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Infection and Immunity, July 2004, p. 3769-3776, Vol. 72, No. 7
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.7.3769-3776.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Adaptation of Campylobacter jejuni NCTC11168 to High-Level Colonization of the Avian Gastrointestinal Tract

Michael A. Jones,1* Kerrie L. Marston,1 Claire A. Woodall,2 Duncan J. Maskell,2 Dennis Linton,3 Andrey V. Karlyshev,4 Nick Dorrell,4 Brendan W. Wren,4 and Paul A. Barrow1

Institute for Animal Health, Compton, Newbury Berkshire RG20 7NN,1 Centre for Veterinary Sciences, University of Cambridge, Cambridge CB3 0ES,2 School of Biological Sciences, University of Manchester, Manchester M13 9PT,3 London School of Hygiene and Tropical Medicine, London WC1A 7HT, United Kingdom4

Received 14 October 2003/ Returned for modification 28 January 2004/ Accepted 31 March 2004


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The genome sequence of the human pathogen Campylobacter jejuni NCTC11168 has been determined recently, but studies on colonization and persistence in chickens have been limited due to reports that this strain is a poor colonizer. Experimental colonization and persistence studies were carried out with C. jejuni NCTC11168 by using 2-week-old Light Sussex chickens possessing an acquired natural gut flora. After inoculation, NCTC11168 initially colonized the intestine poorly. However, after 5 weeks we observed adaptation to high-level colonization, which was maintained after in vitro passage. The adapted strain exhibited greatly increased motility. A second strain, C. jejuni 11168H, which had been selected under in vitro conditions for increased motility (A. V. Karlyshev, D. Linton, N. A. Gregson, and B. W. Wren, Microbiology 148:473-480, 2002), also showed high-level intestinal colonization. The levels of colonization were equivalent to those of six other strains, assessed under the same conditions. There were four mutations in C. jejuni 11168H that reduced colonization; maf5, flaA (motility and flagellation), and kpsM (capsule deficiency) eliminated colonization, whereas pglH (general glycosylation system deficient) reduced but did not eliminate colonization. This study showed that there was colonization of the avian intestinal tract by a Campylobacter strain having a known genome sequence, and it provides a model for colonization and persistence studies with specific mutations.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Campylobacter jejuni is currently the most common single causative agent of bacterial food poisoning in the developed world, and there are up to 55,000 notified cases per annum in the United Kingdom (41); however, this is a gross underestimate of the true disease burden. One of the most important reservoirs of this organism is the intestinal tract of domestic poultry. Several approaches have been employed to reduce the incidence of Campylobacter in commercial poultry production. The introduction of critical control point procedures and trials with competitive exclusion preparations have had little effect on the incidence of flock infection (2, 14, 25, 39). The increase in production of free-range poultry suggests that the incidence of infection is likely to continue to rise. Because of economic and other constraints on improvements in slaughterhouse hygiene, other rational approaches to the biological control of intestinal infection during rearing are therefore being sought. These approaches include vaccination, for which an understanding of the mechanism of intestinal persistence and the bacterial factors involved is desirable.

Chickens in the field are generally colonized with C. jejuni when they are around 2 weeks old (39), and while a number of workers have used 1-day-old chicks as a model for colonization, consideration of the microbiological and physiological differences at these two ages suggests that there is a rational basis for using older birds. During the first 2 weeks after hatching, major physiological changes occur in the intestinal tract, which reflect the changes in the gut flora and the nutritional status of the bird, as early reliance on the yolk sac is replaced by high-protein-content feed. A key factor in colonization is likely to be competition with the natural gut flora. The gut flora in a 1-day-old chick is rudimentary, is still developing, and does not reflect the complexity of the flora present in older birds (44). Furthermore, the period of study with chicks has tended to be short and has not allowed long-term persistence to be assessed (1, 10, 33, 38). There is also evidence that the colonization by bacteria may be less efficient in older birds than in young birds. For example O-antigen mutants of Salmonella, which colonize young birds, are cleared rapidly from older birds (7, 43). The adaptive immune system starts maturing about 1 week before hatching (35) and continually matures during the life of the bird (23). These factors have led us to examine colonization in longer-term studies with 2-week-old birds, which have an established gut flora.

Although C. jejuni NCTC11168 was chosen for genome sequencing (31), it has been described as a poor colonizer or noncolonizer of young chickens (1, 33). In this report we describe in vivo selection of C. jejuni NCTC11168 for increased colonization of the avian gastrointestinal tract. We also show that hypermotile derivatives of C. jejuni NCTC11168 selected in vitro (18) colonize the gastrointestinal tract of 2-week-old birds rapidly. Thus, C. jejuni NCTC11168 has the genes required for colonization of the avian gastrointestinal tract, and changes that increase its motility also make it colonization proficient. We used mutants of the hypermotile strain defective in motility, encapsulation, and protein glycosylation to show that there is time-dependent clearance of noncolonizing mutants and that there is long-term low-level persistence in groups of birds, which validated this model as a model for both colonization and persistence of Campylobacter in the avian intestinal tract.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Bacterial strains and culture conditions. C. jejuni NCTC11168 (31) and NCTC11828 (30) have been described previously. Isolation of the hypermotile variant C. jejuni 11168H was described by Karlyshev et al. (18). C. jejuni strains 49S and 35R and Campylobacter coli DR4 are natural isolates obtained from avian neck swabs and carcass rinses and were a gift from F. Jørgensen and T. Humphrey, University of Bristol. C. jejuni 11168H::kpsM is acapsulate (17); and C. jejuni 11168H::pglH is defective in glycosylation, and construction of the mutation has been described previously (21). Strain G1 was isolated from a patient who went on to develop Guillain-Barré syndrome (22). C. jejuni 81-176 was isolated from an outbreak associated with unpasteurized milk (20).

Mutant 11168H flaA::kan was constructed for this study by using plasmid cam157g10, which is a 1.3-kb pUC18 shotgun clone that was constructed as part of the genome sequencing project and contains a fragment of the flaA gene (cj1339) (http://www.sanger.ac.uk/Projects/C_jejuni/). The Kanr cassette from plasmid pJMK30 (45) was cloned in a nonpolar orientation into the SwaI site at bp 59 of flaA. This construct was electroporated into C. jejuni 11168H, and Kanr colonies resulting from allelic replacement were selected.

Mutant 11168H maf5::kan was constructed by using plasmid cam151b9, which contains a fragment of the maf5 gene (cj1337) in a 2-kb insertion (http://www.sanger.ac.uk/Projects/C_jejuni/). The Kanr cassette was inserted into an EcoRV site at bp 156 of maf5. The maf5::kan construct was electroporated into C. jejuni 11168H, and Kanr colonies resulting from allelic replacement were selected.

Bacterial strains were cultured on sheep blood agar (Oxoid, Basingstoke, United Kingdom). Motility assays were carried out on Mueller-Hinton plates (Oxoid) containing 0.5% agar. Cultures used for inoculation were routinely incubated for 24 h in 10 ml of Mueller-Hinton broth. All cultures and enrichments were incubated under standard microaerobic conditions (5% oxygen, 10% carbon dioxide, 85% nitrogen) at 37°C.

Campylobacter blood-free selective plates were prepared according to the manufacturer's instructions from Campylobacter blood-free selective agar (CCDA; CM739; Oxoid) and CCDA selective supplement (SR155; Oxoid). Enrichment of swab samples was carried out in modified Exeter enrichment broth (6).

Experimental animals. Specific-pathogen-free (SPF) Light Sussex chickens were produced at the Institute for Animal Health. Chickens were inoculated orally on the day of hatching with 0.1 ml of Campylobacter-free adult gut flora preparations. To do this, 1 g of cecal contents was taken from a 50-week-old SPF chicken immediately after the bird had been killed and was used to inoculate 10 ml of Luria-Bertani broth, which was then incubated for 24 h at 37°C. Inoculated birds were housed in separate rooms in a high-biosecurity facility until they were 2 weeks old, after which they were used in colonization trials. The birds were fed a vegetable-based diet (Special Diet Services, Manea, Cambridgeshire, United Kingdom) ad libitum.

Colonization trials. Groups of 20 2-week-old birds with a developed gut flora were inoculated orally with 0.1 ml of a Mueller-Hinton broth culture containing log10 7.0 CFU of the desired Campylobacter strain per bird. Cloacae were sampled at weekly intervals with sterile cotton-wool swabs, and fecal excretion was assessed semiquantitatively by using a standard method for large groups of birds housed together (37). The cloacal swabs were mixed in 1 ml of modified Exeter broth and plated in a standard manner on Campylobacter blood-free selective agar before they were incubated in a microaerobic atmosphere at 37°C for 48 h, at which point colony counts were estimated; the counts obtained were referred to as direct counts. The swabs were also incubated for 48 h in the remaining enrichment broth at 37°C and then plated on Campylobacter blood-free selective agar and scored for the presence of Campylobacter; the resulting counts were referred to as enrichment counts. At the end of the trials five birds were randomly chosen from each group and used postmortem to determine the number of bacteria in the intestinal contents at several points along the alimentary tract. Decimal dilutions of the intestinal contents were made in phosphate-buffered saline (PBS) and plated on Campylobacter blood-free agar to obtain the number of Campylobacter cells per gram of tissue or intestinal contents. The plates were incubated microaerobically for 48 h. Tissue samples were homogenized with PBS in Griffith's tubes. The volume of PBS was made up to 1 ml per g of tissue, and decimal dilution and plating were carried out as described above for intestinal contents.

PFGE. Pulsed-field gel electrophoresis (PFGE) was carried out in 0.7% agarose gels with 0.5% Tris-borate-EDTA buffer. The gels were electrophoresed at 6 V/cm with a switch time of 1 to 6 s for 12 h at 14°C. DNA plugs were generated as described by Gibson et al. (12), and restriction profiles were generated by using the enzyme KpnI (24).

Serology. Serology testing was carried out as described by Penner et al. (32).

DNA microarray hybridization. The currently available annotation of the NCTC11168 genome contains 1,654 annotated C. jejuni open reading frames (ORFs) (http://www.sanger.ac.uk/Projects/C_jejuni/). Primer pairs for each ORF were designed with the Primer 3 software and were selected by BLAST analysis to have minimal cross-homology with all other ORFs (16). A PCR product representing each ORF was amplified from C. jejuni NCTC11168 chromosomal DNA and spotted onto CMT-GAPS II-coated glass slides (Corning Glass Works, Corning, N.Y.) by using a MicroGrid II microgrid robot (BioRobotics, Cambridge, United Kingdom). All of the procedures used, including postprocessing of deposited arrays, have been described previously (15).

Microarray slides were incubated in a prehybridization buffer (3.5x SSC buffer, 0.1% sodium dodecyl sulfate [SDS], 10 mg of bovine serum albumin per ml [1x SSC is 0.15 M NaCl plus 0.015 M sodium citrate]) at 65°C for 20 min. After prehybridization, the slides were washed for 1 min in distilled water and then for 1 min in isopropanol. Cy3- and Cy5-labeled DNA from the control and test genomic DNA samples, respectively, were pooled and purified with a QIAGEN MinElute PCR purification kit by using a two-step washing procedure with 500 µl and then 250 µl of buffer PE and elution of the labeled DNA from the MinElute column with 14 µl of H2O. The columns retained approximately 1 µl, so the final eluted volume was ~13 µl; the volume of the eluate was adjusted to 30 µl by using 4x (final concentration) SSC buffer and 0.3% (final concentration) SDS. The hybridization mixture was denatured at 95°C for 2 min, cooled slowly to room temperature, applied to the microarray, and covered with a LifterSlip coverslip (22 by 25 mm; Erie Scientific Company, Portsmouth, N.H). The slide was placed in a waterproof hybridization chamber (CMT hybridization chamber; Corning, High Wycombe, United Kingdom) for hybridization in a 65°C water bath overnight. After hybridization, the slide was washed in 1x SSC buffer with 0.06% SDS at 65°C for 5 min and then twice in 0.06x SSC buffer for 2 min at room temperature.

Each C. jejuni NCTC11168 gene was represented on the array by duplicate reporter elements. Two microarray hybridizations (technical replicates) were performed for each test sample of genomic DNA in order to compare the gene content with that of sequenced strain NCTC11168.

Data acquisition and analysis. Slides were scanned with an Affymetrix 418 scanner (MWG Biotech) by following the manufacturer's guidelines. Fluorescent spot intensities were quantified by using the ImaGene 5.5 software (BioDiscovery Inc., Los Angeles, Calif.). For each spot, the background fluorescence was subtracted from the average spot fluorescence to produce a channel-specific value. The data were further analyzed by using the GeneSpring 6.1 software (Silicon Genetics, Redwood City, Calif.). The geometric mean of the normalized red/green ratio was calculated for each strain by using data from two array experiments. Spots were excluded if they were flagged by the ImaGene 5.5 software as ABSENT or UNKNOWN. A nominal cutoff for a signal ratio of 0.5 was used to highlight genes that may have been absent or highly divergent (11). To determine which genes were statistically absent or highly divergent, a P value of <0.01 was used to determine whether the normalized signal intensity for each gene in each strain was statistically different from 1.0 by employing a two-sided one-sample t test with the GeneSpring 6.1 software.

Motility test. Motility was assessed in two ways, by growth on Mueller-Hinton agar containing 0.5% agar and by phase-contrast microscopy (Eclipse E400; Nikon). The strains to be tested were grown for 18 h on sheep blood agar, and colonies were picked by using sterile pipette tips and spotted into the agar. Motility was scored visually based on the ability of colonies of a strain to extend through the agar from the point of inoculation. The microscopic assessment of motility was based on a positive score when more than 90% of the bacteria in the field of view showed darting movement.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Adaptation of C. jejuni NCTC11168 to high-level colonization. The abilities of C. jejuni NCTC11168 and NCTC11828 (10) to colonize chickens were compared. NCTC11168 colonized with low efficiency soon after infection, and less than 50% of the chickens were positive at any one time, compared to 80% of the chickens inoculated with NCTC11828 (Fig. 1A and B). The level of colonization by NCTC11168 fluctuated within the group inoculated during the first 5 weeks of infection (Fig. 2), and this strain remained detectable by the enrichment culture method only in less than 50% of the group. However, after this time the excretion rate was consistently high (Fig. 1A), and 85 to 100% of the chickens excreted high levels of Campylobacter for the duration of the experiment (8 weeks). The trial was repeated four times, and the lag in the time necessary for adaptation varied between 2 and 5 weeks (median, 4 weeks). The levels of Campylobacter obtained from pooled cecal and fecal droppings were assessed. When enrichment was required for detection of Campylobacter on swabs, the fecal counts were below detectable limits (<100 CFU/g), whereas when direct counts were obtained from swabs, the values correlated with a significant increase in the counts from droppings in the group (>log10 4.0 CFU/g) (data not shown). These data correlated with the data from postmortem studies carried out in one trial, in which birds with negative swab scores were negative for direct cecal counts, whereas birds that were positive as determined by enrichment only had counts up to log10 4.0 CFU/g and birds with direct counts from swabs had counts greater than log10 4.0 CFU/g.



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  		FIG. 1. Profiles of C. jejuni colonization. (A) C. jejuni NCTC11168, unadapted. (B) C. jejuni NCTC11828. (C) C. jejuni PASS67. (D) C. jejuni 11168H. The dark grey bars indicate the percentage of each group with direct counts of Campylobacter, and the light grey bars indicate the percentage of each group in which the presence of Campylobacter was determined only upon enrichment. Birds were inoculated when they were 2 weeks old. Swab samples were taken at weekly intervals.

 


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  		FIG. 2. Colonization of 20 birds with C. jejuni NCTC11168. The birds were identified by their wing band numbers (3201 to 3220). The birds were infected when they were 2 weeks old, and swab data were taken each week. The open boxes indicate that no Campylobacter was recovered from swab samples. The grey boxes indicate that Campylobacter was identified only after enrichment for 2 days (low level). The black boxes indicate that there was direct enumeration of Campylobacter on blood-free agar. An X indicates that a bird was removed for nonexperimental reasons. The percentages of the group are indicated for both enrichment and direct counts. The data are from a independent experiment, not the experiment whose results are shown in Fig. 1.

 
Several C. jejuni NCTC11168 colonies were reisolated from the cecal swabs taken during week 8 postinoculation from the different trials described above. One isolate was used in further studies and was designated C. jejuni PASS67. This isolate was colony purified on sheep blood agar and was tested for colonization in 2-week-old Light Sussex chickens with a gut flora. C. jejuni PASS67 colonized the chickens at a high level from 1 week postinfection, and this continued for 7 weeks (Fig. 1C). Trials with two other independent isolates produced the same results, and in one trial high-level colonization was observed up to 13 weeks postinoculation, at which point the trial was terminated. C. jejuni PASS67 was passaged every 3 days for 8 weeks on sheep blood agar by streaking from and to single colonies. This serially passaged strain was tested for colonization by using the standard assay. The strain that was passaged in vitro colonized at the same level as the original C. jejuni PASS67 strain (data not shown). Because the chickens were SPF and were kept in a high-biosecurity facility, it is extremely unlikely that the apparent adaptation was a result of external contamination. However, to ensure that the colonization-proficient strains which we recovered were derived from the strain of C. jejuni NCTC11168 introduced, we carried out a PFGE, serotyping, and comparative genomic microarray analysis of both the input and output strains from the persistence trials. The PFGE profiles (Fig. 3) and serotyping and microarray-derived gene complement results for the two strains were indistinguishable, indicating that these strains were clonal (data not shown). C. jejuni 11168H was compared to the original and passaged isolates and was also indistinguishable from NCTC11168 and PASS67 (Fig. 3, lane 4, and data not shown).



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  		FIG. 3. KpnI-generated PFGE profiles for C. jejuni in vivo adaptation strains NCTC11168 and PASS67 and strain 11168H adapted in vitro. Lane 1, C. jejuni NCTC11168, unadapted; lane 2, C. jejuni NCTC11168 isolated at week 8 (see Fig. 1A); lane 3, PASS67 (see Fig. 1C); lane 4, C. jejuni 11168H (see Fig. 1D); lane M, markers.

 
C. jejuni NCTC11168 and PASS67 differ in motility. C. jejuni NCTC11168 has been described as a strain that is poorly motile (18), and motility has been linked to colonization of chickens (26, 27, 46). We compared the motilities of several passaged strains, including PASS67, to those of C. jejuni NCTC11168 and the previously described strain C. jejuni 11168H (see below) (18). The original C. jejuni NCTC11168 strain was completely nonmotile, and there was no halo of motile bacteria on Mueller-Hinton motility plates containing 0.5% agar. C. jejuni PASS67 and C. jejuni 11168H had 1.5-cm halos after 1 day of incubation, and the halos spread to cover a 10-cm petri dish after 2 days of incubation. The other passaged strains all produced halos that were equivalent sizes. The observed motilities of 11168H and PASS67 were equivalent to those of the other isolates used in this study (C. jejuni G1, 81-176, 49S, and C. jejuni 35R and C. coli DR4) when they were assessed on Mueller-Hinton plates containing 0.5% agar.

Isolates were obtained during different weeks during the NCTC11168 colonization experiment, and their morphologies and motilities were assessed by using both phase-contrast microscopy and motility plates. The Campylobacter isolates were vibrioid throughout the experiment, and motility correlated with the colonization profile. Bacteria isolated only by the enrichment procedure were nonmotile, while isolates obtained by the direct count procedure were motile.

A hypermotile derivative of NCTC11168 isolated in vitro colonized chickens to the same extent as PASS67. C. jejuni 11168H is a hypermotile isolate that was derived from the source of C. jejuni NCTC11168 used for genome sequencing (31) by successive culturing from the edges of swarming colonies on motility plates (18). C. jejuni 11168H colonized 2-week-old chickens to the same extent as C. jejuni PASS67 and NCTC11828 (Fig. 1).

Colonization profiles of other Campylobacter strains. Five other Campylobacter strains (C. jejuni G1, 81-176, 49S, and 35R and C. coli DR4) were tested to determine their abilities to colonize and persist within chickens (Fig. 4). All the strains colonized 2-week-old birds at a high level. Interestingly, C. jejuni 81-176 began to be cleared from the birds at 5 to 6 weeks postinoculation (Fig. 4B).



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  		FIG. 4. Profiles of C. jejuni colonization. (A) C. jejuni G1. (B) C. jejuni 81-176. (C) C. jejuni 49S. (D) C. jejuni 35R. (E) C. coli DR4. The dark grey bars indicate percentage of each group with direct counts of Campylobacter, and the light grey bars indicate the percentage of each group in which the presence of Campylobacter was determined only upon enrichment. Birds were inoculated when they were 2 weeks old. Swabs samples were taken at weekly intervals.

 
Intestinal colonization. At the end of each trial a group of five randomly selected birds was assessed postmortem to obtain a quantitative assessment of the level of colonization by Campylobacter in the cecum. Figure 5A shows the levels of colonization by the different strains. Both C. jejuni NCTC11168, which had undergone in vivo adaptation during the trial, and hypermotile strain 11168H showed high levels of colonization (log10 8.5 and log10 8.4 CFU/g of cecal contents, respectively) (Fig. 5, lanes 1 and 3). The values were readily comparable to those for a range of strains tested (Fig. 5). A more detailed study to determine the extents of colonization at various sites along the intestinal tract was carried out with C. jejuni NCTC11168 and G1 at 6 weeks postinoculation. The data show that both C. jejuni NCTC11168, which had adapted to become colonization proficient, and C. jejuni G1 could colonize several sites in the avian intestinal tract (Table 1). It is interesting that both strains exhibited a low level of colonization in the upper intestinal tract and a high level of colonization of the cecum (Table 1). A histopathological examination was carried out with cecal and intestinal sections, but no obvious differences between the infected and uninfected tissues were observed (data not shown).



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  		FIG. 5. Postmortem cecal counts of Campylobacter strains used in this study. Cecal counts of Campylobacter were determined for five random birds at 6 weeks postinoculation (age, 9 weeks). At this time, postmortem examinations were carried out, and the levels of the various Campylobacter strains were expressed as the number of CFU per gram of cecal contents. The values are log10 values (circles), and the averages are indicated by bars. (A) Lane 1, C. jejuni NCTC11168; lane 2, C. jejuni PASS67; lane 3, C. jejuni 11168H; lane 4, C. jejuni NCTC11828; lane 5, C. jejuni G1; lane 6, C. jejuni 81-176; lane 7, C. coli DR4; lane 8, C. jejuni 49S; lane 9, C. jejuni 35R. (B) C. jejuni 11168H pglH (data from 20 birds). The asterisk indicates that the counts are counts for swab-positive birds (swab-negative birds had counts below the detectable limits). A dagger indicates that nine sample points had values below the threshold value (100 CFU/g of cecal contents).

 

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  		TABLE 1. CFU of Campylobacter per gram of tissue

 
Motility is required for colonization. We assessed the abilities of two nonmotile mutants, 11168H maf5::kan (with a mutation in a gene which is required for flagellum formation) and 11168H flaA::kan (with a mutation which eliminates FlaA production), to colonize the avian intestinal tract of 2-week-old birds with a developed gut flora. Both mutant strains failed to colonize the intestinal tract and were cleared by 7 days postinoculation (Table 2).


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  		TABLE 2. CFU per gram of cecal contents

 
Capsulation and glycosylation play a vital role in colonization. Two previously constructed single-gene knockout mutants of 11168H, 11168H kpsM::kan and 11168H pglH::kan (with mutations in genes required for capsule formation and the general protein glycosylation pathway, respectively [17, 21]), were similarly tested to determine their abilities to colonize and persist in chickens. C. jejuni 11168H kpsM::kan was cleared rapidly after inoculation, indicating that capsulation is important in colonization (Fig. 6A). C. jejuni 11168H pglH::kan was a poor colonizer. In the first week postinoculation only 50% of the birds were colonized, as detected by direct counting from cloacal swabs. At 2 weeks postinoculation the inoculated bacteria were detectable only after enrichment of cloacal swabs (Fig. 6B). At the end of the trial, a postmortem analysis was carried out for all the birds in the group to determine the levels of inoculated bacteria in the cecal contents. There was wide variation in the levels of colonization by C. jejuni 11168H pglH::kan, and nine birds were negative for the inoculated bacteria (the levels were below detectable limits [i.e., <100 CFU/g of cecal contents]). However, 50% of the birds had colonization levels of log10 7 CFU/g of cecal contents or higher (Fig. 5B). The analysis of the cecal contents from the kpsM trial group resulted in no detectable counts postmortem (data not shown).



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  		FIG. 6. Colonization profiles for pglH and kpsM insertion mutants. (A) C. jejuni 11168H::kpsM. (B) C. jejuni 11168H::pglH. The dark grey bars indicate the percentage of each group with direct counts of Campylobacter, and the light grey bars indicate the percentage of each group in which the presence of Campylobacter was determined only upon enrichment. Birds were inoculated when they were 2 weeks old. Swabs samples were taken at weekly intervals.

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
C. jejuni is a commensal in poultry and, in the field, can colonize the intestinal tracts of adult chickens with a fully developed gut flora. Studies directed at understanding the colonization and persistence of Campylobacter in poultry are important for designing intervention strategies to reduce the level of C. jejuni in the food chain. Many previous studies of chicken colonization have been carried out with natural isolates of C. jejuni. There is wide variation in the genomes of natural isolates, and this could lead to difficulties during comparisons of the contributions of particular genes to colonization (11). The genetically characterized strain C. jejuni NCTC11168 (31) is the obvious choice for studying colonization, but it has been described previously as either a poor colonizer (1) or a noncolonizer of day-of-hatching and 2-day-old chickens (33). Here we found that selection for high-level colonization by this strain can be done both in vivo and in vitro. Selection for increased colonization of chickens by Campylobacter has been observed previously (10, 33, 34, 38). However, our study differed in several key respects. Our experiments were carried out with 2-week-old birds with a developed gut flora, and the study was carried out nonsacrificially over a 6-week period, which allowed within-group strain adaptation to occur. This is important given that adaptation of C. jejuni NCTC11168 was not observed in short-term trials (33). We also observed high-level colonization by strain 11168H that was selected in vitro (Fig. 5, lane 3). The length of the trial in this study also allowed adequate time for clearance from the intestine of noncolonizing bacteria (11168H kpsM). In previous reports the workers have described short-term colonization by specific strains over a 7-day period (33). Our data show that clearance of a noncolonizing mutant can take up to 2 weeks (Fig. 6A). Therefore, a clear distinction has to be made between short-term carriage and persistent colonization by using experimental protocols that allow adequate time for clearance.

The data show that C. jejuni NCTC11168 can colonize the avian intestinal tract. Due to the availability of the complete sequence of NCTC11168 (31), this strain is being used extensively in other studies of specific physiological processes, and therefore, the hypermotile or passaged strain is the ideal strain with which to address the link between key metabolic processes and colonization of the avian intestinal tract.

Motility and expression of FlaA flagellin have been repeatedly implicated as colonization factors in humans, mice, rabbits and chickens (8, 9, 26, 27, 29, 46). The importance of changes in the motility of C. jejuni NCTC11168 during this adaptation to high-level colonization of chickens is indicated by the ability of the in vitro hypermotile isolate 11168H to colonize the chicken intestine at a high level (Fig. 5). This strain was isolated under more defined conditions (18) than those which generated PASS67. The improved motility of 11168H is apparently enough to allow C. jejuni NCTC11168 to colonize the avian intestinal tract. Independent mutations in two genes required for the motility of C. jejuni 11168H, maf5 and flaA (cj1337 and cj1339, respectively), were assessed to determine their effects on colonization, and our results clearly show that motility is required for 11168H to colonize the intestinal tract (Table 2). The changes that lead to increased motility could be pleiotropic, so other factors may also be required for optimal colonization.

The in vivo selection and greatly improved colonization occurred during a period of several weeks during which low numbers of Campylobacter were present in the ceca, as confirmed in separate sacrificial experiments. Importantly, the data show the potential of C. jejuni strains to persist in the avian alimentary tract by undergoing adaptation which may lead to an increase in the ability to colonize. The pattern of persistence is illustrated in Fig. 2, which shows that once adapted, C. jejuni NCTC11168 rapidly colonized other birds in a group. These results have implications for the epidemiology of Campylobacter infection in flocks. Previous investigations have suggested that there is selection of a specific subpopulation within the population (38). Given the variation in motility seen in vitro (18), one could hypothesize that there is random low-level production of hypermotile variants in the whole NCTC11168 population and that growth in vivo, which requires motility, favors these variants. In addition, attempts to control Campylobacter by using methods that reduce the level of bacteria but do not clear the bacteria from a group of birds may leave enough bacteria present within a flock to eventually circumvent any negative selection and repopulate at a high level.

While the change in motility was an obvious phenotypic change, we could not rule out the possibility that other adaptations occurred in vivo, due to the undefined and multifactorial nature of the selection. Previous studies of the passage of C. jejuni through cell monolayers showed that there was a correlation between changes in the lipooligosaccharide and the capsular polysaccharide (CPS) and an increased ability to invade and attach to cells (5). The PFGE data indicated that no major rearrangements of the genome occurred. However, the genome of Campylobacter has numerous hypervariable sequences (31), many of which are associated with sugar-nucleotide metabolism and capsule production. We compared the parental NCTC11168 strain with the passaged strain PASS67 to look for changes in the lipooligosaccharide (data not shown), but no obvious difference was observed. C. jejuni NCTC11168, 11168H, and PASS67 were tested with Penner serum 2, which binds the CPS (17, 32). All three strains reacted in the same manner. This strongly suggests that there were no major changes in the CPS during selection for improved colonization of C. jejuni NCTC11168. These results do not address whether more subtle changes occurred in surface structures, and further investigation is required.

We assessed the colonization profiles for a range of Campylobacter strains and found that the profile obtained for C. jejuni NCTC11168 was not an artifact of the protocol which we used. The strains used were C. jejuni G1 (22); C. jejuni 81-176, a well-studied clinical isolate known to contain at least two virulence-associated plasmids (3, 20); and C. jejuni 49S and 35R and C. coli DR4, which are field isolates from a chicken abattoir. C. jejuni G1, 49S, 35R, and 81-176 and C. coli DR4 all colonized chickens at high levels in the week after inoculation, as assessed by cloacal swabbing. C. jejuni G1, 49S, and 35R and C. coli DR4 persisted at high levels in their groups for up to 6 weeks, while C. jejuni 81-176 started to clear from some birds in its test group. This may suggest that there is a fundamental difference between strains in terms of the ability to persist within the gastrointestinal tract. The postmortem data show that 81-176 colonized at a slightly lower level than the other parental strains tested in this study colonized and that the birds with negative swab results had negative cecal counts (data not shown). C. jejuni 81-176 is well characterized in terms of its virulence plasmid, which has been shown to be important in virulence in the ferret model and in in vitro cell analysis (3). The data presented here show that C. jejuni NCTC11168, which does not possess such a plasmid, can colonize the intestinal tract at a high level. Thus, it appears that the plasmid-borne genes identified so far (3, 4) are not required for colonization, suggesting that there is a clear distinction between chicken colonization and plasmid-borne virulence genes. Isolates of C. jejuni 81-176 that have lost the tetracycline resistance plasmid colonize with the same profile as isolates with both plasmids (M. A. Jones, L. T. Tricket, and P. A. Barrow, unpublished data).

We wanted to validate the 2-week-old bird model for the persistence of specific Campylobacter mutants. We found that NCTC11168 has the genetic complement required for high-level colonization of the chicken intestinal tract, but colonization studies carried out with the unadapted strain C. jejuni NCTC11168 are complicated by the variable length of adaptation to high-level colonization. Therefore, we used the hypermotile strain C. jejuni 11168H as a model organism for persistence studies with four mutants. Two nonmotile mutants of C. jejuni, maf5::kan and flaA::kan, failed to colonize. Mutation of the kpsM gene greatly reduced the colonization ability of 11168H, and since this mutation blocks the synthesis of the capsule of Campylobacter (17, 19), the results indicate that the capsule is essential for colonization. The Campylobacter capsule has been implicated in virulence in the ferret diarrheal disease model (5) and in several in vitro assays, including serum sensitivity and cell invasion assays (5). In this respect the serum and colonization sensitivity may be similar to that seen in mutants of Salmonella and Vibrio cholerae lacking O antigen and CPS, respectively (28, 36, 43). It is interesting to hypothesize that there may be functional homology between the Campylobacter capsule and both Salmonella lipopolysaccharide and Vibrio CPS in terms of their resistance to harmful factors during intestinal colonization.

We also investigated the role of protein glycosylation in colonization and persistence in the avian intestinal tract. We used a mutation in pglH (21) in the hypermotile C. jejuni 11168H background. This mutation caused a reduction in, but did not eliminate, the colonization by Campylobacter, suggesting that Campylobacter glycoproteins have a nonessential but important role in colonization. Although the postmortem data for 11168H pglH (Fig. 5B) apparently conflict with the swab data, we argue that the pglH mutant was in the process of adapting to the gut and fecal shedding was in fact increasing at the termination of the experiment (Fig. 6B). However, this mutation affects the glycosylation of numerous proteins with various functions (21). Also, there is evidence that protein glycosylation affects the antigenicity of flagella and the bacterium-host interaction (13, 40, 42). Thus, the reduced colonization could well be multifaceted, and further investigation is required. These two mutations have shown two clear phenotypes, complete clearance of the kpsM mutant over 1 week and prolonged low-level persistence of the pglH mutant in a group of birds, that would not be well described by short-term trials.

In this work we (i) established a robust validated persistence model for Campylobacter colonization of the avian intestinal tract in birds that were a field-relevant age and had a developed gut flora; (ii) showed that C. jejuni NCTC11168 has the genetic potential to colonize the avian host at a high level; (iii) validated the model by construction of mutations in the colonization-proficient organism C. jejuni 11168H, which allowed us to assess the role of specific gene products in a known genetic background in both colonization and persistence; (iv) showed that there was time-dependent clearance of noncolonizing mutants, suggesting that short-term colonization trials should be treated with caution; and (v) provided a clear model that can distinguish short-term colonization, persistence, and long-term but low-level persistence.

Given the well-defined nature of C. jejuni NCTC11168, we suggest that it is a suitable candidate for further studies of the physiology of C. jejuni colonization of the chicken.


   ACKNOWLEDGMENTS
 
This work was supported by the BBSRC, the Wellcome Trust, a DEFRA Senior Fellowship in Veterinary Microbiology (to D.J.M.), and the EU (grant FAIR CT98-4006).

We thank P. Guerry, N. Gregson, F. Jørgensen, and T. Humphrey for strains used in this study.


   FOOTNOTES
 
* Corresponding author. Mailing address: Institute for Animal Health, Compton, Newbury Berkshire RG20 7NN, United Kingdom. Phone: (1635) 578411. Fax: (1635) 577243. E-mail: majones{at}bbsrc.ac.uk. Back

Editor: V. J. DiRita


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Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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Infection and Immunity, July 2004, p. 3769-3776, Vol. 72, No. 7
0019-9567/04/$08.00+0     DOI: 10.1128/IAI.72.7.3769-3776.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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