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Genomic, phenotypic changes of Campylobacter jejuni strains after passage of the chicken gut
I. Hänel, E. Borrmann, J. Müller, W. Müller, B. Pauly, E.M. Liebler-Tenorio, F. Schulze
To cite this version:
I. Hänel, E. Borrmann, J. Müller, W. Müller, B. Pauly, et al.. Genomic, phenotypic changes of Campylobacter jejuni strains after passage of the chicken gut. Veterinary Microbiology, Elsevier, 2009, 136 (1-2), pp.121. �10.1016/j.vetmic.2008.10.018�. �hal-00532525�
Accepted Manuscript
Title: Genomic, phenotypic changes of Campylobacter jejuni strains after passage of the chicken gut
Authors: I. H¨anel, E. Borrmann, J. M¨uller, W. M¨uller, B.
Pauly, E.M. Liebler-Tenorio, F. Schulze
PII: S0378-1135(08)00494-X
DOI: doi:10.1016/j.vetmic.2008.10.018
Reference: VETMIC 4247
To appear in: VETMIC Received date: 27-6-2008 Revised date: 16-10-2008 Accepted date: 21-10-2008
Please cite this article as: H¨anel, I., Borrmann, E., M¨uller, J., M¨uller, W., Pauly, B., Liebler-Tenorio, E.M., Schulze, F., Genomic, phenotypic changes of Campylobacter jejuni strains after passage of the chicken gut, Veterinary Microbiology (2008), doi:10.1016/j.vetmic.2008.10.018
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Accepted Manuscript
Genomic and phenotypic changes of Campylobacter jejunistrains after passage of the
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chicken gut
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I. Hänel1*, E. Borrmann2, J. Müller1, W. Müller1, B. Pauly1, E. M. Liebler-Tenorio2, F. Schulze1
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Friedrich-Loeffler-Institute (1Institute of Bacterial Infections and Zoonoses, Jena, 2Institute of
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Molecular Pathogenesis, Jena,) Naumburger Str. 96a, 07743 Jena Germany
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*Corresponding author. Tel.: +49-3641 804-375; fax: +49-3641 804-228.
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E-mail address: ingrid.haenel@fli.bund.de(I. Hänel)
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Abstract
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The ability to colonize the chicken gut was determined for 17 Campylobacter jejunistrains of
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human and bovine origin. The level of colonization varied according to the strain used for
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experimental infection. Two Campylobacterisolates from patients suffering from
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gastroenteritis were found in the group of non-colonizing strains, suggesting that other
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reservoirs as poultry are also important sources of human Campylobacterinfections. Bovine
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Campylobacter isolates can also effective colonize the chicken intestine and may be a
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source for poultry infection. The invasion ability of the strains as determined in the cell culture
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model using Caco-2 cells correlates with their colonization capacity in the chicken gut. The
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genomic and phenotypic stability of the selected strains were evaluated by analysis of their
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pulsed-field gel electrophoresis (PFGE) patterns, flaA-typing and in vitrodetermination of
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motility, adhesion and invasion abilities after colonizing chickens for up to 21 days. Changes
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were identified in flaA-types of six isolates and three isolates from chicken showed different
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patterns by PFGE using SmaI or KpnI as restriction enzymes. One isolate showed
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phenotypic differences after in vivo passage which were seen in enhancement of adherence
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to eukaryotic cells, decrease of motility and changes in morphology. These phenotypic
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changes were not associated with the observed genomic instabilities.
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Keywords: Campylobacter jejuni, Invasion, Chicken colonization, Typing
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1. Introduction
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The food-borne pathogen Campylobacter (C.) jejuniand, to a smaller extent, the closely
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related C. colihave become recognized as probably the most common cause of sporadic
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bacterial gastroenteritis. Most infections by Campylobacterhave a zoonotic background
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associated with consumption of contaminated animal products or animal contact. Poultry
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products are now widely accepted as a common source of human infection with
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Campylobacter.C. jejunistrains differ markedly in their ability to colonize the chicken gut
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ranging from strains with immediately sustained colonization to completely non-colonizing
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strains (Ringoir and Korolik 2003, Hänel et al., 2004). The differences in colonization types
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may be due to genetic differences, or differences in gene expression of colonization/invasion
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related genes. C. jejuniexhibits several potential virulence properties of which the best
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characterized include flagellum-mediated motility, adhesion and invasion capability (Young et
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al., 2007, Wassenaar and Blaser, 1999). In the chick colonization model could be shown that
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virulence-associated genes such as ciaB, cadF (Ziprin et al., 2001), flhA (Carrillo et al.,
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2004), docA, docB, docC (Hendrixson and DiRita, 2004), and the two-component systems
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racRS (Bras et al., 1999) and dccRS (MacKichan et al., 2004) are involved in colonization of
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the chick gastrointestinal tract by C. jejuni.
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Both phenotypic and genomic changes of C. jejunistrains after experimental infection of
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chickens may occur. An increase of colonization potential for C. jejunistrains after passage
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in vivothrough a chicken was reported (Cawthraw et al., 1996, Ringoir and Korolik, 2003).
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Several reports have described genomic changes of C. jejuniafter passage through chick
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intestine because of genomic rearrangement, insertions, deletions or point mutations using
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pulse-field gel electrophoresis (PFGE) (Hänninen et al. 1999, Wassenaar et al., 1998, de
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Boer et al., 2002). However, there is a lack of information if the genetic instabilities correlates
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with virulence related phenotypic properties such as adhesion or invasion.
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In the present study we investigated the colonization ability of 17 C. jejuniisolates of human
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and bovine origin using the chicken gut model. Further the genomic and phenotypic stability
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of the C. jejunistrains were investigated after passage of the chicken gut. For this purpose
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strains used for inoculation were compared with the corresponding isolates after three weeks
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lasting colonization in the chicken gut with regard to adhesion and invasion in the cell culture
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model using Caco-2 cells and the detection of 23 putative virulence genes by PCR. The
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genomic stability of the strains was studied after passage through chicken intestine by PFGE
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and flagellin gene typing.
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2. Material and methods
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2.1. Bacterial isolates
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17 C. jejuniisolates of human and bovine origin (Table 1) were investigated. The strains
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were characterized by different abilities to adhere to and to invade into Caco-2-cells. All
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strains were cultivated on Müller-Hinton (MH) agar plates supplemented with 10 % bovine
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citrated blood under microaerobic (85 % N2, 10 % CO2, 5 % O2) conditions at 37 oC. In
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investigations of invasion on epithelial cells, the non-invasive E. coliK12 strain was included
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as a negative control. This strain shows a percentage entry in the range of 0.001 % - 0.01 %
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although 21 % of the inoculum was found to adhere to Caco-2 cells.
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2.2. Experiments on colonization of C. jejuniin the chicken gut
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White Leghorn chicks used in this study were hatched from specific pathogen-free eggs
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obtained from the companies Lohmann and Charles River. The groups of animals were
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housed in different, but adjacent rooms under comparable conditions. In each experiment on
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day of hatch chicks were randomly separated into groups of 20 birds each. Chicks were
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reared in cages and fed ad libitum with a commercial starter ration devoid of antibiotic or
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coccidiostat. They were provided with chlorinated drinking water. Cloacal swab samples
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were taken before inoculation to confirm the Campylobacternegative status of the animals at
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the start of the experiment. At 9 days of age, the chickens of a group received approximately
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1 x 108colony forming units (cfu) of one C. jejunistrain via oesophageal gavage. Bacterial
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inoculations were prepared from frozen stock cultures of C. jejuniwhich were thawed,
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inoculated on MH blood agar plates and incubated microaerobically for 48 h at 37 °C.
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Bacteria were harvested and resuspended in sterile phosphate-buffered saline (PBS), pH
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7.4. The suspension was adjusted using spectrophotometry and the mean numbers of cfu
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per ml were determined by performing standard plate counts. 1, 2, 6, 14 and 21 days after
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inoculation, 4 chicks of each group were sacrificed by cervical dislocation and liver and
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faeces were collected for processing. Faecal samples were obtained from intact ceca,
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weighed, homogenized with sterile buffer (stomacher), diluted and inoculated onto CCDA
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medium and modified Skirrow medium supplemented with 30 µg/ml Cefoperazone, 10 µg/ml
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Amphotericin B and 10 % calf blood. The liver samples were processed in the same manner.
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The plates were incubated microaerobically at 37 °C for 48 h, after which time the
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Campylobactercolonies were enumerated. A liquid enrichment based on CCDA medium
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without agar was also used. At the end of each experiment, the colonizing strains were
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reisolated from the ceca of 4 chickens. A plate sweep was recovered from each bird for
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comparison with the corresponding C. jejunistrain used for inoculation.
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The chicken colonization experiments were performed according to the law and approved by
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the ethical committee of Thuringia (04-01/03).
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2.3. Adherence and invasion assay
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Caco-2 cells (ACC 169, German Collection of Microorganisms and Cell Cultures
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Braunschweig) were grown in Dulbecco's Modified Eagle's Medium supplemented with 10 %
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fetal bovine serum (FBS) and 1 % non-essential amino acids without the use of antibiotics at
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37 °C in a 5 % CO2, humidified atmosphere. For the experimental assays, Caco-2 cells were
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grown in 24-well plastic plates. The cells were seeded at 4.5 x 104cells per well and
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incubated for 7 days at 37 °C in a 5 % CO2, humidified atmosphere. Prior the assay, the cell
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monolayers were once washed with PBS, pH 7.2. Adhesion and invasion assays were
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performed as described by Hänel et al. (2004), except that the bacteria were grown 24 h
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before they were harvested from plates. Briefly, for determination of adherent bacteria cells
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infected with approximately 1 x 108cfu/ml were incubated for 3 h at 37 °C in a 5 % CO2,
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humidified atmosphere. In order to quantify bacterial invasion the cells were treated for
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another 2 h with 150 µg/ml gentamicin to kill extracellular bacteria. Adherent or intracellular
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bacteria were evaluated after cell lysis with 1 % Triton X-100 by plating serial dilutions of the
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cell lysates on MH agar plates and by counting the resulting colonies. Significance between
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samples was determined with t test and a Pvalue of < 0.05 was considered significant.
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2.4. Detection of virulence-associated genes by PCR
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DNA preparation from a 24 h culture on MH agar and PCR detection of 18 virulence
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associated genes (flaA, flaB, flhA, flhB, flgB, flgE2, fliM, fliY, ciaB, iamA, virB11, cadF, docA-
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C, cdtA-C) were carried out according to Müller et al. 2006. Additionally, virulence genes
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secD, secF, Cj1470c, Cj1471c, and Cj1474cwere detected using primers and annealing
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temperatures listed in Table 2. For the generation of PCR primers, the Primer Express
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software (Applied Biosystems) was used.
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2.5. Flagellin gene typing and RFLP analysis
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PCR-RFLP profiles of the flaA gene were produced as previously described (Hänel et al.,
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2004) using restriction endonucleases DdeI or AluI (New England Biolabs). Typing and RFLP
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analysis of flaB gene were additionally performed using following primers (Smith et al.,
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1999): FlaB3 (5'-ATA AAC ACC AAC ATC GGT GCA-3') and FlaB4 (5'-GTT ACG TTG ACT
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CAT AGC ATA-3') with the same PCR and restriction conditions such as used forflaA typing.
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2.6. Pulsed-field gel electrophoresis (PFGE)
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Preparation of unsheared DNA for PFGE, digestion using the restriction endonucleases
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SmaI and KpnI (Amersham Biosciences Europe GmbH) and electrophoresis were performed
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as described by CAMPYNET [http://campynet.vetinst.dk/PFGE.html] and Hänel et al. (2007).
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The DNA fragments produced were visualized by ethidium bromide staining.
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A lambda ladder (size range from 48.5 to 727.5 kb, New England Biolabs) used to determine
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fragment size was run on three lanes (both edges and middle) of each gel.
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2.7. Motility assay
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Motility phenotypes of strains were tested in Brucella media containing 0.5 % agar. Bacterial
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cells were harvested from a 24 h culture on MH agar plates incubated at 37 oC under
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microaerophilic conditions into PBS to obtain an optical density at 600 nm of 0.45
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(approximately 1 x 109cfu/ml). 1 µl of a bacterial suspension of approximately 1 x 108cfu/ml
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was stabbed into motility agar. Plates were incubated at 37 oC or 42 oC under microaerophilic
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conditions and the diameter of the resulting swarms was measured after 24 h. Isolates from
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chickens were scored against the motility phenotype of the strain used for inoculation of the
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chickens. The results were the mean of at least six separate determinations of three
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experiments. Significance of differences between samples was determined with t test and a
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Pvalue of < 0.05 was considered significant.
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2.8. Electron microscopy
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For negative staining, 24 h broth-grown cultures of Campylobacterstrains were used. The
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broth was gently swirled, 50 µl of the suspension collected and placed as drop on a glass
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slide. 400-mesh copper grids that had been filmed with formvar, coated with carbon and
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hydrophilisated by glow discharge were floated for 10 min on the suspension. Then grids
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were removed and excess liquid drained with filter paper. Finally grids were contrasted on a
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drop of 2% phosphotungstic acid for 10 minutes. After drying, grids were examined in a
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transmission electron microscope (Tecnai 12, FEI, Netherlands).
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3. Results
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3.1. Colonization of C. jejuniin the chicken gut
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The colonization potential in the chicken gut was assessed for 17 C. jejunistrains of human
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or bovine origin with different abilities to adhere to and to invade into Caco-2 cells. None of
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the control chickens was colonized with Campylobacterat the end of experiment. The
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colonization ability of the strains in 9-day-old chickens differed in a wide range (Table 1). The
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colonization potential was assessed by means of the following criteria: first detection after
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inoculation, number of C. jejuniper gram of sample, duration of excretion (up to the end of
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experiment at three weeks after inoculation), number of positive animals out of the group at
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different investigation times, tendency to invasion of the liver. According to these criteria the
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isolates were divided into the following three groups: (1) No colonization - the isolates could
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not be reisolated. According to this criterion three strains (128/94, 129/94, 972 dJ) were
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arranged in this group. (2) Weak or delayed colonization - the strains only achieved low
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counts per gram caecal content and were intermittently excreted (not all animals positive at
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different investigations). Two strains (292/94, 315/94) corresponded to these criteria. They
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showed a delayed colonization and strain 292/94 could not be reisolated at the end of
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experiment. (3) Strong colonization - these strains caused a strong colonization up to the end
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of experiment at three weeks after inoculation. According to these characteristic features, 12
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strains (167/96, 44/96, 170/96, 288/94, 320/94, 305/94, 158/96, 277/94, 164/96, 165/96,
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163/96, 216/04) were assigned to this group. Already two days after inoculation, the first six
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strains achieved more than 108cfu per gram of ceacal content. Six days after inoculation, the
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other strains were detectable in high concentrations in this organ and all strains remained
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detectable in these high concentrations up to the end of experiment. Although some C. jejuni
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could be reisolated from the liver, the tendency to invade the liver showed no clear
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correlation to the concentration of the pathogen in caeca.
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3.2. Adhesion and invasion abilities of C. jejunistrains
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Adherence and invasion studies were carried out with monolayers of Caco-2 cells. All C.
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jejunistrains examined in the chicken colonization model adhered to Caco-2-cells at a range
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of 0.15 - 2.03 % of the starting viable inoculum (Table 1). The invasiveness of
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Campylobacterstrains into Caco-2 cells was found to be in a wide range of 0.0013 % - 0.75
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% of the starting bacterial inoculum. E. colistrain K12 was used as non-invasive control
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strain in the invasion assay showing 0.001-0.01 % entry into Caco-2 cells. In comparison
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with the non-invasive control-strain we found three of the tested strains (128/94, 129/94 and
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972dJ) which were not invasive for Caco-2 cells. Two strains (292/94 and 315/94) showed a
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minor enhancement of entry into the cells compared to the non-invasive control strain. All
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other Campylobacterstrains included in the study were considered to be invasive, having at
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least 0.1 % intracellular bacteria of the inoculum which survived gentamicin treatment (Table
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1). There was no correlation between adhesion to Caco-2 cells and the colonization groups
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in the chicken model. In contrast a correlation was observed between invasion and
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colonization as all good colonizers invaded Caco-2 cells whereas weak and no colonizers did
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not invade into the cells. A measure of invasiveness is the invasion index, which is the
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number of invaded bacteria as a percentage of the number of adhered bacteria. Hence, the
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invasion index reflects the organism’s ability to adhere to a cell and how likely it is that that
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interaction will lead to internalization. It is a useful parameter comparing invasiveness
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between similar pathogens (Elsinghorst, 1994, Hänel et al., 2004). The invasion index
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formed the basis for the differentiation of the strains into three groups. Strains which were
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characterized by an invasion index below 1 % (128/94, 129/94 and 972dJ) could not be
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reisolated from the chicks. Two strains (292/94 and 315/94) causing a weak or delayed
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colonization were characterized by an invasion index about ten (9.7 % and 10.6 %
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respectively). For most of the strains we determined an invasion index over 20 %. These
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strains showed a strong, 21 days lasting colonization in chickens.
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Adhesion and invasion differences between strains used for inoculation of chickens and the
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isolates from the chickens were not seen except for strain C. jejuni305/94. The results for
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this strain, shown in Fig. 1a, indicated that the isolates from chickens adhered to Caco-2
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cells at significantly (P< 0.05) higher levels compared to the strain used for inoculation. The
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effect on invasion of Caco-2 cells was more modest and not significant. The invasion index
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(Fig.1b), was decreased (below 10 %) in comparison to the inoculation strain (31.5 %).
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3.3. Genotyping
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Described virulence-associated genes (flaA, flaB, flhA, flhB, flgB, flgE2, fliM, fliY, ciaB, iamA,
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virB11, cadF, docA-C, cdtA-C, secD, secF, Cj1470c, Cj1471c, and Cj1474c) were
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investigated by PCR in C. jejunistrains used for inoculation and in the reisolated strains of 4
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chickens (chicken 17 – chicken 20) 21 days after inoculation. Except of differences in flaA-
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types we could not identify any differences of the virulence-associated genes between the
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strain used for inoculation of chickens and reisolated strains of each isolate. Additionally,
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there were no differences in the detection of the selected genes among the three
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colonization and invasion groups of isolates.
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Plate sweeps taken from ceacal isolates recovered at 21 days post inoculation showed
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different flaA-types for six of 17 tested isolates (158/96, 163/96,164/96, 165/96, 216/04 and
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305/94) of at least for one chicken in comparison to the inoculation strain (Fig. 2). In the
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cases of strains 163/96 and 165/96 the isolates of all four chickens had a different flaA-type
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compared to the inoculum strain but the same type among themselves. Plate sweeps of one
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chicken for strains 158/96 and 305/94 and plate sweeps of two chickens for strains 164/96
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and 216/04 showed different flaA-types in comparison to the inoculation strain (Fig. 2). In the
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case of 216/04 the two different reisolated strains (216/04-chicken 17 and 216/04-chicken
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20) had not the same flaA-type. Furthermore, we were not able to detect a flaA PCR product
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in strains 158/96 and 165/96 after reisolation from two chickens.
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The genetic variability of Campylobacterstrains throughout a passage through chicken
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intestine was further investigated by means of PFGE. The genotypes of 14 strains remained
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unchanged after passage through chicken intestines and showed identical patterns by PFGE
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using SmaI and KpnI as restriction enzymes. For three strains (315/94, 288/94, 305/94)
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changed PFGE types were detected in individual chickens indicating that DNA
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rearrangement had occurred in these strains (Fig. 3). The inoculum of strain 315/94 and
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three isolates from chickens showed identical patterns for SmaI, while for the fourth isolate
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(chicken 18) one band at 425.0 kb was missing. For this isolate with novel SmaI PFGE type
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also a distinctKpnI PFGE genotype was obtained. Using the restriction enzyme SmaI, two
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isolates from strain 288/94 showed the same patterns as the inoculum, while the isolates of
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chicken 18 and 20 differed in their SmaI profiles. However, all isolates showed four bands in
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the range between 97.0 and 194.0 kb. UsingKpnI as restriction enzyme, all isolates
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exhibited identical patterns. On the other hand, strain 305/94 showed identical PFGE pattern
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for inoculum and the corresponding chicken isolates usingSmaI, but differences in the
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fingerprints for KpnI for one chicken (305/94-chicken 20).FlaA-typing of these strains with
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changed PFGE types in individual chickens showed no differences in comparison to the
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strains inoculated, demonstrating that observed PFGE changes did not involve this known
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variable region. However, strains that varied in flaA-types had constant PFGE patterns.
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3.4. Motility assays
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The motility phenotypes ofCampylobacterstrains before and after passage through the
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chicken were determined for strains showing alterations in the PFGE pattern or flaA-type.
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Despite absence of genomic changes for strains 305/94-chicken 17 and 19, all isolates from
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chickens infected withCampylobacterstrain 305/94 displayed significantly (P < 0.05)
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reduced motility in Brucella motility agar comparing to the strain used for inoculation (Fig. 4).
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Interestingly, the increase of incubation temperature up to 42 oC resulted in an increase of
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motility only of the inoculum strain. The analysis of growth zones for strains 158/96, 163/96,
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164/96, 165/96, 216/04, 288/94 and 315/94 as well as the corresponding chicken isolates
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showed no differences between the strain used for inoculation and the corresponding
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isolates at 37oC and an enhancement of motility at 42 oC for all strains.
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3.5. Morphology
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Due to the decrease of motility of the chicken isolates from strain 305/94 we assumed a
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possible alteration of the flagella. Strain C. jejuni305/94 used for inoculation into chickens
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and the isolates had one long flagella at each end (Fig. 5) and the isolates were comparable
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in diameter and length. Otherwise the original strain C. jejuni305/94 had the expected spiral
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shape (Fig. 5a, b), but surprisingly all isolates from the chickens presented as straight rods
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(Fig. 5 c, d).
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4. Discussion
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Results from the present study demonstrated that colonization of nine days old chickens with
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C. jejunivaries according to the Campylobacterstrain used. Furthermore, we confirmed
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results of previous investigations (Hänel et al., 2004) suggesting a relationship between
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invasion of C. jejuniin Caco-2 cells and their colonization ability in the chicken gut. Three
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colonization phenotypes were identified ranging from non-colonizing strains to strains
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producing a strong and sustained colonization. Only Campylobacterstrains with strong ability
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to colonize the gut of chickens were found to be invasive in the Caco-2 cell culture model.
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Such differences in colonization of the chick gut may be related to differences in the source
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of isolation (Ringoir and Korolik, 2003). The strains in our study were selected from a
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collection of isolates of human and bovine origin. Campylobacterstrains previously isolated
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from faeces of humans suffering from gastroenteritis were found in the group of non-
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colonizers as well as in the group of isolates successful colonizing chickens (Table 1). Others
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(Korolik et al., 1998, Ringoir and Korolik, 2003) showed that strains isolated from patients
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were less successful in colonizing chickens in comparison with C. jejunistrains isolated from
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chicken faeces. Our results support the opinion that other reservoirs as poultry could be
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important sources of human Campylobacterinfections. Up to now there are only few studies
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of the possible importance of non-poultry sources of human C. jejuniinfections (Siemer,
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2004, Wilson et al. 2008). As shown, bovine Campylobacterisolates can also effective
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colonize the chicken intestine. The importance of cattle as reservoir and potential source of
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Campylobacterinfections in poultry production has not been investigated in detail. Our
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results suggest that bovine Campylobacterstrains may be a source of poultry colonization.
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Campylobacterstrains are considered to have a high frequency of intragenomic
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recombination and other events that can change the pheno- and genotypes (Hänninen et al.,
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1999, Wassenaar et al., 1998, Ridley et al., 2008). Genomic instability has been
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demonstrated to affect both flaAtyping (Harrington et al., 1997) and PFGE (Wassenaar et
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al., 1998, Hänninen et al., 1999). The occurrence of genomic instabilities suggests that there
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are, as yet unknown, selective benefits to such novel subpopulatins (Ridley et al., 2008). Up
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to now, phenotypic changes followed by genomic alterations were not investigated. In the
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present study the genomic and phenotypic stability of C. jejunistrains colonizing the chicken
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gut were studied. We were able to show that novel PFGE genotypes and flaA-types can be
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formed and phenotypic changes occur during a three weeks lasting passage through the
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chicken gut. We also determined a constant bacterial genetic fingerprint by PFGE for 14 and
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by flaA-typing for 11 of 17 Campylobacterstrains at the beginning and the end of
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colonization in chickens. The changes in PFGE profiles seem to be caused by various
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mechanisms, as we determined enzyme specific changes and changed PFGE patterns for
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both enzymes tested. Only strain 315/94 showing a delayed colonization was found to
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change the PFGE type for two restriction enzymes in one chicken (C-18) indicating a large
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genome rearrangement. These genomic changes were not linked with the determined flaA-
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type or changes in adherence, invasion or motility. Any other observed changes in the PFGE
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patterns for the various chicken isolates have never correlated with alterations in phenotypic
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properties like motility, adherence or invasion and did not affect the flaA-gene as indicated by
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determination of a constantflaA-type of inoculation strains and chicken isolates throughout.
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FlaA-type changes detected in one or two of four identical infected chickens with four strains
322
(158/96, 164/96, 216/04, 305/94), suggesting randomly distributed events of nucleotide
323
polymorphism, could possibly induced by the chicken gut environment. The identical change
324
of the flaA-type in isolates from all four chickens (163/96) or from three chickens (165/96)
325
compared to the corresponding inoculation strains could indicate a specific influence of the
326
chicken passage. The detected variations in flaA-types did not cause changes in adherence,
327
invasion or motility of the reisolated strains.
328
Phenotypic changes of the strains in comparison to the respective chicken isolates could be
329
observed only with the invasive strain C. jejuni305/94 where all isolates from the chickens
330
exhibit dramatic differences in morphology, motility and their adhesion ability. The isolates
331
from all four chickens adhered to Caco-2 cells at significantly higher numbers than the
332
inoculation strain. We determined an invasion index below 10 %, showing that the increase
333
Accepted Manuscript
of binding to the epithelial cells was not associated with a subsequent increase of
334
internalization. These results indicated that internalization of adherent bacteria is impaired.
335
While C. jejuni305/94 showed the expected spiral shape with two flagella, one at each end,
336
the isolates from the chickens lost this shape and appeared as a straight rod with two polar
337
flagella. The reisolated strains were significantly impaired in their motility. The results of
338
genotyping analysis of strain 305/94 indicated that no causal relationship exists between the
339
determined genomic changes for several chicken isolates and the uniform changes of
340
phenotypic properties for all isolates after chicken passage. It is possible that the phenotypic
341
differences caused by other more subtle genetic changes detectable only by whole-genome
342
approaches. Gaynor et al. (2004) reported that the genome-sequenced variant of C. jejuni
343
NCTC 11168 and the original clinical isolate from which it was derived exhibit dramatic
344
differences in numerous virulence-associated phenotypes, including colonization, invasion,
345
translocation, and motility. Despite these differences, these clonally derived strains appear
346
indistinguishable by molecular genotyping techniques like AFLP and PFGE. Using
347
sequencing they were able to show that genome-sequenced variant of C. jejuniNCTC 11168
348
contains very subtle genetic changes, which clearly engender marked differences in
349
transcription and phenotype.
350
The flagella of Campylobacterare essential for motility and virulence. Recombinations,
351
deletions and duplications between flagellin gene sequences of Campylobacterhave been
352
demonstrated in chickens (Nuijten et al., 2000). They found that during colonization of the
353
chicken intestine a nonmotile flaAmutant of C. jejuniunderwent rearrangements within its
354
flagellin locus, thereby regaining its motility and colonization capacity. In contrast to these
355
findings we identified loss of motility of the strain 305/94 during colonization of the chicken
356
gut. The most striking finding in this connection was the change in morphology of this strain
357
from a spiral shape to a straight rod without loss of the flagella. Because in addition to the
358
flagella the spiral morphology of Campylobacteris one important factor in the organism’s
359
ability to move (Shigematsu et al., 1998), the morphological change to a straight rod is likely
360
the reason for the observed reduction of motility. Furthermore, some reports describe
361
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flagella as adhesins while others describe flagella as factor for Campylobacterpenetration of
362
host epithelial cells (Grant et al., 1993, Guerry, 2007). These functions of flagella may be
363
linked with the determined changes in in vitroadhesion and invasion properties of the
364
chicken isolates of strain 305/94. Further investigations are required to elucidate how and
365
why the observed phenomenon occurs.
366
Genetic changes, resulting in small differences in restriction patterns were demonstrated
367
during the course of colonization of chickens (Hänninen et al., 1999, Wassenaar et al.,
368
1998), of mice (Mixter et al., 2003) and also during the course of a human infection
369
(Steinbrückner et al., 2001). Otherwise the genetic stability of four C. jejunistrains after
370
gastrointestinal passage through experimentally colonized chickens after seven days was
371
shown using LPS genes (LG) genotyping (Knudson et al., 2005). Nielsen et al. (2001) could
372
show that many Campylobacterstrains are indeed genetically stable during in vitroand mice
373
passage according to a spectrum of typing methods. Our study confirms these results
374
showing that during intestinal colonization in the chicken gut behind apparently genomic
375
stable C. jejunistrains genomic rearrangement may occur. These genomic instabilities were
376
dependent on the individual bird, suggesting the presence of host-specific factors. However,
377
the observed genetic changes were not followed by examined phenotypic changes as in vitro
378
adhesion or invasion. Our data suggest that although there is increasing evidence that
379
genetic recombination can occur in Campylobacterduringin vivopassage, the subtype
380
pattern variations, leading to phenotypic changes, were not common events and occur only
381
occasionally.
382 383 384
Acknowledgements
385
386
We thank Peggy Methner, Waltraud Wilhelm, Susann Bahrmann, Byrgit Hofmann, Christine
387
Muselmann, Sabine Lied and Wolfram Maginot for excellent technical assistance.
388
389
Accepted Manuscript
References
390
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Figure captions
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Fig. 1Adhesion and invasion of C. jejuni305/94 and the corresponding chicken isolates
482
(305/94-17 – 305/94-20), a – adhesion and invasion, b – invasion index
483
484
Fig. 2FlaA-types of C. jejunistrains used for inoculation and isolates after chicken passage;
485
St – 100 bp ladder, WT – inoculation strain, C17-C20 – corresponding chicken isolates, CS –
486
control strain
487
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Fig. 3PFGE patterns of C. jejunistrains used for inoculation and isolates after chicken
489
passage showing changed genotypes for individual chickens; St - molecular size ladder; WT
490
– inoculation strain, C17-C20 – corresponding chicken isolates
491
492
Fig. 4Motility of strain C. jejuni305/94 used for inoculation and corresponding isolates
493
observed after 21 days of chicken passage (305/94-17 – 305/94-20; black column – motility
494
at 37 oC, grey column – motility at 42 oC
495
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Fig. 5Morphology of C. jejuni305/94 and representative morphology of corresponding
497
Campylobacter isolates after chicken passage, a – inoculation strain characterized by a
498
spiral shape, b – one long flagella is present at each end of the spiral bacterium, c – all
499
reisolated bacteria appear as straight rods, b – one long flagella is present at each end of the
500
straight bacterium
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502
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Figure 1
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Figure 2
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Figure 3
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Figure 4
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Figure 5
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Table 1
Colonization ability in the chicken gut, adherence to and invasion of Caco-2 cells
Colonization group
Strain Origin of isolate % of inoculum adhered
% of inoculum internalized
Invasion index
Non-colonizing strains
128/94 Human enteritis 0.5 0.0013 0.17
129/94 Human enteritis 0.67 0.004 0.67
972dJ Calf intestine 0.62 0.0025 0.46
Strains with weak or delayed
colonization
292/94 Dairy cow 0.2 0.019 9.7
315/94 Dairy cow 0.15 0.016 10.6
Strains with strong colonization
ability
277/94 Dairy cow 2.03 0.75 37.8
288/94 Dairy cow 0.94 0.2 21.1
320/94 Dairy cow 1.7 0.52 30.3
305/94 Dairy cow 0.87 0.28 31.5
158/96 Human enteritis 1.92 0.52 26.7
167/96 Human enteritis 1.76 0.4 22.8
44/96 Human enteritis 1.26 0.3 23.5
170/96 Human enteritis 1.22 0.32 27.3
164/96 Human enteritis 1.4 0.58 41.8
165/96 Human enteritis 0.56 0.19 32.9
163/96 Human enteritis 0.65 0.13 20.7
216/04 Human enteritis 0.54 0.14 26.3
Accepted Manuscript
Table 2
List of target genes, PCR primers, and annealing temperatures
Target gene/
ORF
Primers Sequence Annealing
tempera- ture (°C)
Ampli- con lenght
(bp)
Reference
secD CjsecD11 CjsecD22
5'-CCC AAA GTC ACA GCA AAA CC-3' 5'-GCA GTC AGA ACG TGG TGC GAA G-3'
55°C 1371 This study
secF CjsecF11 CjsecF22
5'-TCA TAC ATT GCA CGA TTG CG-3' 5'-ATG AGA ATG CGT TTT GCT GC-3'
50°C 920 This study
Cj1470c Cj1470cF1 Cj1470cF2
5'-CCT GAG CTA AGC TCC CAC ATA G-3' 5'-CAG CAC AAG CAA AAG CCT TAT C-3'
50°C 1089 This study
Cj1471c Cj1471cE1 Cj1471cE2
5'-GAT GAC AAC GCT CAC ATC CT-3' 5'-GAC GGC TTT ATG ATT ATT AG-3'
50°C 1129 This study
Cj1474c Cj1474cD1 Cj1474cD2
5'-GGA GCA TTT ACG CTA TCT AC-3' 5'-GCC ATT ATC TTT ATG CTT TA-3'
45°C 1342 This study
