Campylobacter Jejuni Colonization Research Articles

89. Impact of deoxynivalenol (DON) and its derivate Deepoxy-deoxynivalenol (DOM-1) on broiler performance and Campylobacter jejuni colonization and translocation in broiler chickens

89. Impact of deoxynivalenol (DON) and its derivate Deepoxy-deoxynivalenol (DOM-1) on broiler performance and Campylobacter jejuni colonization and translocation in broiler chickens

Assessment of probiotic Bacillus velezensis supplementation to reduce Campylobacter jejuni colonization in chickens

Campylobacter jejuni continues to be a major public health issue worldwide. Poultry are recognized as the main reservoir for this foodborne pathogen. Implementing measures to decrease C. jejuni colonization on farms has been regarded as the most effective strategy to control the incidence of campylobacteriosis. The probiotics supplementation has been regarded as an attractive approach against C. jejuni in chickens. Here the inhibitory effects of one probiotic B. velezensis isolate CAU277 against C. jejuni was evaluated in vitro and in vivo. The in vitro antimicrobial activity showed that the supernatant of B. velezensis exhibited the most pronounced inhibitory effects on Campylobacter strains compared to other bacterial species. When co-cultured with B. velezensis, the growth of C. jejuni reduced significantly from 7.46 log10 CFU/mL (24 h) to 1.02 log10 CFU/mL (48 h). Further, the antimicrobial activity of B. velezensis against C. jejuni remained stable under a broad range of temperature, pH, and protease treatments. The in vivo experiments demonstrated that oral administration of B. velezensis significantly reduced the colonization of C. jejuni by 2.0 log10 CFU/g of feces in chicken cecum at 15 d postinoculation. In addition, the supplementary of B. velezensis significantly increased microbial species richness and diversity in chicken ileum, especially enhanced the bacterial population of Alistipes and Christensenellaceae, and decreased the existence of Lachnoclostridium. Our study presents that B. velezensis possesses antimicrobial activities against C. jejuni and promotes microbiota diversity in chicken intestines. These findings indicate a potential to develop an effective probiotic additive to control C. jejuni infection in chicken.

Antibiotic-induced microbiome depletion promotes intestinal colonization by Campylobacter jejuni in mice

BackgroundTo establish a method to induce Campylobacter jejuni colonization in the intestines of C57BL/6 mice through antibiotic-induced microbiome depletion.ResultsFifty-four female C57BL/6 mice were divided into the normal, control, and experimental groups. The experimental group was administered intragastric cefoperazone sodium and sulbactam sodium (50 mg/mL) for 2 days; then, the experimental and control mice were intragastrically administered 200 µL C. jejuni, which was repeated once more after 2 days. Animal feces were collected, and the HipO gene of C. jejuni was detected using TaqMan qPCR from day 1 to day 14 after modeling completion. Immunofluorescence was used to detect intestinal C. jejuni colonization on day 14, and pathological changes were observed using hematoxylin and eosin staining. Additionally, 16S rDNA analyses of the intestinal contents were conducted on day 14. In the experimental group, C. jejuni was detected in the feces from days 1 to 14 on TaqMan qPCR, and immunofluorescence-labeled C. jejuni were visibly discernable in the intestinal lumen. The intestinal mucosa was generally intact and showed no significant inflammatory-cell infiltration. Diversity analysis of the colonic microbiota showed significant inter-group differences. In the experimental group, the composition of the colonic microbiota differed from that in the other 2 groups at the phylum level, and was characterized by a higher proportion of Bacteroidetes and a lower proportion of Firmicutes.ConclusionsMicrobiome depletion induced by cefoperazone sodium and sulbactam sodium could promote long-term colonization of C. jejuni in the intestines of mice.

Fecal Microbiota Transplantation Reduces Campylobacter jejuni Colonization in Young Broiler Chickens Challenged by Oral Gavage but Not by Seeder Birds.

Campylobacter spp., particularly C. jejuni and C. coli, are major food safety concerns, transmitted to humans mainly via contaminated poultry meat. In a previous study, we found that some commercial broiler farms consistently produced Campylobacter-free flocks while others consistently reared Campylobacter-colonized flocks, and significant differences in the gut microbiota compositions between the two types of farm categories were revealed. Therefore, we hypothesized that gut microbiota influences Campylobacter colonization in poultry and that the microbiota from Campylobacter-free flocks may confer colonization resistance to Campylobacter in the chicken intestine. In this study, two fecal microbiota transplantation (FMT) trials were performed to test the hypothesis. Newly hatched chicks were given FMT via oral gavage of the cecal content of Campylobacter-free adult chickens (treatment groups) or PBS (control groups) before the feed consumption. Approximately two weeks after the FMT, the birds were challenged with C. jejuni either by oral gavage (trial 1) or by co-mingling with Campylobacter-colonized seeder birds (trial 2) to evaluate the potential protective effect of the FMT. Cecal contents were collected (3 times, 5 days apart) to determine the Campylobacter colonization levels via culture and microbiota compositions via 16S rRNA gene sequencing. FMT reduced cecal Campylobacter colonization significantly (log10 1.2-2.54 CFU/g) in trial 1 but not in trial 2, although FMT significantly impacted the diversity and compositions of the gut microbiota in both trials. Several genera, such as Butyricimonas, Parabacteroides, Parasutterella, Bilophila, Fournierella, Phascolarctobacterium, and Helicobacter, had increased abundance in the FMT-treated groups in both trials. Furthermore, Campylobacter abundance was found to be negatively correlated with the Escherichia and Ruminococcus_torques_group genera. These findings indicate that even though FMT with adult cecal microbiota can positively affect the subsequent development of the gut microbiota in young broilers, its inhibitory effect on Campylobacter colonization varies and appears to be influenced by the challenge models.

Evaluation of N-glycan-decorated live attenuated Escherichia coli and outer membrane vesicles as vaccines against Campylobacter jejuni colonisation in chickens

Campylobacter jejuni is a leading global cause of bacterial gastroenteritis in humans, and poultry are a major reservoir. Glycoconjugate vaccines containing the conserved C. jejuni N-glycan have previously been reported to be effective at reducing caecal colonisation of chickens by C. jejuni. These include recombinant subunit vaccines, live E. coli strains expressing the N-glycan on the surface as well as outer membrane vesicles (OMVs) derived from these E. coli strains. In this study, we evaluated the efficacy of live E. coli expressing the C. jejuni N-glycan from a plasmid and glycosylated OMVs (G-OMVs) derived from them against colonisation by different C. jejuni strains. Despite the C. jejuni N-glycan being expressed on the surface of the live strain and the OMVs, no reduction in caecal colonisation by C. jejuni was observed and N-glycan-specific responses were not detected.

Immunopathogenesis of Campylobacter jejuniinfection in a small animal model

Abstract We set out to develop and characterize a small animal model of Campylobacter jejuni(CJ) infection that recapitulates human campylobacteriosis. Adult C57BL/6J mice are rendered susceptible to colonization and disease by pre-treatment with a zinc deficient diet and a broad-spectrum antibiotic cocktail. We have established this model with four strains with diverse capsular serotypes and flagellar groups, two key virulence factors for CJ pathogenesis. We measured colonization, weight loss, diarrhea, fecal inflammatory markers, and cytokine production by mesenteric lymphocytes and splenocytes. Diarrhea containing visible mucous and/or blood and degree of weight loss vary in severity depending on the strain and dose. Interestingly, we identified an inverse relationship between inoculum dose and levels of fecal inflammatory markers, with lower inoculum doses inducing significantly higher inflammation. We also observed production of IFNγ and IL-17 at day 9 post infection and despite no decrease in CJ colonization, IFNγ and IL-17 levels decreased by day 21 with a subsequent increase in IL-10 production. We also observed higher levels of IFNγ and IL-17 in mice challenged with strain CG8486 relative to those challenged with strain 81–176 pointing to potential strain differences. These differences observed were more striking in mesenteric lymphocytes versus splenocytes, indicating that local cellular responses differed from systemic responses. We have developed a model of inflammatory diarrhea in adult mice that exhibits hallmarks of CJ infection and further identified significant shifts in cytokine expression associated with the duration of infection, bacterial strain utilized, and therapeutic treatment. Research reported in this presentation is supported by Navy work unit number: 6000.RAD1.DA3.A0308 and CARB-X. CARB-X’s funding for this project is sponsored by the Cooperative Agreement Number IDSEP160030 from ASPR/BARDA and by awards from Wellcome Trust, the UK Global Antimicrobial Resistance Innovation Fund (GAMRIF) funded by the UK Government Department of Health and Social Care (DHSC) and the Bill & Melinda Gates Foundation. The content is solely the responsibility of the authors and does not necessarily represent the official views of CARB-X or any of its funders. Disclaimers: The views expressed in this work are those of the authors and do not necessarily reflect the official policy or position of the Department of the Navy, Department of Defense, nor the U.S. Government. F. Poly is an employee of the U.S. Government. This work was prepared as part of official duties. Title 17 U.S.C. §105 provides that ‘Copyright protection under this title is not available for any work of the United States Government.’ Title 17 U.S.C. §101 defines a U.S. Government work as a work prepared by a military service member or employee of the U.S. Government as part of that person’s official duties.The animal study protocol was reviewed and approved by the Naval Medical Research Center IACUC in compliance with all applicable Federal regulations governing the protection of animals in research.

Effects of dietary yeast cell wall supplementation on growth performance, intestinal Campylobacter jejuni colonization, innate immune response, villus height, crypt depth, and slaughter characteristics of broiler chickens inoculated with Campylobacter jejuni at d 21

A study was conducted to assess the effects of a dietary yeast cell wall (YCW) with and without a Campylobacter jejuni (CJ) challenge. A total of 2,240-day-old Ross 708 males were randomly assigned within 8 treatments with a 4×2 factorial design, with 4 diets (negative control, positive control, YCW constant dose (400 g/ton), and YCW step-down dose (800/400/200 g/ton in the starter/grower/finisher diets, respectively) and with and without d 21 CJ oral gavage challenge at 5.2×107 CFU/mL. At d 0, 14, 28, and 41 body weights and feed consumption were measured to determine performance. At d 14, 28, and 42, 8 jejunal and ileal histology samples per treatment were collected for villi morphology measurements. At d 22 and 28 (1- and 7-days postinoculation), 24 ileal tissue samples per treatment were collected for relative gene expression analysis. At d 42, 24 cecal content samples per treatment were collected for CJ enumeration. Finally, on d 44, 96 birds per treatment were processed to determine carcass yield and 16 carcass rinses per treatment were collected to determine CJ prevalence after processing. Diet or inoculation did not impact broiler performance (P > 0.05). Limited differences were observed in intestinal morphology, and villus height and crypt depth were different only in the ileum at d 42 (P=0.0280 and P=0.0162, respectively). At d 1 postinoculation, differences between treatments inoculated with CJ and PBS were observed in the expression of avian beta defensin 10 (AvBD10), interleukin 1ß (IL-1ß), and interleukin 10 (IL-10) (P < 0.05). At d 7 postinoculation, expression of AvBD10, IL-1ß, and IL-10 was similar among all treatments (P > 0.05). At d 42, all birds, regardless the inoculation, had similar levels of CJ recovered from cecal contents (P > 0.05). After processing, carcass yield and CJ prevalence postchilling was similar in all treatments (P > 0.05). Overall, under the conditions of this study, the addition of YCW during a CJ challenge did not have an impact in growth performance, innate immune response, cecal colonization, carcass yield, or CJ prevalence after processing.

Prospective Application of Nanoencapsulated Bacillus amyloliquefaciens on Broiler Chickens’ Performance and Gut Health with Efficacy against Campylobacter jejuni Colonization

Probiotics as novel antibiotics' substitutes are verified to provide barriers for hindering the colonization of enteric bacterial pathogens with nutritional benefits. For enhancement of the probiotics' effectiveness, their integration within nanomaterials is a paramount tool to support the progress of new compounds with functional features. Therefore, we addressed the impact of effective delivery of probiotics (Bacillus amyloliquefaciens) loaded nanoparticles (BNPs) on performance and Campylobacter jejuni (C. jejuni) shedding and colonization in poultry. Two hundred Ross broiler chickens were divided into four groups fed various BNP levels: BNPs I, BNPs II, BNPs III, and BNPs-free diets for 35 days. Nanoparticles delivery of probiotics within broiler diets improved growth performance as reflected by higher body weight gain and superior feed conversion ratio, especially in BNPs II- and BNPs III-fed groups. In parallel, the mRNA expression levels of digestive enzymes encoding genes (AMY2a, PNLIP, CELA1, and CCK) achieved their peaks in BNPs III-fed group (1.69, 1.49, 1.33, and 1.29-fold change, respectively) versus the control one. Notably, with increasing the levels of BNPs, the abundance of beneficial microbiota, such as Bifidobacterium and Lactobacillus species, was favored over harmful ones, including Clostridium species and Enterobacteriaceae. Birds fed higher levels of BNPs displayed significant improvement in the expression of barrier functions-linked genes including DEFB1, FABP-2, and MUC-2 alongside substantial reduction in cecal colonization and fecal shedding of C. jejuni. From the aforementioned positive effects of BNPs, we concluded their potential roles as growth promoters and effective preventive aids for C. jejuni infection in poultry.

Tryptanthrin Reduces Campylobacter jejuni Colonization in the Chicken Gut by a Bactericidal Mechanism.

Campylobacter jejuni is a leading cause of foodborne bacterial gastroenteritis worldwide, and raw or undercooked chicken meat is considered the major source of human campylobacteriosis. In this study, we identified 36 compounds that showed inhibitory effects on C. jejuni growth at low concentrations by screening a chemical compound library. Three of the 36 compounds were herbal compounds, including tryptanthrin (TRP), an indoloquinazoline alkaloid. TRP has been reported to have a variety of biological properties, such as antimicrobial, anti-inflammatory, and antitumor activities, but there was previously no information about its anti-C. jejuni activity. We further conducted in vitro and in vivo experiments to evaluate the potential of TRP for the control of C. jejuni in chicken farms. The MIC of TRP for C. jejuni was much lower than that of 13 other herbal compounds that were previously reported to have anti-C. jejuni activities. Time-kill assays under growing and nongrowing conditions demonstrated that TRP has bactericidal activity against C. jejuni. In addition, TRP showed a narrow-spectrum antimicrobial effect against C. jejuni, and there was little potential for the development of TRP-resistant C. jejuni during serially passaged culture. In chick infection experiments, the administration of TRP in drinking water significantly reduced the cecal colonization of C. jejuni when TRP was used either before or after C. jejuni infection. These data suggest that TRP is effective for the control of C. jejuni in chicken farms. IMPORTANCE Campylobacter is a widespread pathogen in the food chain of chickens. Once chickens become infected, large numbers of Campylobacter cells are excreted in their feces. The development of an effective material for reducing the amount of Campylobacter in the chicken intestinal tract will make it possible to reduce the contamination of the food chain with Campylobacter and to produce safe and secure chicken meat. In the present study, in vivo experiments revealed that the use of an herbal compound, tryptanthrin, significantly reduced the number of Campylobacter cells in the chicken gut by a bactericidal mechanism. Furthermore, our in vitro experiments demonstrated that, compared with the other herbal compounds, tryptanthrin achieved antimicrobial activity against C. jejuni at the lowest concentration. The use of tryptanthrin may lead to the development of a novel control measure for reducing the colonization of C. jejuni in the food chain.

Bacillus subtilis PS-216 Spores Supplemented in Broiler Chicken Drinking Water Reduce Campylobacter jejuni Colonization and Increases Weight Gain

Campylobacter jejuni is the leading cause of bacterial gastroenteritis, or campylobacteriosis, in humans worldwide, and poultry serves as a major source of infection. To reduce the risk associated with C. jejuni transmission via poultry meat, effective interventions during poultry production are needed, and the use of probiotics is a promising approach. In this study, 15 Bacillus subtilis strains were initially screened for their anti-Campylobacter activities. B. subtilis PS-216 strain demonstrated the best anti-Campylobacter activity against 15 C. jejuni isolates when examined using in vitro co-cultures. To evaluate the suitability of B. subtilis PS-216 for probiotic use, its susceptibility to eight clinically important antimicrobials and simulated gastric conditions was investigated. B. subtilis PS-216 was sensitive to all of the tested antibiotics. Although vegetative cells were sensitive to gastric conditions, B. subtilis PS-216 spores were highly resistant. We further evaluated the use of a B. subtilis PS-216 spore preparation (2.5 × 106 CFU/mL water) to prevent and/or reduce C. jejuni colonization in broiler chickens in vivo. Compared to the untreated group, significantly lower Campylobacter counts were detected in caeca of broilers continuously treated with B. subtilis PS-216 spores in their drinking water. Furthermore, broilers continuously treated with B. subtilis PS-216 spores showed improved weight gain, compared to the control group. Together, these results demonstrate the potential of B. subtilis PS-216 for use in poultry to reduce C. jejuni colonization and improve weight gain.

Research Note: Effect of a phlorotannin extract of the brown seaweed Ascophyllum nodosum as a potential control strategy against Campylobacter in broilers

Poultry is generally recognized as the main source of human campylobacteriosis and Campylobacter is highly prevalent at the farm level. To reduce the relative risk of human campylobacteriosis attributable to broiler meat, it is necessary to reduce Campylobacter loads in broiler ceca but to date, no effective, reliable and practical strategy is available. The marine environment is a rich source of original natural compounds exhibiting different biological activities. The objective of this study was to test a phlorotannin extract of the brown seaweed Ascophyllum nodosum as a potential control strategy against Campylobacter in broilers. Bactericidal activity has been demonstrated in vitro, on several Campylobacter spp. strains at a range of 0.06 to 0.47 mg/mL. Therefore, an in vivo trial in experimental facilities was performed to evaluate addition of 0.2% (w/w) of an A. nodosum extract to feed distributed at the end of rearing from day 31 to day 35, and to assess the effect on artificial Campylobacter jejuni colonization. No statistical differences in Campylobacter enumeration were observed between the treated and control groups. Another trial was performed in a commercial broiler flock. Feed containing the extract at 0.2% (w/w) (2 kg/t) was distributed during the last 5 days of rearing (day 33–day 38). No significant effects on Campylobacter colonization and on growth parameters were observed compared to the control group. Additional studies are needed to assess whether active polyphenols are found in the cecum.

Bioinformatic Analysis for Iron-Uptake System of Campylobacter spp. in humans and chickens

Asymptomatic Campylobacter jejuni colonisation is highly prevalent in chickens, however, it’s a major cause of bacterial foodborne disease in humans. Iron is an essential co-factor in many physiological processes and Campylobacter strains employ several non-redundant iron acquisition systems for survival and colonization. The outer membrane receptors CfrA and CfrB are involved in iron acquisition fromcatecholamine siderophores and are required for colonisation of the chicken GI tract. In contrast, ChuAB are part of an uptake system, enabling the utilization of iron in haem and is not required for colonisation. This study aims to compare differences in the iron-uptake systems present in Campylobacter strains isolated from chickens or humans. Analysis of Campylobacter jejuni/coli genome sequences in PubMLST (https://pubmlst.org/organisms/campylobacter-jejunicoli [https://pubmlst.org/organisms/campylobacter-jejunicoli]) added from the UK in 2018 reveals differences in sequence type (ST) and chuA, chuB, cfrA and cfrB between isolates from chicken (CI) or human (HI) sources. ST828 was the most common ST for the C. coliCI (45%) and ST827 (32%) for the HI. C. jejuniST5136 was the most common of the HI (10%) and CI (13%), ST50 (8%) for the HI and ST21 (7%) for the CI. &gt;95% of isolates contained chuAand chuB, we also found the Chu system to be highly conserved. For cfrA81% of HI and 74% of CI contained the gene, and 92% of HI and 96% of CI harboured cfrB.&lt;3% of all isolates had neither cfrAor cfrB, cfrBmore associated with CI than HI. This data analysis reveals potential gene targets towards vaccine development against Campylobacter strains.

Deepoxy-deoxynivalenol (DOM-1), a derivate of deoxynivalenol (DON), exhibits less toxicity on intestinal barrier function, Campylobacter jejuni colonization and translocation in broiler chickens

BackgroundIntestinal epithelial cells are challenged by mycotoxins and many bacterial pathogens. It was previously shown that the mycotoxin deoxynivalenol (DON) as well as Campylobacter (C.) jejuni have a negative impact on gut integrity. Recently, it was demonstrated that DON increased the load of C. jejuni in the gut and inner organs. Based on this finding, it was hypothesized the DON metabolite (deepoxy-deoxynivalenol, DOM-1) should be able to reduce the negative effects of DON on colonization and translocation of C. jejuni in broilers, since it lacks the epoxide ring, which is responsible for the toxicity of DON.MethodsA total of 180 broiler chickens were housed in floor pens on wood shavings with feed and water provided ad libitum. Birds were divided into six groups (n = 30 with 5 replicates/group): 1. Control, 2. DOM-1, 3. DON, 4. DOM-1 + C. jejuni, 5. DON + C. jejuni, 6. C. jejuni. At day 14, birds of groups 4, 5 and 6 were orally inoculated via feeding tube (gavage) with 1-ml of a PBS suspension containing 1 × 108 CFU of C. jejuni NCTC 12744. The performance parameters: body weight (BW), body weight gain (BWG), and feed intake of the birds were determined. At 7, 14, and 21 days post infection, samples from liver, spleen, duodenum, jejunum and cecum were aseptically collected and processed for bacteriological investigations. Finally, at each killing time point, segments of duodenum, jejunum and cecum were harvested and prepared for Ussing chamber studies to measure the paracellular mannitol fluxes.ResultsA significant decrease in body weight was observed for chickens receiving the DON diet with or without C. jejuni compared to the other groups. Furthermore, it was found that the co-exposure of birds to DON and C. jejuni resulted in a higher C. jejuni load not only in the gut but also in liver and spleen due to increased paracellular permeability of the duodenum, jejunum and cecum. On the contrary, DOM-1 supplementation in the feed improved the birds’ performance and led to a better feed conversion ratio throughout the trial. Furthermore, DOM-1 did not negatively affect gut permeability and decreased the C. jejuni counts in the intestine and internal organs.ConclusionAltogether, the presence of DOM-1 in the feed as a result of the enzymatic biotransformation of DON leads to a lower C. jejuni count in the intestine and better feed conversion ratio. Moreover, this study demonstrates that the detoxification product of DON, DOM-1, does not have negative effects on the gastrointestinal tract and reduces the Campylobacter burden in chickens and also the risk for human infection.

Long-Term Grow-Out Affects Campylobacter jejuni Colonization Fitness in Coincidence With Altered Microbiota and Lipid Composition in the Cecum of Laying Hens.

Campylobacter jejuni is one of the leading causes of gastrointestinal illness worldwide and is mainly transmitted from chicken through the food chain. Previous studies have provided increasing evidence that this pathogen can colonize and replicate in broiler chicken during its breeding; however, its temporal kinetics in laying hen are poorly understood. Considering the possible interaction between C. jejuni and gut microbiota, the current study was conducted to address the temporal dynamics of C. jejuni in the cecum of laying hen over 40 weeks, with possible alteration of the gut microbiota and fatty acid (FA) components. Following oral infection with C. jejuni 81-176, inocula were stably recovered from ceca for up to 8 weeks post-infection (p.i.). From 16 weeks p.i., most birds became negative for C. jejuni and remained negative up to 40 weeks p.i. 16S rRNA gene sequencing analyses revealed that most of the altered relative rRNA gene abundances occurred in the order Clostridiales, in which increased relative rRNA gene abundances were observed at >16 weeks p.i. in the families Clostridiaceae, Ruminococcaceae, Lachnospiraceae, and Peptococcaceae. Lipidome analyses revealed increased levels of sterols associated with bile acid metabolisms in the cecum at 16 and/or 24 weeks p.i. compared with those detected at 8 weeks p.i., suggesting that altered microbiota and bile acid metabolism might underlie the decreased colonization fitness of C. jejuni in the gut of laying hens.

Characterizing the immune response of chickens to Campylobacter jejuni (Strain A74C).

Campylobacter is one of the major foodborne pathogens causing bacterial gastroenteritis worldwide. The immune response of broiler chickens to C. jejuni is under-researched. This study aimed to characterize the immune response of chickens to Campylobacter jejuni colonization. Birds were challenged orally with 0.5 mL of 2.4 x 108 CFU/mL of Campylobacter jejuni or with 0.5 mL of 0.85% saline. Campylobacter jejuni persisted in the ceca of challenged birds with cecal colonization reaching 4.9 log10 CFU/g on 21 dpi. Campylobacter was disseminated to the spleen and liver on 7 dpi and was cleared on 21 dpi from both internal organs. Challenged birds had a significant increase in anti-Campylobacter serum IgY (14&21 dpi) and bile IgA (14 dpi). At 3 dpi, there was a significant suppression in T-lymphocytes derived from the cecal tonsils of birds in the challenge treatment when compared to the control treatment after 72 h of ex vivo stimulation with Con A or C. jejuni. The T-cell suppression on 3 dpi was accompanied by a significant decrease in LITAF, K60, CLAU-2, IL-1β, iNOS, and IL-6 mRNA levels in the ceca and an increase in nitric oxide production from adherent splenocytes of challenged birds. In addition, on 3 dpi, there was a significant increase in CD4+ and CD8+ T lymphocytes in the challenge treatment. On 14 dpi, both pro and anti-inflammatory cytokines were upregulated in the spleen, and a significant increase in CD8+ T lymphocytes in Campylobacter-challenged birds’ ceca was observed. The persistence of C. jejuni in the ceca of challenged birds on 21 dpi was accompanied by an increase in IL-10 and LITAF mRNA levels, an increase in MNC proliferation when stimulated ex-vivo with the diluted C. jejuni, an increase in serum specific IgY antibodies, an increase in both CD4+ and CD8+ cells, and a decrease in CD4+:CD8+ cell ratio. The balanced Th1 and Th2 immune responses against C. jejuni might explain the ceca’s bacterial colonization and the absence of pathology in Campylobacter-challenged birds. Future studies on T lymphocyte subpopulations should elucidate a pivotal role in the persistence of Campylobacter in the ceca.

Influence of Glycyrrhiza glabra Extract on Growth, Gene Expression of Gut Integrity, and Campylobacter jejuni Colonization in Broiler Chickens.

Phytogenic feed additives have been gaining considerable interest due to their ability to improve gut health and thereby performance of broiler chickens. The impact of Glycyrrhiza glabra (licorice) extract (GE) on expression of genes coding for tight junction proteins and gut protection and Campylobacter jejuni colonization in broilers has not been discussed until now. Thus, the current study assessed the effective dose of GE for maximum growth in broiler chickens, clear-cut molecular mechanisms related to integrity and health of intestine, and controlling C. jejuni colonization. Over a 35-day feeding period, a total of 500 Ross broiler chicks were allocated to five groups; the first group was fed a control diet without GE and the second group to the fifth group were fed a control diet with GE (0.25, 0.5, 1, and 2 g/kg of diet); each group comprised 100 chicks with 10 replicates (10 birds/replicate). Birds fed GE had an improved body weight gain and feed conversion ratio. Furthermore, the highest body weight gain was observed in the group that received 1 g/kg of GE (P < 0.05). The expression of genes coding for tight junction proteins [occludin and junctional adhesion molecules (JAM)] was upregulated in all groups supplemented with GE. Moreover, birds fed 1 g/kg of GE exhibited the maximum gene expression of occludin and JAM [0.2 and 0.3 fold change, respectively (P < 0.05)]. In relation to enterocyte protective genes [glucagon-like peptide (GLP-2) and fatty acid-binding protein (FABP-6)], use of GE significantly upregulated expression of GLP-2 gene with 0.8 fold change in 2 g/kg of the GE supplemented group (P < 0.05) while the expression of FABP-6 gene was not affected by GE supplementation (P > 0.05). After challenge with C. jejuni, the expression of mucin (MUC-2) gene was upregulated and the inflammatory markers such as Toll-like receptors (TLR-4) and interleukin (IL-1β) were downregulated with increasing level of supplemented GE (P < 0.05). The mean log10 count of C. jejuni in cecal samples after 7 days post-infection by culture and real-time qPCR was decreased in groups fed GE in a dose-dependent manner (P < 0.05). In addition, the highest reduction of C. jejuni count in cecal samples by culture and real-time qPCR was observed in the group fed 2 g/kg of GE (2.58 and 2.28 log10 CFU/g, respectively). Results from this study suggested that G. glabra extract (1 g/kg) improved growth performance of broiler chickens, as well as influenced the maintenance of intestinal integrity and reduced C. jejuni shedding from infected birds.

Competitive reduction of poultry-borne enteric bacterial pathogens in chicken gut with bioactive Lactobacillus casei

In this study, the effect of sustainable probiotics on Campylobacter jejuni colonization and gut microbiome composition was evaluated using chicken as a model organism. Chickens were given Lactobacillus casei over-expressing myosin-cross-reactive antigen (LC+mcra). LC+mcra can generate bioactive compounds in larger quantity including conjugated linoleic acid. A total of 120 chickens were used in duplicate trials to investigate the effectiveness of LC+mcra in decreasing C. jejuni colonization by means of kanamycin resistant strain compared to the control group. We observed that LC+mcra can efficiently colonize various parts of the chicken gut and competitively reduce colonization of natural and challenged C. jejuni and natural Salmonella enterica. LC+mcra was found to reduce C. jejuni colonization in cecum, ileum and jejunum, by more than one log CFU/g when compared to the no-probiotic control group. Furthermore, 16S rRNA compositional analysis revealed lower abundance of Proteobacteria, higher abundance of Firmicutes, along with enriched bacterial genus diversity in gut of LC+mcra fed chicken. Decreased contamination of drinking water by C. jejuni and S. enterica was also observed, suggesting a potential function of reducing horizontal transfer of enteric bacteria in poultry. Outcomes of this study reveal high potential of LC+mcra as sustainable approach to decrease colonization of C. jejuni and S. enterica in poultry gut along with other beneficial attributes.

Evaluation of Glycosylated FlpA and SodB as Subunit Vaccines Against Campylobacter jejuni Colonisation in Chickens.

Campylobacter jejuni is the leading bacterial cause of human gastroenteritis worldwide and the handling or consumption of contaminated poultry meat is the key source of infection. C. jejuni proteins FlpA and SodB and glycoconjugates containing the C. jejuni N-glycan have been separately reported to be partially protective vaccines in chickens. In this study, two novel glycoproteins generated by protein glycan coupling technology—G-FlpA and G-SodB (with two and three N-glycosylation sites, respectively)—were evaluated for efficacy against intestinal colonisation of chickens by C. jejuni strain M1 relative to their unglycosylated variants. Two independent trials of the same design were performed with either a high challenge dose of 107 colony-forming units (CFU) or a minimum challenge dose of 102 CFU of C. jejuni M1. While antigen-specific serum IgY was detected in both trials, no reduction in caecal colonisation by C. jejuni M1 was observed and glycosylation of vaccine antigens had no effect on the outcome. Our data highlight inconsistencies in the outcome of C. jejuni vaccination trials that may reflect antigen-, challenge strain-, vaccine administration-, adjuvant- and chicken line-specific differences from previously published studies. Refinement of glycoconjugate vaccines by increasing glycosylation levels or using highly immunogenic protein carriers could improve their efficacy.

(-)-α-Pinene reduces quorum sensing and Campylobacter jejunicolonization in broiler chickens.

Campylobacter jejuni is one of the most prevalent causes of bacterial gastroenteritis worldwide, and it is largely associated with consumption of contaminated poultry. Current Campylobacter control measures at the poultry production level remain insufficient, and hence there is the need for alternative control strategies. We evaluated the potential of the monoterpene (-)-α-pinene for control of C. jejuni in poultry. The antibacterial and resistance-modulatory activities of (-)-α-pinene were also determined against 57 C. jejuni strains. In addition, the anti-quorum-sensing activity of (-)-α-pinene against C. jejuni NCTC 11168 was determined for three subinhibitory concentrations (125, 62.5, 31.25 mg/L) over three incubation times using an autoinducer-2 bioassay based on Vibrio harveyi BB170 bioluminescence measurements. The effects of a subinhibitory concentration of (-)-α-pinene (250 mg/L) on survival of C. jejuni, and in combination with enrofloxacin on fluoroquinolone resistance development in C. jejuni, were determined in a broiler chicken model, by addition of (-)-α-pinene to the broiler water supply. The reduction of C. jejuni numbers by (-)-α-pinene was further determined in broiler chickens that were colonized with either fluoroquinolone-susceptible or -resistant strains, by direct gavage treatment. We observed weak in vitro antimicrobial activity for (-)-α-pinene alone (MIC >500 mg/L), but strong potentiating effects on antibiotics erythromycin and ciprofloxacin against different Campylobacter strains (>512 fold change). After 24 h of treatment of C. jejuni with (-)-α-pinene, its quorum-sensing signaling was reduced by >80% compared to the untreated control. When given in the drinking water, (-)-α-pinene did not show any significant inhibitory effects on the level of C. jejuni in the colonized chickens, and did not reduce fluoroquinolone resistance development in combination with enrofloxacin. Conversely, when (-)-α-pinene was administered by direct gavage, it significantly reduced the number of fluoroquinolone susceptible C. jejuni in the colonized broiler chickens. These results demonstrate that (-)-α-pinene modulates quorum-sensing in Campylobacter, potentiates antibiotics against different Campylobacter strains, and reduces Campylobacter colonization in broiler chickens.

Research Note: Lyophilization of hyperimmune egg yolk: effect on antibody titer and protection of broilers against Campylobacter colonization

Oral administration of antibodies is a promising strategy against various infectious diseases. Previously, it was demonstrated that passive immunization by providing hyperimmune egg yolk through the feed reduces Campylobacter jejuni colonization in broilers. Campylobacteriosis is the most commonly reported bacterial foodborne zoonosis worldwide, and poultry products are the number one origin of these bacteria for human infection. To date, no effective control measures exist to limit Campylobacter colonization in the chicken's intestinal tract. Here, the effect of lyophilization of hyperimmune egg yolk on protection of broilers against C. jejuni was investigated. During an in vivo trial, broiler chickens were prophylactically given feed with lyophilized hyperimmune or non-immunized egg yolk powder starting from day 1 after hatch. At day 11, broilers were inoculated with C. jejuni according to a seeder model. Five days later, all broilers were euthanized and cecal content was examined for C. jejuni colonization. No decrease in C. jejuni colonization was found. The freeze-drying resulted in a 16-fold decrease of the antibody titer in the yolk powder compared to the fresh yolks, presumably caused by structural changes in the antibodies. In conclusion, applying freeze-dried hyperimmune egg yolk failed to protect broilers against C. jejuni colonization, possibly because lyophilization affected the antibodies' functionality.