adherent and non-invasive mutant ofC.jejuni 81–176 generated by a site-specific insertionalmutagenesis was characterized byYao et al. (1997).They demonstrated that the chemotaxis response regu-lator protein (CheY) was responsible for the observednon-adherent and non-invasive phenotype. The mu-tant, although able to colonize mice, was attenuated inthe ferret disease model.Bacon et al. (2000)showedthat a mutation in a plasmid gene (virB11) in C. jejuni81–176 resulted in a statistically significant reductionof adherence and invasion in vitro compared to thewild type. The isogenicvirB11 mutant of this strainalso showed significantly reduced virulence in the fer-ret diarrheal disease model. However, the present datado not specify if the primary defect is in adherence,invasion or other undefined factors.

The role of flagella as putative adhesin inC. jejuniis currently undefined. The flagella may be essential tothe preadherence process by providing motility for thebacteria to approach the host cells and in postbindingevents such as internalization and dissemination fromthe intestinal epithelia (Konkel et al., 2000). The 11C. jejuni strains used in this study were discriminatedby characterization of the flagellin gene. We identifiedeight differentflaA-types in the 11 selected strains. ThesameflaA type was found in a non-colonizing strainas well as in a strong colonizing strain. Each of thenon-colonizing strains had a differentflaA type. Therewas no correlation between colonization, invasion andflaA typing.Chen and Stern (2001)comparedC. jejuniisolates from chicken and human in their colonizationability andflaA type. They could not find a clear cor-relation between colonization and flagellin type alsoby sequencing of the short variable region (SVR) oftheflaA gene.

Our present data from 11C. jejuni strains investi-gated clearly demonstrates the relationship betweenthe invasion in an in vitro model with Caco-2 cellsand colonization in the chick gut. When interpretingour data, it should be considered that the study wasconducted on a low number of strains. Therefore,

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these results are preliminary and further work shouldbe undertaken to substantiate the putative correlationbetween Campylobacter colonization in the chickgut and pathogenicity investigated in the cell culturemodel.

Acknowledgements

We thank Peggy Methner, Waltraud Wilhelm,Sieglinde Drexler and Byrgit Hofmann for excellenttechnical assistance.

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