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The Children's Research Centre, Our Lady's Children's Hospital, Crumlin, Dublin 12, and The Conway Institute of Biomolecular and Biomedical Research, UCD School of Medicine and Medical Science, University College Dublin, Ireland
Correspondence
Billy Bourke
billy.bourke{at}ucd.ie
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Guarner & Malagelada, 2003). Although the faecal microbiota conventionally is viewed as having a symbiotic relationship with the host, when viewed individually many members of the faecal flora are better described as commensals or opportunists [i.e. existing for their own benefit, but without conferring beneficial (symbiotic) or detrimental (pathogenic) effects on the host (Falk et al., 1998)]. It is well recognized that some commensal enteric organisms become pathogenic under specific conditions (Falk et al., 1998; Farthing, 2004) (e.g. Clostridium difficile) and that the pathogenic effect of some enteric organisms is potentiated in a restricted host range or age group (e.g. Escherichia coli O157 : H7, Salmonella and Campylobacter) (Moxley, 2004; Rabsch et al., 2002).
Campylobacter are the principal bacterial cause of food-borne gastrointestinal disease in developed countries and probably also in the developing world (Blaser, 1997; Crushell et al., 2004; Takkinen et al., 2003). Additionally, Campylobacter can cause invasive disease and extra-intestinal sequelae including bacteraemia and Guillain–Barré syndrome (Hannu et al., 2002; Reed et al., 1996). Epidemiological studies identify poultry to be a major reservoir of human infection (Frost, 2001; Hanninen et al., 2000; Nadeau et al., 2003). Chickens are commonly colonized by Campylobacter jejuni, with some studies showing up to 98 % of point-of-sale product contaminated (Hanninen et al., 2000; Jacobs-Reitsma, 2000; Madden et al., 1998; Pearson et al., 2000). Despite the high prevalence of C. jejuni in chickens, intestinal disease does not appear to occur following naturally acquired infection, avian intestinal inflammation is absent, and no cellular attachment or invasion is demonstrated in the intestine of colonized birds (Beery et al., 1988; Meinersmann et al., 1991).
Therefore, although pathogenic to humans, C. jejuni behaves as a commensal in avian hosts. Knowledge of the mechanisms underlying these differences in disease outcome following Campylobacter exposure could serve as a useful model for understanding pathogen–commensal relationships in the gastrointestinal tract, and might provide a better understanding of how Campylobacter colonizes hosts and causes enteric disease. We speculated that failure of C. jejuni to attach to or invade avian intestinal cells in vivo (Beery et al., 1988; Meinersmann et al., 1991) reflects a species-specific tissue tropism. To explore this hypothesis we used a primary intestinal cell model to examine the interaction of C. jejuni with a variety of host intestinal cells ex vivo. C. jejuni readily invaded primary human and avian intestinal epithelial cells. We demonstrate that chicken-derived intestinal mucus, but not human mucus, attenuated C. jejuni invasion in vitro. From these data we conclude that specific avian intestinal luminal factor(s) rather than tissue tropism underlie Campylobacter commensalism in chickens. These findings provide an important basis for further exploration of the effect of mucin and other constituents of the mucus layer on bacterial pathogenicity during enteric infection.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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); C. jejuni NCTC 11168, the first sequenced strain (Parkhill et al., 2000); C. jejuni F1882 and F1998, low-passage human isolates (Byrne et al., 2001); and CB123, a fresh human isolate. C. jejuni 1646DF and 1449BF were fresh chicken isolates. Organisms, including most clinical isolates, were stored at –70 °C in 10 % (v/v) glycerol, 10 % (v/v) fetal calf serum (FCS), 80 % (v/v) Brucella broth. They were recovered on Columbia agar (Oxoid) containing 7 % (v/v) defibrinated horse blood, incubated at 37 °C under microaerophilic conditions generated by Campygen gas packs (Oxoid). C. jejuni CB123 was freshly isolated on CCDA selective medium (Oxoid) and plated onto Columbia blood agar. Replicate experiments were conducted within two to four subcultures of primary isolation over a time frame of a week. For infection assays Campylobacter isolates were grown in a biphasic medium for approximately 18 h. Biphasic medium was prepared using a Columbia agar slant overlaid with 7 ml cell culture medium in 25 cm2 culture flasks. Enumeration of bacteria following invasion assays was achieved by dilution in phosphate-buffered saline (PBS) and plating onto Campylobacter blood-free selective agar base (Oxoid CM739), without selective supplement but with an additional 0.1 % (w/v) agar to minimize the swarming of mucoid isolates. Clinical isolates were identified using an API Campy system (bioMerieux). E. coli HB101, a non-invasive control organism, was grown on Columbia blood agar at 37 °C in air for 18–24 h. Post-infection assays were plated to MacConkey agar.
Primary cell isolation and culture.
Biopsy material was obtained from children undergoing endoscopy at Our Lady's Children's Hospital, Dublin, Ireland. Ethical permission for this study was obtained from the ethics committee of the hospital. Informed, signed consent was obtained from parents/guardians of all children enrolled in the study. Mucosal biopsy specimens were taken from the first part of the duodenum using a standard endoscopic biopsy forceps. Only grossly normal tissue was sampled. Chickens (Cobb 500, commercial broiler) aged between 6 and 12 weeks were killed by injection of Euthatal (Merial Animal Health). Sections of distal small intestine were excised, slit longitudinally, fixed in position and rinsed in cold PBS. Biopsies were taken using endoscopy forceps and placed into primary cell culture medium at 4 °C for transport. Cells were isolated essentially as described by Clyne & Drumm (1993) for isolating human gastric cells. Briefly, biopsies were incubated in 15 ml Hanks' Balanced Salt Solution (HBSS) without Ca2+ or Mg2+, containing 0.1 mM EDTA and 0.1 mM DTT, with vigorous shaking for 15 min at 37 °C. Collagenase, 0.05 % (w/v) (Sigma) in RPMI, was applied to the biopsies, which were reincubated with shaking for periods of 10 min until digestion was complete. The supernatant containing released cells and crypt fragments was aspirated to a tube containing 5 ml primary cell medium (see below) containing 10 % (v/v) FCS to halt digestion. The suspension was centrifuged at 800 g for 5 min at 4 °C. Cellular harvests were suspended in fresh medium and held on ice until further digestion of biopsies was complete. The combined harvests were again washed in primary medium and resuspended in a small volume (2–4 ml) of medium. The harvested crypts were examined using an inverted microscope and seeded onto 13 mm plastic Thermanox coverslips (Nunc, Thermanox 174950) in 24-well trays. Harvested primary human and chicken intestinal cells were grown in medium comprising 50 : 50 DMEM-F12, 10 % (v/v) FCS, 8 µg insulin ml–1, 50 µg hydrocortisone ml–1 (Sigma), 10 µg gentamicin ml–1, 2.5 µg amphotericin ml–1, 100 µg penicillin ml–1 and 100 U streptomycin ml–1 (BioWittaker). The medium was supplemented at 18 h without disturbing the attaching cells and culture continued. Preparations were used approximately 42–48 h post-seeding. Except where stated, all cell cultures were grown at 37 °C in a humidified atmosphere containing 5 % CO2. Chicken epithelial harvests were initially cultured at both 37 °C and 42 °C. Prior to assays, cells were rinsed twice in pre-warmed antibiotic-free primary medium to remove non-adherent cells and reincubated in antibiotic-free medium for a minimum of 2 h prior to infection.
Collection of mucus.
A sterile universal container was used to scrape gently along the surface of the excised chicken intestine in order to collect crude intestinal mucus. This was then emulsified with 
15 % (v/v) PBS to facilitate mixing and dispensing and stored at –20 °C. Crude human mucus, obtained in a similar manner from human intestinal specimens, was a generous gift from Professor Prem Puri, The Children's Research Centre, Dublin. Mucus was confirmed free of enteropathogens, including Salmonella, Shigella and Campylobacter, using culture to appropriate medium. Control experiments verified that the mucus used in these experiments did not exhibit antibacterial activity against C. jejuni 81-176. Attendant normal microflora was present.
Antibodies and staining.
C. jejuni polyclonal antiserum was raised in rabbits. C. jejuni 81-176, 11168 and F1882 were harvested from agar plates. The suspensions were pooled and washed in PBS. Bacteria were suspended in PBS containing 0.5 % (v/v) formaldehyde at a concentration of 1 g (wet wt) per 100 ml of suspension. The immunization protocol was as previously described for the generation of Helicobacter pylori antiserum (Clyne & Drumm, 1993). Vital staining (red) of bacteria was achieved by incubating organisms in PBS containing 10 µg carboxytetramethylrhodamine ml–1 (TAMRA T-6105, Molecular Probes) for 30 min at 37 °C in the dark, followed by washing four times in PBS (Mooney et al., 2003).
Cell cultures were fixed using methanol or 4 % formaldehyde in PBS as appropriate. Where required, cells were permeabilized using 0.1 % (v/v) Triton X-100 (Sigma). Anti-pancytokeratin (Sigma C-2562) revealed by anti-mouse FITC (Sigma F8771) was used to confirm the epithelial origin of human intestinal cells (Mooney et al., 2003). An FITC-conjugated cytokeratin antibody (Sigma F 0397), which includes chicken in the species range, was used to stain chicken intestinal cells. Methanol-fixed cells were stained with Giemsa (BDH) and mounted in DPX. Primary cells, invaded with red-labelled C. jejuni, were fixed and stained as follows. Preparations were blocked with 10 % (v/v) goat serum in PBS, incubated in C. jejuni antiserum, 1 : 200 in bovine serum albumin, washed, and incubated in 1 : 200 goat-anti-rabbit IgG Alexa Fluor 488 (Molecular Probes A-11070), labelling external organisms green. Cells were then permeabilized using 0.1 % (v/v) Triton X-100 for 4 min and stained with 1 : 6000 TRITC-phalloidin (Sigma P1951) or 1 : 2000 FITC-phalloidin (Sigma P 5282) for 6 min, permitting clear discrimination between organisms and cellular morphology. Nuclei were visualized by including DAPI (Sigma) at a dilution of 1 : 4000. Stained preparations were mounted in fluorescent mounting medium (Dako) and sealed with clear nail polish.
Adherence assays.
Cell culture medium was replaced with 4x107 TAMRA-labelled E. coli or C. jejuni (strains listed above) in 500 µl antibiotic-free medium containing 1 % (v/v) FCS and centrifuged at 250 g for 5 min at room temperature. Trays were incubated at 37 °C for 2 h in a microaerophilic environment unless otherwise stated. At the end of incubation, coverslips were washed four times in PBS, fixed and stained.
Invasion assays.
Microscopic discrimination of internalization into primary human and primary chicken cells was established by differential staining of adherent organisms. Following infection assays, TAMRA-labelled adherent organisms (red) were stained green using a specific C. jejuni antibody revealed by a green probe (Alexa Fluor 488), leaving the internalized organisms single stained (red) while adherent organisms were double labelled (red and green). In addition to growing and infecting cells at 37 °C, primary chicken cells also were grown and infected at 42 °C using TAMRA-labelled organisms. Additionally, gentamicin protection was used to evaluate internalization of Campylobacter into primary human and chicken cells. These invasion assays were set up as for adherence, omitting the bacteria-labelling step. The invasion of a number of strains was examined at the same organism density, under the same conditions. At the end of the invasion period, cells were washed in PBS to remove non-adherent organisms and reincubated in medium containing 400 µg gentamicin ml–1 (BioWittaker) for a further 2 h to kill adherent organisms (Mooney et al., 2003). Cells were washed in PBS, and incubated in 200 µl PBS with 0.1 % (v/v) Triton X-100 for 15 min at 37 °C to release intracellular organisms. Dilutions of replicate wells in PBS were plated and colonies enumerated after 48 h incubation. Primary cells grew as discontinuous islets at 104–105 per well. As primary cell numbers are substantially lower than those conventionally employed, results are expressed as c.f.u. per well rather than percentage inoculum internalized.
Effect of inhibitors on primary cell invasion.
The effect of cytochalasin B (Sigma) (inhibitor of actin polymerization), vincristine (Sigma) (inhibitor of microtubule polymerization) and filipin (Sigma) (caveolin inhibitor) on internalization of C. jejuni 81-176 into primary human intestinal cells was assessed using the gentamicin protection assay. Inhibitors were added to primary cells 1 h prior to the addition of bacteria and maintained throughout the assay. Control experiments verified that inhibitors did not affect cell viability using trypan blue exclusion or bacterial viability by plating experiments.
Primary cell invasion assays in the presence of mucus.
The effect of various mucus preparations on internalization of C. jejuni 81-176 into primary human intestinal cells was examined. Primary intestinal cells were challenged using 200 µl C. jejuni suspended in medium at a concentration of 8x107 c.f.u. ml–1 (control) or in medium containing different mucus preparations at 15–20 % (v/v). Preparations containing organisms and mucus were mixed briefly using a vortex and applied to cultured cells. Trays were centrifuged at 250 g for 5 min at room temperature and incubated at 37 °C in microaerophilic conditions for 2 h. Internalization was measured using gentamicin protection.
Effect of mucus concentration on total cell association.
Given the relatively restricted availability of primary human intestinal cells, the concentration of chicken mucus required to reduce Campylobacter cell association was examined using confluent Caco-2 cells grown in 96-well trays. Cells were seeded at 2.5x104 per well and cultured for 11 days in Eagle's Minimal Essential Medium (EMEM) (Bio-Whittaker) with 10 % (v/v) FCS, 1 % (v/v) non-essential amino acids and 2 mM L-glutamine. Chicken small intestinal mucus was dispensed into sterile tubes to give a range of concentrations starting at 1 % (w/v). Some mucus preparations were liquefied using a DTT-containing agent (Sputasol, Oxoid SR0233A). C. jejuni 81-176 was added to each preparation to a final concentration of 8x107 c.f.u. ml–1. Cells were challenged by C. jejuni in medium or in chicken mucus-containing medium in 100 µl volumes and incubated for 2 h at 37 °C in a microaerophilic atmosphere. Mucus-containing supernatants were aspirated carefully and the cells washed four times in PBS. Intracellular organisms were released by incubation in 100 µl PBS with 0.1 % (v/v) Triton X-100 for 15 min. Total cell-associated organisms were enumerated by plating.
Imaging.
A Nikon TMS inverted microscope and Olympus camera were used to examine and record patterns of primary cell growth. Stained slides were examined using blue, green and red filters with a Leica DML fluorescent microscope. Images were acquired using a Leica DC camera and manipulated using Adobe Photoshop 6. Confocal images were acquired using a Zeiss LSM UV 510 META confocal microscope. Merged images show internal organisms in red, external organisms in yellow (i.e. combined red and green) and nuclei in blue. Cellular actin is stained faintly in red in some images. XYZ series were taken through areas of interest.
Statistical analysis.
Experiments were performed on three or more occasions using cells harvested from different patients or chickens. Graphs were drawn using Microsoft Excel. Data from experiments were analysed using Minitab. Statistical significance was estimated using the non-parametric Mann–Whitney test when comparing primary cell infections or two-tailed Student's t test when examining the influence of mucus concentration. In all cases differences were considered significant at P
0.05.
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RESULTS |
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). They could be seen to emanate from harvested intestinal crypts, and some coalesced to yield areas of confluent growth. Primary human intestinal cells were maintained in culture for up to 3 weeks. Cytokeratin staining was used to confirm epithelial lineage. Initial chicken harvests were cultured at both 37 °C and 42 °C and maintained for up to 10 days. No difference was noted in extent of growth, appearance, size of cells or duration in culture. Thereafter, chicken cells were cultured at 37 °C only. Chicken cells were marginally smaller than human-derived cells. In addition to islets of epithelial lineage, chicken biopsies initially yielded small numbers of fibroblasts and macrophages (rarely observed in human cultures). Contamination with non-epithelial cell lineages was reduced by decreasing the biopsy depth (to avoid sampling the mucosa of the thinner avian intestine) and by allowing crypt fragments to sediment naturally in a conical tube for 5–7 min, removing the supernatant, containing isolated cells and using the crypt-enriched deposit to seed wells.
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). In contrast to C. jejuni, TAMRA-labelled E. coli HB101 did not associate with primary human intestinal cells.
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As shown in previous studies (Biswas et al., 2000; Konkel et al., 1992; Schwartz et al., 1996; Tay et al., 1996) using conventional tissue culture, levels of invasiveness varied considerably among strains. Invasion of primary human intestinal cells using a variety of C. jejuni isolates is shown in Table 1. With one exception (F1889), fresh isolates from both humans and chickens tended to invade more efficiently than C. jejuni 81-176. All differences were significant at P<0.05 compared to C. jejuni 81-176 (n=6).
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Attachment to and invasion of primary chicken intestinal cells by C. jejuni
As Campylobacter does not attach to or invade chicken epithelial cells in vivo (Beery et al., 1988; Meinersmann et al., 1991), we hypothesized that C. jejuni would not invade primary avian intestinal cells in vitro. Primary chicken intestinal cells were challenged with a number of different TAMRA-labelled strains of C. jejuni. Because avian intestinal core temperature ranges between 41.5 and 42 °C, preliminary cell cultures and invasions with TAMRA-labelled C. jejuni were conducted at both 37 °C and 42 °C. Microscopic examination showed no detectable difference in cell growth or bacterial attachment and entry at these two temperatures (data not presented); therefore subsequent incubations and assays were conducted at 37 °C. Microscopic examination clearly showed that C. jejuni attached to and entered cultured primary chicken intestinal cells (Fig. 2d
). The extent and pattern of C. jejuni invasion of chicken intestinal cells were comparable to those observed with primary human cells (Fig. 2a, b, c). E. coli HB101 did not associate with chicken epithelial cells to any significant extent.
Similar to invasion of primary human intestinal cells, invasiveness varied between C. jejuni isolates; in general, fresh isolates showed slightly higher rates of invasion than C. jejuni 81-176 (Table 1; n=6). Apart from the chicken isolate 1646DF, the differences were significant (P<0.05) compared to C. jejuni 81-176.
Effect of mucus on Campylobacter internalization into primary intestinal cells
Efficient internalization of C. jejuni into primary chicken intestinal cells was unexpected given its lack of association with chicken crypt epithelium in vivo. Therefore, we postulated that the failure of bacteria to associate with cells in vivo might result from avian luminal factor(s) that differ from those of humans. Colonization of the enteric mucus layer is a key early step in enteric pathogenesis. As avian mucus is readily and persistently colonized by C. jejuni (Stern et al., 1988) (without cellular association) we explored the influence of human- and chicken-derived mucus on primary human intestinal cell invasiveness. As the relative colonization of the proximal small intestine by C. jejuni is lower than that of the large intestine in chickens (Beery et al., 1988; Hendrixson & DiRita, 2004), the effects of small and large intestinal mucus were examined. Control experiments showed that neither organism nor cell viability was affected in the presence of mucus under the experimental conditions. Infection of primary cells in the presence of intestinal mucus modified internalization, but its effect differed depending on the source of the mucus. Crude human mucus increased internalization of C. jejuni by 35±22 % (P=0.61). In contrast, chicken large intestinal mucus reduced C. jejuni entry into primary human intestinal cells by 54±18 % and chicken small intestinal mucus reduced entry by 98±2 % (P=0.0350 and 0.0000 respectively).
Effect of mucus concentration on total cell association
To explore in more detail the influence of chicken mucus on the ability of Campylobacter to infect intestinal cells, infection assays were carried out over a range of mucus concentrations. Chicken small intestinal mucus reduced cell association of C. jejuni with Caco-2 cells in a dose-dependent manner (Fig. 3). As little as 2 % chicken mucus significantly reduced Campylobacter recovery in these infection assays (P<0.05). Interestingly, liquefaction of chicken mucus using Sputasol did not abrogate the inhibitory effect of mucus on cell association.
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DISCUSSION |
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; Meinersmann et al., 1991). Although immature birds challenged by large C. jejuni inocula may get diarrhoea and some reports allude to a form of vibrionic hepatitis, there is no evidence that naturally acquired C. jejuni causes infection in avian intestinal epithelium cells (Newell, 2000; Skirrow, 1994). Recently, Smith et al. (2005) used an avian kidney cell model of Campylobacter infection and showed adherence to and invasion of these cells but the interaction of Campylobacter with avian intestinal cells has never been studied directly. In this study we have developed a primary cell model of intestinal campylobacteriosis to investigate the host-specific tropism of these organisms. Contrary to expectation, C. jejuni efficiently adhered to and entered avian intestinal epithelium. Chicken (but not human) intestinal mucus effectively attenuated C. jejuni invasiveness and reduced cell association in a dose-dependent manner, suggesting that a component of chicken mucus may be responsible for the divergent disease outcomes in humans and poultry following Campylobacter exposure.
In previous studies of Helicobacter it has been shown that primary cells obtained from human endoscopic biopsy samples provide a biologically relevant model of gastrointestinal infection that may reflect more precisely the intestinal milieu than conventional tissue culture (Clyne & Drumm, 1993; Clyne & Drumm, 1997). In a previous report we showed that Campylobacter upsaliensis adheres to and invades primary intestinal cells (Mooney et al., 2003). More recently, primary cells have been used to model the tissue tropism seen in vivo with Cryptosporidium species (Hashim et al., 2004). In this study we have further explored this ex vivo model of enteric infection. We have shown that primary human intestinal cells remain viable for prolonged periods and are not overgrown with non-epithelial lineages. Using inhibitors of cellular processes previously described to affect Campylobacter pathogenicity in immortalized cell lines, we have confirmed the importance of host microtubules, microfilaments and lipid rafts for cellular uptake of these organisms.
Some strains of C. jejuni, most notably the well characterized 81-176 isolate, previously have been described as being dependent on microtubule polymerization, but independent of actin, for effective entry into epithelial cells (Hu & Kopecko, 1999; Oelschlaeger et al., 1993). In contrast, other investigators have shown that microfilament inhibitors effectively reduce host cell entry by C. jejuni isolates and other campylobacters (Biswas et al., 2000; Mooney et al., 2003). In the present study, C. jejuni 81-176 was inhibited from invading primary cells previously exposed to inhibitors of actin accumulation. These results are in keeping with those recently reported for C. jejuni invasion of INT 407 cells by Monteville et al. (2003). The contribution to cell entry of a variety of different cell processes, including lipid rafts and cytoskeleton components, is an emerging paradigm among enteric pathogens (Cossart & Sansonetti, 2004). The use of primary cells can provide important information regarding the role in vivo of proposed pathogenic mechanisms without necessitating animal or human challenge. Primary cell models also serve to validate the performance of existing cell line models (Pageot et al., 2000). We believe that they will have an important role in future exploration of the cellular microbiology of Campylobacter and other enteric pathogens.
Given the evidence for lack of interaction between C. jejuni and avian epithelium in vivo (Beery et al., 1988; Meinersmann et al., 1991), it was surprising that a number of C. jejuni isolates (of both human and poultry origin) invaded chicken cells with an efficiency that was comparable to invasion of primary human intestinal cells in vitro. Clearly, then, the lack of chicken intestinal epithelial cell association in vivo does not reflect absence on these cells of the appropriate receptor-ligand(s) to effect Campylobacter adhesion and invasion. Our experiments suggested that the temperature differences that exist between humans and chickens (37 °C versus 42 °C) did not influence significantly the pathogenicity of the organisms (data not shown). In common with intestinal commensals in general, C. jejuni colonizes the intestinal mucus layer in chickens (Beery et al., 1988; Meinersmann et al., 1991) and components of mucus act as chemoattractants (Hugdahl et al., 1988). Therefore, we examined the relative effects of intestinal mucus derived from humans and poultry. Chicken mucus markedly reduced invasion of primary human cells, without affecting organism viability. In contrast, human mucus tended to promote C. jejuni invasion although the results did not achieve statistical significance (P=0.61).
The nature of the relationship between the host and its faecal microflora and the role of this relationship in health and disease currently is the focus of intense research. The present view is that the intestinal microbiota are maintained in a dynamic relationship with the host involving a complex ‘trialogue’ between micro-organism, epithelium and mucosal immune system (Bourlioux et al., 2003; Falk et al., 1998). Recent work has started to uncover some important bacterial factors necessary for successful avian colonization by C. jejuni (Hendrixson & DiRita, 2004; Jones et al., 2004; Karlyshev et al., 2004; Nachamkin et al., 1993). However, the specific host factors responsible for establishing and maintaining the affiliation of micro-organisms with the intestinal environment remain largely unknown. Species differences in mucus or associated factors may be sufficient to transform the behaviour of a pathogen in humans to commensal behaviour in another species. This would have substantial implications for furthering our understanding of the ecology of the intestinal tract.
At present it is not clear what component(s) of mucus that differ(s) between humans and poultry attenuated Campylobacter virulence in this study. An early study of intestinal mucus by McSweegan et al. (1987) suggested a role for rabbit secretory IgA in the blocking of Campylobacter adherence to INT 407 cells. Recently, it was shown that avian kidney cells and macrophages express pro-inflammatory cytokines and chemokines in response to Campylobacter infection (Smith et al., 2005). These studies raise the possibility that the avian immune system could influence Campylobacter infectivity in vivo. However, the chickens used in our experiments were housed in bio-secure facilities and were confirmed not to harbour Campylobacter before use. Furthermore, although chicken mucus attenuated virulence in our experiments, no effect on bacterial viability was noted. Therefore, we feel it is unlikely that the observed reduction in bacterial virulence, in our experiments, following exposure to mucus was caused by a specific avian immune response or antibacterial metabolites.
The pH of the chicken intestinal tract is lower (pH 5.7–6.4) (Denbow, 2000) than that of humans, a factor that potentially could affect virulence. However, the mucus used in these experiments was buffered to a neutral pH, and therefore increased acidity was not the cause of the altered pathogenicity profile demonstrated in these experiments. Chicken intestinal flora was present in the crude mucus and previously has been shown to reduce or delay chick colonization in vivo (Stern et al., 2001). Although a role for chicken microflora in attenuating virulence in our experiments cannot be discounted, it is noteworthy that intestinal pathology does not usually occur when day-of-hatch chicks are experimentally infected (Beery et al., 1988), suggesting that the acquisition of normal gut flora alone is unlikely to account for the absence of pathology in poultry.
The nature or composition of the avian mucus itself provides perhaps the most attractive candidate for the effect on the pathogenicity of Campylobacter observed in this study. Chicken mucins differ considerably from those of humans (Smirnov et al., 2004; Verma et al., 1994). Differences in mucus structure, folding, glycosylation, charge or associated molecules such as electrolytes could account for a ‘trapping’ effect of chicken compared with human mucus. Sputasol liquefaction failed to reverse the mucus-induced inhibition of bacterial cell association, suggesting the effect is not simply related to viscosity. However, previous work on C. jejuni (Szymanski et al., 1995), and more recent experiments using Pseudomonas (Wolfgang et al., 2004), provide strong support for the concept that mucus can have a direct effect on motility and virulence. Furthermore, it has been shown that the related gastrointestinal pathogen, H. pylori, binds specifically to TFF1, a human trefoil peptide found in gastric mucus (Clyne et al., 2004). Further exploration of the composition and constituents of chicken mucus is warranted to explain how, in avian hosts, an intestinal mucosal environment has evolved that not only tolerates, but possibly even promotes the growth of Campylobacter while simultaneously defending the avian host from pathological effects.
In summary, using a primary cell model, we have shown that the lack of adherence of C. jejuni to chicken intestinal epithelium in vivo does not result from lack of cell surface receptors. Some extra cellular factor acting to interrupt attachment in colonized birds may explain the lack of cell association in vivo. We found chicken intestinal mucus markedly attenuated C. jejuni virulence compared with human mucus. Knowledge of the basis of the differential effect of chicken and human mucus on pathogenic behaviour in Campylobacter has implications for understanding the nature of commensalism in the gastrointestinal tract and may provide novel opportunities to interrupt the transmission of this zoonotic pathogen to humans.
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ACKNOWLEDGEMENTS |
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Edited by: P. H. Everest
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Received 1 August 2006;
revised 5 November 2006;
accepted 7 November 2006.
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