Prebiotic Driven Increases in IL-17A Do Not Prevent Campylobacter jejuni Colonization of Chickens.
ABSTRACT: Worldwide Campylobacter jejuni is a leading cause of foodborne disease. Contamination of chicken meat with digesta from C. jejuni-positive birds during slaughter and processing is a key route of transmission to humans through the food chain. Colonization of chickens with C. jejuni elicits host innate immune responses that may be modulated by dietary additives to provide a reduction in the number of campylobacters colonizing the gastrointestinal tract and thereby reduce the likelihood of human exposure to an infectious dose. Here we report the effects of prebiotic galacto-oligosaccharide (GOS) on broiler chickens colonized with C. jejuni when challenged at either an early stage in development at 6 days of age or 20 days old when campylobacters are frequently detected in commercial flocks. GOS-fed birds had increased growth performance, but the levels of C. jejuni colonizing the cecal pouches were unchanged irrespective of the age of challenge. Dietary GOS modulated the immune response to C. jejuni by increasing cytokine IL-17A expression at colonization. Correspondingly, reduced diversity of the cecal microbiota was associated with Campylobacter colonization in GOS-fed birds. In birds challenged at 6 days-old the reduction in microbial diversity was accompanied by an increase in the relative abundance of Escherichia spp. Whilst immuno-modulation of the Th17 pro-inflammatory response did not prevent C. jejuni colonization of the intestinal tract of broiler chickens, the study highlights the potential for combinations of prebiotics, and specific competitors (synbiotics) to engage with the host innate immunity to reduce pathogen burdens.
Project description:Colonization of broiler chickens by the enteric pathogen Campylobacter jejuni is widespread and difficult to prevent. Bacteriophage therapy is one possible means by which this colonization could be controlled, thus limiting the entry of campylobacters into the human food chain. Prior to evaluating the efficacy of phage therapy, experimental models of Campylobacter colonization of broiler chickens were established by using low-passage C. jejuni isolates HPC5 and GIIC8 from United Kingdom broiler flocks. The screening of 53 lytic bacteriophage isolates against a panel of 50 Campylobacter isolates from broiler chickens and 80 strains isolated after human infection identified two phage candidates with broad host lysis. These phages, CP8 and CP34, were orally administered in antacid suspension, at different dosages, to 25-day-old broiler chickens experimentally colonized with the C. jejuni broiler isolates. Phage treatment of C. jejuni-colonized birds resulted in Campylobacter counts falling between 0.5 and 5 log10 CFU/g of cecal contents compared to untreated controls over a 5-day period postadministration. These reductions were dependent on the phage-Campylobacter combination, the dose of phage applied, and the time elapsed after administration. Campylobacters resistant to bacteriophage infection were recovered from phage-treated chickens at a frequency of <4%. These resistant types were compromised in their ability to colonize experimental chickens and rapidly reverted to a phage-sensitive phenotype in vivo. The selection of appropriate phage and their dose optimization are key elements for the success of phage therapy to reduce campylobacters in broiler chickens.
Project description:Campylobacter is one of the major foodborne pathogens that result in severe gastroenteritis in humans, primarily through consumption of contaminated poultry products. Chickens are the reservoir host of Campylobacter, where the pathogen colonizes the ceca, thereby leading to contamination of carcass during slaughter. A reduction in cecal colonization by Campylobacter would directly translate into reduced product contamination and risk of human infections. With increasing consumer demand for antibiotic free chickens, significant research is being conducted to discover natural, safe and economical antimicrobials that can effectively control Campylobacter colonization in birds. This study investigated the efficacy of in-feed supplementation of a phytophenolic compound, ?-resorcylic acid (BR) for reducing Campylobacter colonization in broiler chickens. In two separate, replicate trials, day-old-chicks (Cobb500; n = 10 birds/treatment) were fed with BR (0, 0.25, 0.5, or 1%) in feed for a period of 14 days (n = 40/trial). Birds were challenged with a four-strain mixture of Campylobacter jejuni (?106 CFU/ml; 250 ?l/bird) on day 7 and cecal samples were collected on day 14 for enumerating surviving Campylobacter in cecal contents. In addition, the effect of BR on the critical colonization factors of Campylobacter (motility, epithelial cell attachment) was studied using phenotypic assay, cell culture, and real-time quantitative PCR. Supplementation of BR in poultry feed for 14 days at 0.5 and 1% reduced Campylobacter populations in cecal contents by ?2.5 and 1.7 Log CFU/g, respectively (P < 0.05). No significant differences in feed intake and body weight gain were observed between control and treatment birds fed the compound (P > 0.05). Follow up mechanistic analysis revealed that sub-inhibitory concentration of BR significantly reduced Campylobacter motility, attachment to and invasion of Caco-2 cells. In addition, the expression of C. jejuni genes coding for motility (motA, motB, fliA) and attachment (jlpA, ciaB) was down-regulated as compared to controls (P < 0.05). These results suggest that BR could potentially be used as a feed additive to reduce Campylobacter colonization in broilers.
Project description:Despite reducing the prevalent foodborne pathogen Campylobacter jejuni in chickens decreases campylobacteriosis, few effective approaches are available. The aim of this study was to use microbial metabolic product bile acids to reduce C. jejuni chicken colonization. Broiler chicks were fed with deoxycholic acid (DCA), lithocholic acid (LCA), or ursodeoxycholic acid (UDCA). The birds were also transplanted with DCA modulated anaerobes (DCA-Anaero) or aerobes (DCA-Aero). The birds were infected with human clinical isolate C. jejuni 81-176 or chicken isolate C. jejuni AR101. Notably, C. jejuni 81-176 was readily colonized intestinal tract at d16 and reached an almost plateau at d21. Remarkably, DCA excluded C. jejuni cecal colonization below the limit of detection at 16 and 28 days of age. Neither chicken ages of infection nor LCA or UDCA altered C. jejuni AR101 chicken colonization level, while DCA reduced 91% of the bacterium in chickens at d28. Notably, DCA diet reduced phylum Firmicutes but increased Bacteroidetes compared to infected control birds. Importantly, DCA-Anaero attenuated 93% of C. jejuni colonization at d28 compared to control infected birds. In conclusion, DCA shapes microbiota composition against C. jejuni colonization in chickens, suggesting a bidirectional interaction between microbiota and microbial metabolites.
Project description:Using laboratory challenge experiments, we examined whether Campylobacter-specific maternal antibody (MAB) plays a protective role in young chickens, which are usually free of Campylobacter under natural production conditions. Kinetics of C. jejuni colonization were compared by infecting 3-day-old broiler chicks, which were naturally positive for Campylobacter-specific MAB, and 21-day-old broilers, which were negative for Campylobacter-specific MAB. The onset of colonization occurred much sooner in birds challenged at the age of 21 days than it did in the birds inoculated at 3 days of age, which suggested a possible involvement of specific MAB in the delay of colonization. To further examine this possibility, specific-pathogen-free layer chickens were raised under laboratory conditions with or without Campylobacter infection, and their 3-day-old progenies with (MAB(+)) or without (MAB(-)) Campylobacter-specific MAB were orally challenged with C. jejuni. Significant decreases in the percentage of colonized chickens were observed in the MAB(+) group during the first week compared with the MAB(-) group. These results indicate that Campylobacter-specific MAB plays a partial role in protecting young chickens against colonization by C. jejuni. Presence of MAB in young chickens did not seem to affect the development of systemic immune response following infection with C. jejuni. However, active immune responses to Campylobacter occurred earlier and more strongly in birds infected at 21 days of age than those infected at 3 days of age. Clearance of Campylobacter infection was also observed in chickens infected at 21 days of age. Taken together, these findings (i) indicate that anti-Campylobacter MAB contributes to the lack of Campylobacter infection in young broiler chickens in natural environments and (ii) provide further evidence supporting the feasibility of development of immunization-based approaches for control of Campylobacter infection in poultry.
Project description:Four wild-type Campylobacter jejuni strains isolated from the cecal contents of broiler chickens were sequenced. The average genome size was 1,622,170?bp, with 1,667 to 1,761 coding sequences and 47 to 51 RNAs. Multiple genes encoding motility, intestinal colonization, toxin production, stress tolerance, and multidrug resistance were present in all the strains.
Project description:Source attribution studies report that the consumption of contaminated poultry is the primary source for acquiring human campylobacteriosis. Oral administration of an engineered Escherichia coli strain expressing the Campylobacter jejuni N-glycan reduces bacterial colonization in specific-pathogen-free leghorn chickens, but only a fraction of birds respond to vaccination. Optimization of the vaccine for commercial broiler chickens has great potential to prevent the entry of the pathogen into the food chain. Here, we tested the same vaccination approach in broiler chickens and observed similar efficacies in pathogen load reduction, stimulation of the host IgY response, the lack of C. jejuni resistance development, uniformity in microbial gut composition, and the bimodal response to treatment. Gut microbiota analysis of leghorn and broiler vaccine responders identified one member of Clostridiales cluster XIVa, Anaerosporobacter mobilis, that was significantly more abundant in responder birds. In broiler chickens, coadministration of the live vaccine with A. mobilis or Lactobacillus reuteri, a commonly used probiotic, resulted in increased vaccine efficacy, antibody responses, and weight gain. To investigate whether the responder-nonresponder effect was due to the selection of a C. jejuni "supercolonizer mutant" with altered phase-variable genes, we analyzed all poly(G)-containing loci of the input strain compared to nonresponder colony isolates and found no evidence of phase state selection. However, untargeted nuclear magnetic resonance (NMR)-based metabolomics identified a potential biomarker negatively correlated with C. jejuni colonization levels that is possibly linked to increased microbial diversity in this subgroup. The comprehensive methods used to examine the bimodality of the vaccine response provide several opportunities to improve the C. jejuni vaccine and the efficacy of any vaccination strategy.IMPORTANCE Campylobacter jejuni is a common cause of human diarrheal disease worldwide and is listed by the World Health Organization as a high-priority pathogen. C. jejuni infection typically occurs through the ingestion of contaminated chicken meat, so many efforts are targeted at reducing C. jejuni levels at the source. We previously developed a vaccine that reduces C. jejuni levels in egg-laying chickens. In this study, we improved vaccine performance in meat birds by supplementing the vaccine with probiotics. In addition, we demonstrated that C. jejuni colonization levels in chickens are negatively correlated with the abundance of clostridia, another group of common gut microbes. We describe new methods for vaccine optimization that will assist in improving the C. jejuni vaccine and other vaccines under development.
Project description:Campylobacteriosis is the leading foodborne bacterial diarrheal illness in many countries, with up to 80% of human cases attributed to the avian reservoir. The only control strategies currently available are stringent on-farm biosecurity and carcass treatments. Heritable differences in the resistance of chicken lines to Campylobacter colonization have been reported and resistance-associated quantitative trait loci are emerging, although their impact on colonization appears modest. Recent studies indicated a protective role of the microbiota against colonization by Campylobacter in chickens. Furthermore, in murine models, differences in resistance to bacterial infections can be partially transferred between lines by transplantation of gut microbiota. In this study, we investigated whether heritable differences in colonization of inbred chicken lines by Campylobacter jejuni are associated with differences in cecal microbiota. We performed homologous and heterologous cecal microbiota transplants between line 61 (resistant) and line N (susceptible) by orally administering cecal contents collected from 3-week-old donors to day-of-hatch chicks. Recipient birds were challenged (day 21) with C. jejuni 11168H. In birds given homologous microbiota, the differential resistance of lines to C. jejuni colonization was reproduced. Contrary to our hypothesis, transfer of cecal microbiota from line 61 to line N significantly increased C. jejuni colonization. No significant difference in the overall composition of the cecal microbial communities of the two lines was identified, although line-specific differences for specific operational taxonomic units were identified. Our data suggest that while heritable differences in avian resistance to Campylobacter colonization exist, these are not explained by significant variation in the cecal microbiota.IMPORTANCE Campylobacter is a leading cause of foodborne diarrheal disease worldwide. Poultry are a key source of human infections, but there are currently few effective measures against Campylobacter in poultry during production. One option to control Campylobacter may be to alter the composition of microbial communities in the avian intestines by introducing beneficial bacteria, which exclude the harmful ones. We previously described two inbred chicken lines which differ in resistance to intestinal colonization by Campylobacter Here, we investigated the composition of the microbial communities in the gut of these lines and whether transferring gut bacteria between the resistant and susceptible lines alters their resistance to Campylobacter No major differences in microbial populations were found, and resistance or susceptibility to colonization was not conferred by transferring gut bacteria between lines. The data suggest that gut microbiota did not play a role in resistance to Campylobacter colonization, at least in the lines used.
Project description:The Campylobacter jejuni-host interaction may be affected by the host's gut microbiota through competitive exclusion, metabolites, or modification of the immune response. To understand this interaction, C. jejuni colonization and local immune responses were compared in chickens with different gut microbiota compositions. Birds were treated with an antibiotic cocktail (AT) (experiments 1 and 2) or raised under germfree (GF) conditions (experiment 3). At 18 days posthatch (dph), they were orally inoculated either with 104 CFU of C. jejuni or with diluent. Cecal as well as systemic C. jejuni colonization, T- and B-cell numbers in the gut, and gut-associated tissue were compared between the different groups. Significantly higher numbers of CFU of C. jejuni were detected in the cecal contents of AT and GF birds, with higher colonization rates in spleen, liver, and ileum, than in birds with a conventional gut microbiota (P < 0.05). Significant upregulation of T and B lymphocyte numbers was detected in cecum, cecal tonsils, and bursa of Fabricius of AT or GF birds after C. jejuni inoculation compared to the respective controls (P < 0.05). This difference was less clear in birds with a conventional gut microbiota. Histopathological gut lesions were observed only in C. jejuni-inoculated AT and GF birds but not in microbiota-colonized C. jejuni-inoculated hatchmates. These results demonstrate that the gut microbiota may contribute to the control of C. jejuni colonization and prevent lesion development. Further studies are needed to identify key players of the gut microbiota and the mechanisms behind their protective role.
Project description:Reducing colonization of poultry flocks by Campylobacter spp. is a key strategy in the control and prevention human campylobacteriosis. Horizontal transmission of campylobacters, from in and around the farm, is the presumed route of flock colonization. However, the identification and prioritization of sources are confounded by the ubiquitous nature of these organisms in the environment, their poor rates of recovery by standard culture methods, and the need for cost-effective and timely methods for strain-specific comparison. A real-time PCR screening test for the strain-specific detection of campylobacters in environmental samples has been developed to address this issue. To enable this approach, fluorescently labeled PCR oligonucleotide probes suitable for a LightCycler-based assay were designed to match a highly variable DNA segment within the flaA short variable region (SVR) of Campylobacter jejuni or C. coli. The capacity of such probes to provide strain-specific tools was investigated by using bacterial cultures and spiked and naturally contaminated poultry fecal and environmental samples. The sensitivity of two representative probes was estimated, by using two different C. jejuni strains, to be 1.3 x 10(2) to 3.7 x 10(2) CFU/ml in bacterial cultures and 6.6 x 10(2) CFU/ml in spiked fecal samples. The specificity of the SVR for C. jejuni and C. coli was confirmed by using a panel of strains comprising other Campylobacter species and naturally contaminated samples. The approach was field tested by sampling the environment and feces of chickens of two adjacently located poultry houses on a conventional broiler farm throughout the life of one flock. All environmental samples were enriched for 2 days, and then DNA was prepared and stored. Where feasible, campylobacter isolates were also recovered and stored for subsequent testing. A strain-specific probe based on the SVR of the strain isolated from the first positive chicken fecal sample was developed. This probe was then used to screen the stored environmental samples by real-time PCR. Pulsed-field gel electrophoresis was used to compare recovered environmental and fecal isolates to assess the specificity of the method. The results established the proof of principle that strain-specific probes, based on the SVR of flaA, can identify a flock-colonizing strain in DNA preparations from enriched environmental cultures. Such a novel strategy provides the opportunity to investigate the epidemiology of campylobacters in poultry flocks and allows targeted biosecurity interventions to be developed. The strategy may also have wider applications for the tracking of specific campylobacter strains in heavily contaminated environments.
Project description:This study describes the development and use of bacteriophage cocktails to control Campylobacter in broiler chickens, in a commercial setting, in Queensland Australia, following the birds from farm to the processing plant. The components of the bacteriophage cocktails were selected to be effective against the maximum number of Campylobacter jejuni and Campylobacter coli isolates encountered on SE Queensland farms. Farms were identified that had suitable Campylobacter target populations and phage were undetectable 1 week prior to the intended treatment. Cocktails of phages were administered at 47 days of age. Groups of study birds were slaughtered the following day, on-farm, at the end of flock transport to the plant, and at processing (approximately 28 h post-treatment). On Farm A, the phage treatment significantly reduced Campylobacter levels in the ceca at the farm in the range of 1-3 log10 CFU/g (p = 0.007), compared to mock treated controls. However, individual birds sampled on farm (1/10) or following transport (2/10) exhibited high cecal Campylobacter counts with low phage titers, suggesting that treatment periods > 24 h may be required to ensure phage replication for effective biocontrol in vivo. At the time of the trial the control birds in Farm B were phage positive despite having been negative one week earlier. There was no significant difference in the cecal Campylobacter counts between the treatment and control groups following treatment but a fall of 1.7 log10 CFU/g was observed from that determined from birds collected the previous week (p = 0.0004). Campylobacter isolates from both farms retained sensitivity to the treatment phages. These trials demonstrated bacteriophages sourced from Queensland farms have the potential to reduce intestinal Campylobacter levels in market ready broiler chickens.