Evaluation of Two Recombinant Protein-Based Vaccine Regimens against Campylobacter jejuni: Impact on Protection, Humoral Immune Responses and Gut Microbiota in Broilers.

Campylobacter infections in humans are traced mainly to poultry products. While vaccinating poultry against Campylobacter could reduce the incidence of human infections, no vaccine is yet available on the market. In our previous study using a plasmid DNA prime/recombinant protein boost vaccine regimen, vaccine candidate YP437 induced partial protective immune responses against Campylobacter in broilers. In order to optimise vaccine efficacy, the vaccination protocol was modified using a protein prime/protein boost regimen with a different number of boosters. Broilers were given two or four intramuscular protein vaccinations (with the YP437 vaccine antigen) before an oral challenge by C. jejuni during a 42-day trial. The caecal Campylobacter load, specific systemic and mucosal antibody levels and caecal microbiota in the vaccinated groups were compared with their respective placebo groups and a challenge group (Campylobacter infection only). Specific humoral immune responses were induced, but no reduction in Campylobacter caecal load was observed in any of the groups (p > 0.05). Microbiota beta diversity analysis revealed that the bacterial composition of the groups was significantly different (p ≤ 0.001), but that vaccination did not alter the relative abundance of the main bacterial taxa residing in the caeca. The candidate vaccine was ineffective in inducing a humoral immune response and therefore did not provide protection against Campylobacter spp. infection in broilers. More studies are required to find new candidates.

Plasmid DNA Prime/Protein Boost Vaccination against Campylobacter jejuni in Broilers: Impact of Vaccine Candidates on Immune Responses and Gut Microbiota.

Campylobacter infections, traced to poultry products, are major bacterial foodborne zoonoses, and vaccination is a potential solution to reduce these infections. In a previous experimental trial using a plasmid DNA prime/recombinant protein boost vaccine regimen, two vaccine candidates (YP437 and YP9817) induced a partially protective immune response against Campylobacter in broilers, and an impact of the protein batch on vaccine efficacy was suspected. This new study was designed to evaluate different batches of the previously studied recombinant proteins (called YP437A, YP437P and YP9817P) and to enhance the immune responses and gut microbiota studies after a C. jejuni challenge. Throughout the 42-day trial in broilers, caecal Campylobacter load, specific antibodies in serum and bile, the relative expression of cytokines and ß-defensins, and caecal microbiota were assessed. Despite there being no significant reduction in Campylobacter in the caecum of vaccinated groups, specific antibodies were detected in serum and bile, particularly for YP437A and YP9817P, whereas the production of cytokines and ß-defensins was not significant. The immune responses differed according to the batch. A slight change in microbiota was demonstrated in response to vaccination against Campylobacter. The vaccine composition and/or regimen must be further optimised.

Simultaneous Detection of Salmonella spp. and Quantification of Campylobacter spp. in a Real-Time Duplex PCR: Myth or Reality?

In Europe, there is a process hygiene criterion for Salmonella and Campylobacter on broiler carcasses after chilling. The criterion gives indicative contamination values above which corrective actions are required by food business operators. The reference methods for verifying compliance with the criterion for Salmonella and Campylobacter are international standards EN ISO 6579-1 (2017) and EN ISO 10272-2 (2017), respectively. These methods are time-consuming and expensive for food business operators. Therefore, it would be advantageous to simultaneously detect Salmonella spp. and quantify Campylobacter in the same analysis, using the same sample after the pre-enrichment step for Salmonella recovery. A duplex PCR for Salmonella detection and Campylobacter spp. enumeration was developed. Considering the method as a whole, the LOD and LOQ for Campylobacter enumeration were slightly over the limit of 3 log CFU/g set by the process hygiene criterion. A comparison of the duplex PCR method developed with the ISO method on artificially contaminated bacterial suspensions and on naturally contaminated samples demonstrated a good correlation of the results for Campylobacter enumeration when the duplex PCR was performed on samples taken before or after the pre-enrichment step, but revealed a slight bias with a large standard deviation resulting in widely spaced limits of agreement.

Research Note: Analysis of immune responses in broilers after vaccination against Campylobacter jejuni.

Campylobacter infections traced mainly to poultry products are major bacterial foodborne zoonoses. Among the many control strategies evaluated at primary poultry level to reduce these infections, vaccination could be a solution, but no effective vaccines are available to date. A better understanding of the immune mechanisms involved in protection against Campylobacter would be helpful for designing novel vaccine strategies. The present study was designed to analyze in more depth the immune responses developed in broilers in order to potentially identify which immune parameters may be important for establishing protection against Campylobacter by comparing the immune responses obtained here with those obtained in a previous study performed on vaccinated specific-pathogen-free Leghorn chickens that presented a partial reduction of Campylobacter after experimental challenge. The protection against Campylobacter colonization was evaluated at different time points over 40 d of rearing, by measuring specific IgY levels in serum and IgA antibodies in bile reflecting the systemic and mucosal humoral responses respectively and the relative expressions of 9 cecal immune marker genes (cytokines and antimicrobial peptides), which reflect the innate and cellular immune responses. Despite no reduction of Campylobacter in the cecum, a systemic immune response over time characterized by the production of specific anti-flagellin IgY was observed, in addition to upregulation of the antimicrobial peptide avian ß-defensin (AvBD) 12 gene expression in the cecum of vaccinated broilers compared with the placebo group. However, the levels of specific anti-flagellin mucosal IgA antibodies in the bile as well as the relative expression of other cecal cytokines studied was underexpressed in the vaccinated group or similar in both groups.

Survival of Campylobacter jejuni Co-Cultured with Salmonella spp. in Aerobic Conditions.

Campylobacter and Salmonella are responsible for the two major foodborne zoonotic diseases in Europe; poultry is the main infection source. Campylobacter cannot grow under aerobic conditions, but can show aerobic survival when co-cultured with other microorganisms; however, its interaction with Salmonella has not been studied yet. In this study, these two bacteria were co-cultured under controlled aerobic conditions. Different concentrations and strains of C. jejuni were incubated with or without different Salmonella serotypes (10 CFU) at 37 °C for 16 h. C. jejuni did not grow after incubation with or without Salmonella. The survival of C. jejuni was observed only for the highest initial concentration of 6 log CFU/mL with or without Salmonella. However, its survival was significantly higher when co-cultured with Salmonella. No survival was observed at lower concentrations. C. jejuni survival was positively affected by the presence of Salmonella but depended on the Salmonella serotype, the C. jejuni strain and the initial concentration. On the other hand, the Salmonella enumerations were not affected by C. jejuni. Our results suggest potential interactions between Salmonella and C. jejuni that require further investigations for a clearer understanding of their behavior in natural habitats.

Impact of DNA Prime/Protein Boost Vaccination against Campylobacter jejuni on Immune Responses and Gut Microbiota in Chickens.

Campylobacteriosis is reported to be the leading zoonosis in Europe, and poultry is the main reservoir of Campylobacter. Despite all the efforts made, there is still no efficient vaccine to fight this bacterium directly in poultry. Recent studies have reported interactions between the chicken immune system and gut microbiota in response to Campylobacter colonisation. The present study was designed to analyse in more depth the immune responses and caecal microbiota following vaccination with a DNA prime/protein boost flagellin-based vaccine that induces some protection in specific-pathogen-free White Leghorn chickens, as shown previously. These data may help to improve future vaccination protocols against Campylobacter in poultry. Here a vaccinated and a placebo group were challenged by C. jejuni at the age of 19 days. A partial reduction in Campylobacter loads was observed in the vaccinated group. This was accompanied by the production of specific systemic and mucosal antibodies. Transient relatively higher levels of Interleukin-10 and antimicrobial peptide avian ß-defensin 10 gene expressions were observed in the vaccinated and placebo groups respectively. The analysis of caecal microbiota revealed the vaccination's impact on its structure and composition. Specifically, levels of operational taxonomic units classified as Ruminococcaceae and Bacillaceae increased on day 40.

Effect of Litter Treatment on Campylobacter jejuni in Broilers and on Cecal Microbiota.

Since 2018, when a process hygiene criterion for Campylobacter in broilers at the slaughterhouse was implemented across Europe, efforts to reduce Campylobacter at farm level have increased. Despite numerous studies aiming to reduce Campylobacter colonization in broilers, no efficient control strategy has been identified so far. The present work assessed first the efficacy of a commercial litter treatment to reduce Campylobacter colonization in broilers during two in-vivo trials and second, its impact on cecal microbiota. The treatment does not affect broiler growth and no effect on Campylobacter counts was observed during the in-vivo trials. Nevertheless, cecal microbiota were affected by the treatment. Alpha and beta diversity were significantly different for the control and litter-treated groups on day 35. In addition, several taxa were identified as significantly associated with the different experimental groups. Further work is needed to find a suitable control measure combining different strategies in order to reduce Campylobacter.

Ruminant and chicken: important sources of campylobacteriosis in France despite a variation of source attribution in 2009 and 2015.

Pathogen source attribution studies are a useful tool for identifying reservoirs of human infection. Based on Multilocus Sequence Typing (MLST) data, such studies have identified chicken as a major source of C. jejuni human infection. The use of whole genome sequence-based typing methods offers potential to improve the precision of attribution beyond that which is possible from 7 MLST loci. Using published data and 156 novel C. jejuni genomes sequenced in this study, we performed probabilistic host source attribution of clinical C. jejuni isolates from France using three types of genotype data: comparative genomic fingerprints; MLST genes; 15 host segregating genes previously identified by whole genome sequencing. Consistent with previous studies, chicken was an important source of campylobacteriosis in France (31-63% of clinical isolates assigned). There was also evidence that ruminants are a source (22-55% of clinical isolates assigned), suggesting that further investigation of potential transmission routes from ruminants to human would be useful. Additionally, we found evidence of environmental and pet sources. However, the relative importance as sources varied according to the year of isolation and the genotyping technique used. Annual variations in attribution emphasize the dynamic nature of zoonotic transmission and the need to perform source attribution regularly.

Prevalence of Thermophilic Campylobacter in Cattle Production at Slaughterhouse Level in France and Link Between C. jejuni Bovine Strains and Campylobacteriosis.

Campylobacter is the leading cause of bacterial gastroenteritis in industrialized countries, with poultry reservoir as the main source of infection. Nevertheless, a recent study on source attribution showed that cattle could be a source of human contamination in France (Thépault et al., 2017). However, few data are available on thermophilic Campylobacter epidemiology in cattle in France. The aim of this study is to collect new data of thermophilic Campylobacter prevalence in these animals and to subtype C. jejuni isolates to assess the potential implication of cattle in campylobacteriosis. A 6-month survey was carried out in one of the largest European slaughterhouse of cattle. Based on a statistical representative sampling plan, 959 intestinal content samples (483 adult cattle and 476 calves) were collected. An adapted version of the ISO 10272 standard and Maldi-Tof were used for detection and speciation of thermophilic Campylobacter isolates. Within more than 2000 thermophilic Campylobacter isolates collected, a selection of 649 C. jejuni isolates was typed with Comparative Genomic Fingerprinting (CGF40) and a subset of 77 isolates was typed using Multilocus Sequence Typing (MLST). Simultaneously, clinical isolates occurred in France were genotyped. Prevalence of thermophilic Campylobacter in the global cattle population was 69.1% (CI95% = 66.1, 72.1) at slaughterhouse level. In adult cattle, the prevalence was 39.3%, while 99.4% of calves were contaminated, and C. jejuni was the most prevalent species with prevalence of 37.3 and 98.5%, respectively and a higher genetic diversity in adult cattle. The prevalence of C. coli was lower with 3% in adult cattle and 12.5% in calves. MLST and CGF40 genotyping did not showed a high number of clusters within cattle isolates but the predominance of few clusters accounted for a large part of the population (CC-21, CC-61, CC-48, and CC-257). By comparison with clinical genotypes, genetic diversity was significantly lower in cattle. Moreover, significant overlap was observed between genotypes from both origins, with 3 of the 4 main cattle clusters present in human isolates. This study provides new insights on the epidemiology of thermophilic Campylobacter and C. jejuni in cattle production in France and their potential implication in human infection.

A representative overview of the genetic diversity and lipooligosaccharide sialylation in Campylobacter jejuni along the broiler production chain in France and its comparison with human isolates.

Campylobacter jejuni is the most common cause of bacterial gastroenteritis worldwide and is associated with post-infectious neuropathies. Moreover, the chicken reservoir is described as the main source of human infection and C. jejuni sialylated lipooligosaccharides seem to play an important role in the pathogenesis of neuropathies. In this study, MultiLocus Sequence Typing (MLST) and Comparative Genomic Fingerprinting using 40 assay genes (CGF40) were used to describe C. jejuni populations within clinical isolates and a representative collection of isolates from French poultry production. In addition, the sialylation of C. jejuni LOS was assessed. Here, we report high levels of genetic diversity among both chicken and human disease C. jejuni populations. The predominance of the ST-21, ST-45, and ST-464 complexes in chicken isolates and of the ST-21, ST-206, and ST-48 complexes in the clinical isolates was observed as were correlations between some MLST and CGF40 genotypes. Furthermore, some C. jejuni genotypes were frequently isolated among clinical cases as well as all along the broiler production chain, suggesting a potentially high implication of chicken in human campylobacteriosis in France. Finally, the LOS classes A, B and C were predominant within clinical C. jejuni isolates supporting the hypothesis of a benefit in infectivity for C. jejuni isolates showing sialylated LOS.

A DNA prime/protein boost vaccine protocol developed against Campylobacter jejuni for poultry.

Vaccination of broilers is one of the potential ways to decrease Campylobacter intestinal loads and therefore may reduce human disease incidence. Despite many studies, no efficient vaccine is available yet. Using the reverse vaccinology strategy, we recently identified new vaccine candidates whose immune and protective capacities need to be evaluated in vivo. Therefore, the goal of the present study was to develop and evaluate an avian subunit vaccine protocol for poultry against Campylobacter jejuni. For this, flagellin was used as vaccine antigen candidate. A DNA prime/protein boost regimen was effective in inducing a massive protective immune response against C. jejuni in specific pathogen free Leghorn chickens. Contrastingly, the same vaccine regimen stimulated the production of antibodies against Campylobacter in conventional Ross broiler chickens harbouring maternally derived antibodies against Campylobacter, but not the control of C. jejuni colonization. These results highlight the strength of the vaccine protocol in inducing protective immunity and the significance of the avian strain and/or immune status in the induction of this response. Nevertheless, as such the vaccine protocol is not efficient in broilers to induce protection and has to be adapted; this has been done in one of our recent published work.

Effect of Feed Additives on Productivity and Campylobacter spp. Loads in Broilers Reared under Free Range Conditions.

The poultry reservoir, especially broiler meat, is generally recognized as one of the most-important sources for human Campylobacteriosis. The measures to control Campylobacter targeted essentially the primary production level. The aim of this work was to evaluate the effectiveness of different treatments against natural Campylobacter colonization in a French experimental farm of free-range broilers during the whole rearing period. Five commercial products and a combination of two of them were tested and all the products were added to feed or to water at the dose recommended by the suppliers. Campylobacter loads in caeca and on carcasses of broilers at the slaughter were determined by culture methods. Natural contamination of the flock occurred at the end of the indoor rearing period between day 35 and day 42. At day 42, the multispecies probiotic added to the feed reduced the contamination of 0.55 log10 CFU/g (p = 0.02) but was not significant (p > 0.05) at the end of rearing at day 78. However, another treatment, a combination of a cation exchange clay-based product in feed and an organic acid mixture (formic acid, sodium formate, lactic acid, propionic acid) in water, led to a slight but significant reduction of 0.82 ± 0.25 log10 CFU/g (p = 0.02) compared to the control group at day 78. Testing this combination in field conditions in several flocks is needed to determine if it is biologically relevant and if it could be a valuable measure to reduce Campylobacter in broiler flocks.

Use of the potential probiotic strain Lactobacillus salivarius SMXD51 to control Campylobacter jejuni in broilers.

Campylobacteriosis is the most frequently reported zoonotic disease in humans in the EU since 2005. As chicken meat is the main source of contamination, reducing the level of Campylobacter in broiler chicken will lower the risk to consumers. The aim of this project was to evaluate the ability of Lactobacillus salivarius SMXD51 to control Campylobacter jejuni in broilers and to investigate the mechanisms that could be involved. Thirty broilers artificially contaminated with C. jejuni were treated by oral gavage with MRS broth or a bacterial suspension (107CFU) of Lb. salivarius SMXD51 (SMXD51) in MRS broth. At 14 and 35days of age, Campylobacter and Lb. salivarius loads were assessed in cecal contents. The impact of the treatment on the avian gut microbiota at day 35 was also evaluated. At day 14, the comparison between the control and treated groups showed a significant reduction (P<0.05) of 0.82 log. After 35days, a significant reduction (P<0.001) of 2.81 log in Campylobacter loads was observed and 73% of chickens treated with the culture exhibited Campylobacter loads below 7log10CFU/g. Taxonomic analysis revealed that SMXD51 treatment induced significant changes (P<0.05) in a limited number of bacterial genera of the avian gut microbiota and partially limited the impact of Campylobacter on Anaerotruncus sp. decrease and Subdoligranulum sp. increase. Thus, SMXD51 exhibits an anti-Campylobacter activity in vivo and can partially prevent the impact of Campylobacter on the avian gut microbiota.

Prevalence and characterization of Campylobacter jejuni from chicken meat sold in French retail outlets.

Campylobacter was detected in 76% of broiler meat products collected in retail outlets during a monitoring plan carried out in France throughout 2009. Campylobacter jejuni was the most prevalent species (64.7% of products being contaminated). The 175 C. jejuni isolates collected were characterized. MLST typing results confirmed substantial genetic diversity as the 175 C. jejuni isolates generated 76 sequence types (STs). The ST-21, ST-45 and ST-464 complexes predominated accounting for 43% of all isolates. A class-specific PCR to screen the sialylated lipooligosaccharide (LOS) locus classes A, B and C showed that 50.3% of the C. jejuni isolates harbored sialylated LOS. The antimicrobial resistance profiles established using a subset of 97 isolates showed that resistance to tetracycline was the most common (53.6%), followed with ciprofloxacin and nalidixic acid (32.9%, and 32.0% respectively). All the tested isolates were susceptible to erythromycin, chloramphenicol and gentamicin. Clear associations were demonstrated between certain clonal complexes and LOS locus classes and between certain clonal complexes and antimicrobial resistance. This work paints a representative picture of C. jejuni isolated from poultry products circulating in France, providing data on STs, LOS locus classes and antibiotic resistance profiles in isolates recovered from products directly available to the consumer.

European validation of Real-Time PCR method for detection of Salmonella spp. in pork meat.

The classical microbiological method for detection of Salmonella spp. requires more than five days for final confirmation, and consequently there is a need for an alternative methodology for detection of this pathogen particularly in those food categories with a short shelf-life. This study presents an international (at European level) ISO 16140-based validation study of a non-proprietary Real-Time PCR-based method that can generate final results the day following sample analysis. It is based on an ISO compatible enrichment coupled to an easy and inexpensive DNA extraction and a consolidated Real-Time PCR assay. Thirteen laboratories from seven European Countries participated to this trial, and pork meat was selected as food model. The limit of detection observed was down to 10 CFU per 25 g of sample, showing excellent concordance and accordance values between samples and laboratories (100%). In addition, excellent values were obtained for relative accuracy, specificity and sensitivity (100%) when the results obtained for the Real-Time PCR-based methods were compared to those of the ISO 6579:2002 standard method. The results of this international trial demonstrate that the evaluated Real-Time PCR-based method represents an excellent alternative to the ISO standard. In fact, it shows an equal and solid performance as well as it reduces dramatically the extent of the analytical process, and can be easily implemented routinely by the Competent Authorities and Food Industry laboratories.

Campylobacter contamination of broiler caeca and carcasses at the slaughterhouse and correlation with Salmonella contamination.

In order to estimate the prevalence of Campylobacter spp. and Salmonella spp. on broiler chicken carcasses and the prevalence of Campylobacter spp. in caeca, 58 French slaughterhouses were investigated in 2008. Enumeration of Campylobacter spp. was also performed in order to study the relation between caeca and carcass contamination. A pool of 10 caeca and one carcass were collected from 425 different batches over a 12-month period in 2008. Salmonella was isolated on 32 carcasses leading to a prevalence of 7.5% ([5.0-10.0](95%CI)). The prevalence of Campylobacter was 77.2% ([73.2-81.2](95%CI)) in caeca and 87.5% ([84.4-90.7](95%CI)) on carcasses. No significant correlation was found between Campylobacter and Salmonella. Positive values of Campylobacter were normally distributed and the average level was 8.05 log(10) cfu/g ([7.94-8.16](95%CI)) in caeca and 2.39 cfu/g ([2.30-2.48](95%CI)) on carcasses. A positive correlation (r = 0.59) was found between the mean of Campylobacter in caeca and on carcasses (p < 0.001). Thus, carcasses from batches with Campylobacter-positive caeca had significantly (p < 0.001) higher numbers of Campylobacter per gram than batches with negative caeca. These results show that Campylobacter can be present in both matrices and reduction in caeca could be a possible way to reduce the amount of bacteria on carcasses. Of the 2504 identifications performed, 3 species of Campylobacter (Campylobacter jejuni, Campylobacter coli and Campylobacter lari) were identified. The main species recovered were C. jejuni and C. coli, which were isolated in 55.3% and 44.5% of positive samples, respectively. These two species were equally represented in caeca but C. jejuni was the most frequently isolated on carcasses with 57.1% and 42.5% of positive carcasses for C. jejuni and C. coli, respectively. This study underlines that target a reduction of Campylobacter on final products requires a decrease of contamination in caeca.

Prevalence of and risk factors for Campylobacter spp. contamination of broiler chicken carcasses at the slaughterhouse.

A study was conducted in 2008 to estimate the prevalence and identify the risk factors for Campylobacter spp. contamination of broiler carcasses during the slaughtering process. A pool of 10 caeca and one carcass were collected from 425 batches of broiler chickens slaughtered in 58 French slaughterhouses over a 12-month period. Potential risk factors were identified according to the Campylobacter contamination status of carcasses and processing variables identified from questionnaires. The statistical analysis took into account confounding factors that have already been associated with the presence of Campylobacter on carcasses such as the slaughter age of the chicken or seasonal variations. Campylobacter spp. were isolated from 77.2% of caeca (95% CI 73.2 to 81.2) and from 87.5% of carcasses (95% CI 84.4 to 90.7). A multiple logistic regression showed 4 parameters as significant risk factors (p < 0.05) for contamination: (I) batches were not the first to be slaughtered in the logistic schedule (OR = 3.5), (II) temperature in the evisceration room was higher than 15 °C (OR = 3.1), (III) dirty marks on carcasses after evisceration were visible (OR = 2.6) and (IV) previous thinning of the flocks, from which slaughtered batches came, had occurred at the farm (OR = 3.3). This last result highlighted the need for sanitary precautions to be taken when catching birds for transport. At the slaughterhouse, evisceration seemed to be the operation contributing most to the spread of contamination. Effective risk management solutions could include the systematic external rinsing of carcasses after evisceration and the implementation of slaughtering schedules according to the Campylobacter contamination status of flocks.