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Abstract Since the ban or reduction on the use of antibiotic growth promoters (AGPs) in commercial broiler chickens in many countries, avian necrotic enteritis (NE) caused by Clostridium perfringens has re-emerged as one of the biggest threats for the poultry industry worldwide. While the toolbox for controlling NE in the absence of antibiotics consists of a limited number of alternatives for which the overall effectiveness has yet proven to be suboptimal, an effective vaccine would represent the best control strategy for this often-deadly disease. Using a comparative and subtractive reverse vaccinology approach, we previously identified 14 putative antigenic proteins unique to NE-causing strains of C. perfringens. In the current work, the in silico findings were confirmed by PCR and sequencing, and five vaccine candidate proteins were produced and purified subsequently. Among them, two candidates were hypothetical proteins, two candidates were prepilin proteins which are predicted to form the subunits of a pilus structure, and one candidate was a non-heme iron protein. Western blotting and ELISA results showed that immunization of broiler chickens with five of these proteins raised antibodies which can specifically recognize both the recombinant and native forms of the protein in pathogenic C. perfringens.

The Canadian Food Inspection Agency (CFIA) is developing an establishment-based risk assessment model to categorize rendering plants that produce livestock feed ingredients (ERA-Renderer model) according to animal and human health risks (i.e., feed safety risks) and help in determining the allocation of inspection resources based on risk. The aim of the present study was to identify and select feed-safety-related factors and assessment criteria for inclusion in the ERA-Renderer model. First, a literature review was performed to identify evidence-based factors that impact the feed safety risk of livestock feed during its rendering processes. Secondly, a refinement process was applied to retain only those that met the inclusion conditions, such as data availability, lack of ambiguity, and measurability. Finally, an expert panel helped in selecting factors and assessment criteria based on their knowledge and experience in the rendering industry. A final list of 32 factors was developed, of which 4 pertained to the inherent risk of a rendering plant, 8 were related to risk mitigation strategies, and 20 referred to the regulatory compliance of a rendering plant. A total of 179 criteria were defined to assess factors based on practices in the Canadian rendering industry. The results of this study will be used in the next step of the model development to estimate the relative risks of the assessment criteria considering their impact on feed safety. Once implemented, the CFIA’s ERA-Renderer model will provide an evidence-based, standardized, and transparent approach to help manage the feed safety risks in Canada’s rendering sector.

Avian necrotic enteritis is an enteric disease of broiler chickens caused by certain pathogenic strains of Clostridium perfringens in combination with predisposing factors. A vaccine offering complete protection against the disease has not yet been commercialized. In a previous study, we produced five recombinant proteins predicted to be surface-exposed and unique to necrotic enteritis-causing C. perfringens and the immunogenicity of these potential vaccine candidates was assessed in broiler chickens. In the current work, the relative contribution of the antibodies raised by these putative antigens to protect broiler chickens was evaluated using an experimental necrotic enteritis induction model. Additionally, the link between the immune response elicited and the gut microbiota profiles in immunized birds subjected to infection with virulent C. perfringens was studied. The ELISA results showed that the IgY antibody titers in vaccinated birds on days 21 and 33 were significantly higher than those on days 7 and 14 and those in birds receiving the adjuvant alone, while the relative contribution of the specific immunity attributed to these antibodies could not be precisely determined using this experimental necrotic enteritis induction model. In addition, 16S rRNA gene amplicon sequencing showed that immunization of birds with recombinant proteins had a low impact on the chicken caecal microbiota.

Abstract Background Avian necrotic enteritis (NE) caused by Clostridium perfringens is a disease with a major economic impact, generating losses estimated to 6 billion of dollars annually for the poultry industry worldwide. The incidence of the disease is particularly on the rise in broiler chicken flocks eliminating the preventive use of antibiotics. To date, no alternative allows for the efficient prevention of NE and a control of the disease using a vaccinal strategy would be mostly prized. For this purpose, comparative and subtractive reverse vaccinology identifying putative immunogenic bacterial surface proteins is one of the most promising approaches. Results A comparative genomic study was performed on 16 C. perfringens strains isolated from healthy broiler chickens and from broilers affected with necrotic enteritis. Results showed that the analyzed genomes were composed of 155,700 distinct proteins from which 13% were identified as extracellular, 65% as cytoplasmic and 22% as part of the bacterial membrane. The evaluation of the immunogenicity of these proteins was determined using the prediction software VaxiJen®. Conclusions For the most part, proteins with the highest scores were associated with an extracellular localisation. For all the proteins analyzed, the combination of both the immunogenicity score and the localisation prediction led to the selection of 12 candidate proteins that were mostly annotated as hypothetical proteins. We describe 6 potential candidates of higher interest due to their antigenic potential, their extracellular localisation, and their possible role in virulence of C. perfringens.

Abstract Background The aim of this study was to characterize the genomes of 30 Listeria monocytogenes isolates collected at a pig slaughterhouse to determine the molecular basis for their persistence. Results Comparison of the 30 L. monocytogenes genomes showed that successive isolates (i.e., persistent types) recovered from thew sampling site could be linked on the basis of single nucleotide variants confined to prophage regions. In addition, our study revealed the presence among these strains of the bcrABC cassette which is known to produce efflux pump-mediated benzalkonium chloride resistance, and which may account for the persistence of these isolates in the slaughterhouse environment. The presence of the bcrABC cassette was confirmed by WGS and PCR and the resistance phenotype was determined by measuring minimum inhibitory concentrations. Furthermore, the BC-resistant strains were found to produce lower amounts of biofilm in the presence of sublethal concentrations of BC. Conclusions High resolution SNP-based typing and determination of the bcrABC cassette may provide a means of distinguishing between resident and sporadic L. monocytogenes isolates, and this in turn will support better management of this pathogen in the food industry.

Tail-biting (TB) in pigs is a serious behavioral disorder. It is an important challenge in swine production as it impacts animal welfare and health and the economics and safety of the pork meat supply chain. To prevent TB, approaches including enrichment material and tail docking are proposed but none are optimal. Nutrition appears to be an important factor in TB behavior, perhaps by modulating the intestinal microbiota (IM). Our aim was to assess the association between TB behavior and IM in pigs through comparisons of IM in groups of biter, bitten and non-biter/non-bitten pigs. Each group composed of 12 pigs was formed at the beginning of the growing/finishing phase based on a target behavior analysis centered on TB behavior for the biter group and a score of damages caused to the tail for the bitten group. Blood and fecal samples were collected from each pig during a TB episode, at time 0, t0, and when the TB episode was considered finished, 4 weeks later, at time 1, t1. Serum cortisol level was determined by ELISA and used as an indicator of stress. The pig's fecal microbiota was analyzed from DNA extracted from freshly collected fecal matter using amplicon sequencing of the V4 hypervariable region of the 16S rRNA gene. Serum cortisol levels were significantly higher in either the biter or bitten pig groups compared to the negative control group (p = 0.02 and p = 0.01, respectively). The microbiota alpha-diversity was not significantly different between all groups, biter, bitten and negative control. Analyses of beta-diversity, however, revealed a significant difference between either the biter or the bitten group in comparison to the non-biter/non-bitten negative control group in terms of structure and composition of the microbiota. Lactobacillus were significantly more abundant in the negative control group compared to the two other groups (p = 0.001). No significant difference was revealed between the biter and bitten groups. Quantitative real-time PCR (qPCR) confirmed that lactobacilli were more abundant in the negative control group. Our study indicates that TB behavior is associated with the IM composition in pigs.

The ever-increasing problem of antibiotic resistance makes routine use of antibiotics in animal production no longer considered as a reasonable and viable practice. The Chicken Farmers of Canada have developed and are implementing an Antimicrobial Use Reduction Strategy, which has the ultimate goal of eliminating the preventive use of medically important antibiotics in broiler chicken and turkey production. However, very little is known about the real overall impact of an antibiotic use reduction strategy in complex ecosystems, such as the bird intestine or the commercial broiler chicken farm. The main objectives of the present study were to compare the abundance of antibiotic resistance-encoding genes, characterize the intestinal microbiota composition, and evaluate the presence of Clostridium perfringens, in six commercial poultry farms adopting short-term antibiotic withdrawal and long-term judicious use strategy. Implementing an antibiotic-free program over a 15-months period did not reduce the abundance of many antibiotic resistance-encoding genes, whereas the judicious use of antibiotics over 6 years was found effective. The short-term antibiotic withdrawal and the long-term judicious use strategy altered the intestinal microbiota composition, with the Ruminococcaceae and Lachnospiraceae families being negatively impacted. These findings are in agreement with the lower production performance and with the increased C. perfringens populations observed for farms phasing out the use of antibiotics. Adopting a conventional rearing program on commercial broiler chicken farms selected for specific antibiotic resistance-encoding genes in many barns. This study highlights the potential impacts of different rearing programs in poultry production and will help guide future policies in order to reduce the use of antibiotics while maintaining production performance.

Bacterial pathogens, such as Listeria monocytogenes, can show resistance to disinfection and persistence on working surfaces, permitting them to survive and contaminate food products. Persistence—a complex phenomenon involving interactions between many bacteria within a biofilm—is modulated by in situ characteristics. This study aimed to describe, in silico, the microbiota identified in a swine slaughterhouse after sanitation procedures to better understand the presence of L. monocytogenes on these surfaces. Molecular tools for characterization of microbial communities were used to assess the relative contribution of different bacteria resulting from this phenomenon, and the 16S rRNA sequencing method was used on samples from meat conveyor belt surfaces collected on four sampling visits to study the co-occurrence between L. monocytogenes and other bacteria. From the background microbiota, a total of six genera were found to be negatively correlated with Listeria spp., suggesting Listeria growth inhibition, competition, or at least an absence of shared habitats. Based on these results, a complete scenario of interactions of Listeria with components of background microbiota was established. This work contributes to identifying avenues that could prevent the growth and persistence of L. monocytogenes on food-processing surfaces.

Listeria monocytogenes is the etiological agent of listeriosis, a major foodborne disease and an important public health concern. Contamination of meat with L. monocytogenes occurs frequently at the slaughterhouse. Our aims were; 1) to investigate the distribution of L. monocytogenes in the processing areas of four swine slaughterhouses; 2) to describe the diversity of L. monocytogenes strains by pulsed-field gel electrophoresis; 3) to identify persistent L. monocytogenes strains and describe their distribution; 4) to investigate the associations between persistence of strains and their following characteristics: detection in food isolates, detection in human clinical isolates, and the presence of benzalkonium chloride (BAC) resistance genes. Various operation areas within the four swine slaughterhouses were sampled on four occasions. A total of 2496 samples were analyzed, and L. monocytogenes was successfully isolated from 243 samples. The proportion of positive samples ranged from 32 to 58% in each slaughterhouse and from 24 to 68% in each operation area. Fifty-eight different pulsotypes were identified and eight pulsotypes, present in samples collected during 4 visits, were considered persistent. The persistent pulsotypes were significantly more likely to be detected in food (P < 0.01, exact χ²) and human clinical cases (P < 0.01, exact χ²), respectively. Among pulsotypes harboring the BAC bcrABC resistance cassette or the emrE multidrug transporter gene, 42.8% were persistent compared to 4.5% for pulsotypes without these resistance genes (P < 0.01, exact χ²). Our study highlights the importance of persistent L. monocytogenes strains in the environmental contamination of slaughterhouses, which may lead to repeated contamination of meat products. It also shows that the presence of disinfectants resistance genes is an important contributing factor.

Clostridium perfringens ranks among the three most frequent bacterial pathogens causing human foodborne diseases in Canada, and poultry meat products are identified as a source of infection for humans. The objective of the current study was to estimate the proportion of broiler chicken flocks, carcasses and various environmental samples from critical locations of the slaughter plant positive for the presence of C. perfringens enterotoxin encoding gene (cpe). From the 16 visits conducted, 25% of the 79 flocks sampled, 10% of the 379 carcasses sampled and 5% of the 217 environmental samples collected were found positive for cpe. The proportion of cpe-positive carcasses was statistically different between surveyed plants, with 17.0% for one abattoir and 2.2% for the other. For the most contaminated plant, cpe-positive carcasses were identified at each step of the processing line, with prevalence varying between 10.0% and 25.0%, whereas this prevalence varied between 0% and 25.0% for the environmental surfaces sampled. Based on the results obtained, enterotoxigenic C. perfringens strains could potentially represent a risk for the consumer.

The intestinal microbiota plays several important roles in pig health and growth. The aim of the current study was to characterize the changes in the fecal microbiota diversity and composition of weaned piglets following an oral challenge with an ETEC: F4 strain and/or a treatment with colistin sulfate (CS). Twenty-eight piglets were used in this experiment and were divided into four groups: challenged untreated, challenged treated, unchallenged treated, and unchallenged untreated. Rectal swab samples were collected at five sampling times throughout the study. Total genomic DNA was used to assess the fecal microbiota diversity and composition using the V4 region of the 16S rRNA gene. The relative abundance, the composition, and the community structure of piglet fecal microbiota was highly affected by the ETEC: F4 challenge throughout the experiment, while the oral treatment with CS, a narrow spectrum antibiotic, resulted in a significant decrease of E. coli/Shigella populations during the treatment period only. This study was the first to identify some gut microbiota subgroups (e.g., Streptococcus, Lachnospiraceae) that are associated with healthy piglets as compared to ETEC: F4 challenged animals. These key findings might contribute to the development of alternative strategies to reduce the use of antimicrobials in the control of post-weaning diarrhea in pigs.

Extensive use of antibiotic growth promoters (AGPs) in food animals has been questioned due to the globally increasing problem of antibiotic resistance. For the poultry industry, digestive health management following AGP withdrawal in Europe has been a challenge, especially the control of necrotic enteritis. Much research work has focused on gut health in commercial broiler chicken husbandry. Understanding the behavior of Clostridium perfringens in its ecological niche, the poultry barn, is key to a sustainable and cost-effective production in the absence of AGPs. Using polymerase chain reaction and pulsed-field gel electrophoresis, we evaluated how the C. perfringens population evolved in drug-free commercial broiler chicken farms, either healthy or affected with recurring clinical necrotic enteritis outbreaks, over a 14-month period. We show that a high genotypic richness was associated with an increased risk of clinical necrotic enteritis. Also, necrotic enteritis-affected farms had a significant reduction of C. perfringens genotypic richness over time, an increase in the proportion of C. perfringens strains harboring the cpb2 gene, the netB gene, or both. Thus, necrotic enteritis occurrence is correlated with the presence of an initial highly diverse C. perfringens population, increasing the opportunity for the selective sweep of particularly virulent genotypes. Disease outbreaks also appear to largely influence the evolution of this bacterial species in poultry farms over time.

Biofilm formation by the pathogen Listeria monocytogenes is a major concern in food industries. The aim of this work was to elucidate the effect of nutrient limitation on both biofilm architecture and on the viability of the bacteria in microfluidic growth conditions. Biofilm formation by two L. monocytogenes strains was performed in a rich medium (BHI) and in a 10-fold diluted BHI (BHI/10) at 30°C for 24 h by using both static conditions and the microfluidic system Bioflux. In dynamic conditions, biofilms grown in rich and poor medium showed significant differences as well in structure and in the resulting biovolume. In BHI/10, biofilm was organized in a knitted network where cells formed long chains, whereas in the rich medium, the observed structure was homogeneous cellular multilayers. Biofilm biovolume production in BHI/10 was significantly higher than in BHI in these dynamic conditions. Interestingly, biovolume of dead cells in biofilms formed under limited nutrient conditions (BHI/10) was significantly higher than in biofilms formed in the BHI medium. In the other hand, in static conditions, biofilm is organized in a multilayer cells and dispersed cells in a rich medium BHI and poor medium BHI/10 respectively. There was significantly more biomass in the rich medium compared to BHI/10 but no difference was noted in the dead/damaged subpopulation showing how L. monocytogenes biofilm could be affected by the growth conditions. This work demonstrated that nutrient concentration affects biofilm structure and the proportion of dead cells in biofilms under microfluidic condition. Our study also showed that limited nutrients play an important role in the structural stability of L. monocytogenes biofilm by enhancing cell death and liberating extracellular DNA.

Zoonotic infectious diseases have been an important concern to humankind for more than 10,000 years. Today, approximately 75% of newly emerging infectious diseases (EIDs) are zoonoses that result from various anthropogenic, genetic, ecologic, socioeconomic, and climatic factors. These interrelated driving forces make it difficult to predict and to prevent zoonotic EIDs. Although significant improvements in environmental and medical surveillance, clinical diagnostic methods, and medical practices have been achieved in the recent years, zoonotic EIDs remain a major global concern, and such threats are expanding, especially in less developed regions. The current Ebola epidemic in West Africa is an extreme stark reminder of the role animal reservoirs play in public health and reinforces the urgent need for globally operationalizing a One Health approach. The complex nature of zoonotic diseases and the limited resources in developing countries are a reminder that the need for implementation of Global One Health in low-resource settings is crucial. The Veterinary Public Health and Biotechnology (VPH-Biotec) Global Consortium launched the International Congress on Pathogens at the Human-Animal Interface (ICOPHAI) in order to address important challenges and needs for capacity building. The inaugural ICOPHAI (Addis Ababa, Ethiopia, 2011) and the second congress (Porto de Galinhas, Brazil, 2013) were unique opportunities to share and discuss issues related to zoonotic infectious diseases worldwide. In addition to strong scientific reports in eight thematic areas that necessitate One Health implementation, the congress identified four key capacity-building needs: (1) development of adequate science-based risk management policies, (2) skilled-personnel capacity building, (3) accredited veterinary and public health diagnostic laboratories with a shared database, and (4) improved use of existing natural resources and implementation. The aim of this review is to highlight advances in key zoonotic disease areas and the One Health capacity needs.

The population genetics of pathogenic bacteria has been intensively studied in order to understand the spread of disease and the evolution of virulence and drug resistance. However, much less attention has been paid to bacterial carriage populations, which inhabit hosts without producing disease. Since new virulent strains that cause disease can be recruited from the carriage population of bacteria, our understanding of infectious disease is seriously incomplete without knowledge on the population structure of pathogenic bacteria living in an asymptomatic host. We report the first extensive survey of the abundance and diversity of a human pathogen in asymptomatic animal hosts. We have found that asymptomatic swine from livestock productions frequently carry populations of Salmonella enterica with a broad range of drug-resistant strains and genetic diversity greatly exceeding that previously described. This study shows how agricultural practice and human intervention may lead and influence the evolution of a hidden reservoir of pathogens, with important implications for human health.