Quantification of indicator and pathogenic bacteria in manures and digestates from three agricultural biogas plants over a one-year period.

Interest in the conversion of manure in biogas via anaerobic digestion (AD) is growing, but questions remain about the biosafety of digestates. For a period of one year, we monitored the impact of three mesophilic agricultural biogas plants (BPs) mainly fed with pig manure (BP1, BP3) or bovine manure (BP2) on the physicochemical parameters, the composition of the microbial community and the concentration of bacteria (E. coli, enterococci, Salmonella, Campylobacter, Listeria monocytogenes, Clostridium perfringens, Clostridium botulinum and Clostridioides difficile). The BP2 digestate differed from those of the two other BPs with a higher nitrogen content, more total solids and greater abundance of Clostridia MBA03 and Disgonomonadacea. Persistence during digestion ranked from least to most, was: Campylobacter (1.6 to >2.9 log10 reduction, according to the BP) < E. coli (1.8 to 2.2 log10) < Salmonella (1.1 to 1.4 log10) < enterococci (0.2 to 1.2 log10) and C. perfringens (0.2 to 1 log10) < L. monocytogenes (-1.2 to 1.6 log10) < C. difficile and C. botulinum (≤0.5 log10). No statistical link was found between the reduction in the concentration of the targeted bacteria and the physicochemical and operational parameters likely to have an effect (NH3, volatile fatty acids and total solids contents, hydraulic retention time, presence of co-substrates), underlining the fact that the fate of the bacteria during mesophilic digestion depends on many interacting factors. The reduction in concentrations varied significantly over the sampling period, underlining the need for longitudinal studies to estimate the impact of AD on pathogenic microorganisms.

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.

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.

Asymptomatic Carriage of C. botulinum Type D/C in Broiler Flocks as the Source of Contamination of a Massive Botulism Outbreak on a Dairy Cattle Farm.

In winter 2018, a massive type D/C cattle botulism outbreak occurred on a mixed dairy and broiler farm in France. An investigation was conducted based on the hypothesis of asymptomatic carriage in poultry. We set out to identify the source of contamination of the dairy cattle and to monitor the contamination of broilers over time, including the hatchery delivering chicks to the farm. Environmental samples were collected on the farm during the cattle outbreak (n = 40), after the outbreak for three successive broiler flocks (n = 128), and once in the hatchery delivering the chicks (n = 58). These samples were analyzed using real-time PCR after an enrichment step to detect Clostridium botulinum type D/C. The results showed contamination in the manure from the broilers raised just before the onset of the cattle outbreak (5 + /5), as well as in some of the components of the cattle ration (3 + /17). This latter contamination is likely due to the use of the same tractor bucket to remove litter from the poultry house and to prepare the cattle ration on the same day. Contamination monitoring over several months revealed continuous asymptomatic carriage in the broilers (4 + /20 and 17 + /20 cloacal swabs in 2 successive flocks), a persistence of C. botulinum type D/C in the ventilation system of the poultry house (8 + /14), and contamination of the equipment coming from the hatchery used for delivering the chicks (3 + /18). Further investigations conducted in the hatchery demonstrated contamination in the hatchery by C. botulinum type D/C (6 + /58). Comparison of samples using a multilocus variable number tandem repeat analysis showed the same profile for samples collected on broilers, cattle and in the hatchery. This study highlighted the crucial role of the implementation of biosecurity measures in mixed farms to avoid cross-contamination between production units given the potential asymptomatic carriage of poultry. This study also revealed the contamination of the poultry hatchery. Further investigations are required to better understand the role of hatcheries in the epidemiology of animal botulism.

Manure contamination with Clostridium botulinum after avian botulism outbreaks: management and potential risk of dissemination.

BACKGROUND: Persistence of Clostridium botulinum in the environment is well known. Getting rid of it after animal botulism outbreaks is so tricky, especially as far as manure concerns. This study aimed at 1. describing manure management on 10 poultry farms affected by botulism and 2. assessing the persistence of C botulinum in poultry manure after the outbreak. METHODS: Each farm was visited twice at two different manure storage times (two weeks after manure removal and two months later). Fifteen samples of manure were collected on each visit and C botulinum was detected using real-time PCR. RESULTS: Management of manure varied among poultry farms (classical storage, addition of quicklime, bacterial flora or incineration). C botulinum was detected in the manure of all 10 farms, 56.5per cent of samples being positive. C botulinum was detected significantly more frequently at the second visit (65.8per cent vs 49.7per cent, P<0.01) and on the surface of the pile (63.1per cent vs 50per cent, P=0.025). CONCLUSION: This study shows the persistence of C botulinum in poultry manure over time after a botulism outbreak and highlights manure management as a key health issue in preventing spore dissemination in the environment and recurrence of the disease.

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.

Characterization of Clostridioides difficile strains isolated from manure and digestate in five agricultural biogas plants.

Clostridioides difficile strains were isolated from manure and digestate samples from five biogas plants in France. The objective of this study was to characterize these isolates using PCR ribotyping, wgMLST, a multiplex PCR targeting genes encoding for the main virulence factors, i.e. tcdA, tcdB, cdtA and cdtB, and antimicrobial susceptibility assays. The 54 strains characterized were all positive for tcdA and tcdB and 83% (45/54) were positive for the binary toxin genes. PCR ribotypes 126 (59%) and 078 (37%) were predominant, and wgMLST analysis of 18 isolates showed close proximity of strains within a single biogas plant. Samples from the biogas plant supplied with cattle and poultry manure displayed the largest variety in PCR ribotypes. The in vitro activities of nine antimicrobial agents were determined. All the strains were susceptible to vancomycin and metronidazole, which are currently considered first-line treatments for C. difficile infection in humans. All the strains were resistant to clindamycin. The results of this study show that a high percentage of C. difficile strains present in the French biogas plants investigated are toxigenic strains from PCR ribotypes also commonly found in humans.

Development of An Innovative and Quick Method for the Isolation of Clostridium botulinum Strains Involved in Avian Botulism Outbreaks.

Avian botulism is a serious neuroparalytic disease mainly caused by a type C/D botulinum neurotoxin produced by Clostridium botulinum group III, one of the entwined bacterial species from the Clostridiumnovyisensulato genospecies. Its isolation is very challenging due to the absence of selective media and the instability of the phage carrying the gene encoding for the neurotoxin. The present study describes the development of an original method for isolating C. botulinum group III strains. Briefly, this method consists of streaking the InstaGene matrix extraction pellet on Egg Yolk Agar plates and then collecting the colonies with lipase and lecithinase activities. Using this approach, it was possible to isolate 21 C. novyi sensu lato strains from 22 enrichment broths of avian livers, including 14 toxic strains. This method was successfully used to re-isolate type C, D, C/D, and D/C strains from liver samples spiked with five spores per gram. This method is cheap, user-friendly, and reliable. It can be used to quickly isolate toxic strains involved in avian botulism with a 64% success rate and C. novyi sensu lato with a 95% rate. This opens up new perspectives for C. botulinum genomic research, which will shed light on the epidemiology of avian botulism.

A Case Report of a Botulism Outbreak in Beef Cattle Due to the Contamination of Wheat by a Roaming Cat Carcass: From the Suspicion to the Management of the Outbreak.

We report a botulism outbreak in Charolais cattle fed with wheat flour contaminated by Clostridium botulinum type C and the management of the outbreak at each step from the clinical suspicion to the cleaning and disinfection operations. Diagnosis was based on typical suggestive clinical signs and detection of C. botulinum type C using real-time PCR in samples collected from three young affected bulls. All young exposed bulls and cows (18 animals) eventually died, but three young bulls and one cow were recovering when it was decided to euthanize them. C. botulinum type C was detected in the liver of these four animals. Analysis of the ration components demonstrated that wheat flour, wheat, and the mill used to make flour were positive for C. botulinum type C. A dead cat positive for C. botulinum type C was discovered in the silo where wheat grain was stored and was considered the source of contamination. The cat's entire body was found mummified, well preserved, and not rotting in the silo. Specific measures, in particular, vaccination of the rest of the herd and cleaning and disinfection operations, were implemented to prevent any recurrence of the outbreak. The presence of wild animal carcasses in feed harboring anaerobic conditions like silage, in particular during harvesting, are known to be at risk for the initiation of a botulism outbreak. This outbreak is a reminder that the presence of an animal carcass in feed, regardless of the kind of feed and whenever the contamination occurs, either during harvesting or storage, is sufficient to induce a botulism outbreak.

Evaluation of the occurrence of sporulating and nonsporulating pathogenic bacteria in manure and in digestate of five agricultural biogas plants.

The number of agricultural biogas plants has been increasing in the past decades in some European countries. Digestates obtained after anaerobic digestion (AD) of manure are usually spread on agricultural land; however, their hygiene status regarding pathogens posing public health and/or animal health challenges has been poorly characterized up to now in France. In this study, three replicates of manure and digestate were collected from five farm biogas plants receiving animal manure in order to assess the occurrence and concentrations of sporulating (Clostridium botulinum, Clostridioides difficile, Clostridium perfringens) and nonsporulating (Listeria monocytogenes, thermotolerant Campylobacter spp., Salmonella, Escherichia coli, enterococci) bacteria. Concentrations of E. coli, enterococci, and C. perfringens in digestates ranged from 102 to 104 , 104 to 105 , and <103 to 7 × 105  CFU/g, respectively. Salmonella and C. difficile were detected in manure and digestate from the five biogas plants at concentrations ranging from <1.3 to >7 × 102  MPN/g and from 1.3 to 3 × 102  MPN/g, respectively. Thermotolerant Campylobacter, detected in all the manures, was only found in two digestates at a concentration of cells ranging from <10 to 2.6 × 102  CFU/g. Listeria monocytogenes and C. botulinum were detected in three manures and four digestates. The bacterial counts of L. monocytogenes and C. botulinum did not exceed 3 × 102 and 14 MPN/g, respectively. C. botulinum type B was detected at very low level in both the manure and digestate of farm biogas plants with no botulism history. The levels of pathogenic bacteria in both manure and digestate suggested that some bacteria can persist throughout AD.

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 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.

Promising new vaccine candidates against Campylobacter in broilers.

Campylobacter is the leading cause of human bacterial gastroenteritis in the European Union. Birds represent the main reservoir of the bacteria, and human campylobacteriosis mainly occurs after consuming and/or handling poultry meat. Reducing avian intestinal Campylobacter loads should impact the incidence of human diseases. At the primary production level, several measures have been identified to reach this goal, including vaccination of poultry. Despite many studies, however, no efficient vaccine is currently available. We have recently identified new vaccine candidates using the reverse vaccinology strategy. This study assessed the in vivo immune and protective potential of six newly-identified vaccine antigens. Among the candidates tested on Ross broiler chickens, four (YP_001000437.1, YP_001000562.1, YP_999817.1, and YP_999838.1) significantly reduced cecal Campylobacter loads by between 2 and 4.2 log10 CFU/g, with the concomitant development of a specific humoral immune response. In a second trial, cecal load reductions results were not statistically confirmed despite the induction of a strong immune response. These vaccine candidates need to be further investigated since they present promising features.

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.

Development and Validation of a New Reliable Method for the Diagnosis of Avian Botulism.

Liver is a reliable matrix for laboratory confirmation of avian botulism using real-time PCR. Here, we developed, optimized, and validated the analytical steps preceding PCR to maximize the detection of Clostridium botulinum group III in avian liver. These pre-PCR steps included enrichment incubation of the whole liver (maximum 25 g) at 37°C for at least 24 h in an anaerobic chamber and DNA extraction using an enzymatic digestion step followed by a DNA purification step. Conditions of sample storage before analysis appear to have a strong effect on the detection of group III C. botulinum strains and our results recommend storage at temperatures below -18°C. Short-term storage at 5°C is possible for up to 24 h, but a decrease in sensitivity was observed at 48 h of storage at this temperature. Analysis of whole livers (maximum 25 g) is required and pooling samples before enrichment culturing must be avoided. Pooling is however possible before or after DNA extraction under certain conditions. Whole livers should be 10-fold diluted in enrichment medium and homogenized using a Pulsifier® blender (Microgen, Surrey, UK) instead of a conventional paddle blender. Spiked liver samples showed a limit of detection of 5 spores/g liver for types C and D and 250 spores/g for type E. Using the method developed here, the analysis of 268 samples from 73 suspected outbreaks showed 100% specificity and 95.35% sensitivity compared with other PCR-based methods considered as reference. The mosaic type C/D was the most common neurotoxin type found in examined samples, which included both wild and domestic birds.

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.

Livers provide a reliable matrix for real-time PCR confirmation of avian botulism.

Diagnosis of avian botulism is based on clinical symptoms, which are indicative but not specific. Laboratory investigations are therefore required to confirm clinical suspicions and establish a definitive diagnosis. Real-time PCR methods have recently been developed for the detection of Clostridium botulinum group III producing type C, D, C/D or D/C toxins. However, no study has been conducted to determine which types of matrices should be analyzed for laboratory confirmation using this approach. This study reports on the comparison of different matrices (pooled intestinal contents, livers, spleens and cloacal swabs) for PCR detection of C. botulinum. Between 2013 and 2015, 63 avian botulism suspicions were tested and 37 were confirmed as botulism. Analysis of livers using real-time PCR after enrichment led to the confirmation of 97% of the botulism outbreaks. Using the same method, spleens led to the confirmation of 90% of botulism outbreaks, cloacal swabs of 93% and pooled intestinal contents of 46%. Liver appears to be the most reliable type of matrix for laboratory confirmation using real-time PCR analysis.