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.

Closed Genome Sequence of a Salmonella enterica Serovar Bovismorbificans Strain Isolated from Dried Pork Sausage Associated with an Outbreak in France.

We report here the closed genome sequence of one Salmonella enterica subsp. enterica serovar Bovismorbificans strain isolated from dried pork sausage consumed by a patient suffering from salmonellosis.

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.

Occurrence of Salmonella in the Cattle Production in France.

Salmonella is among the most common foodborne pathogens worldwide, and can lead to acute gastroenteritis. Along with poultry, cattle production is recognized as an important source of human infection. Salmonella transmission from cattle to humans can occur through the environment, or through close contact with sick animals or their derived products. This study aimed to investigate the intestinal carriage of Salmonella spp. within French cattle production. A total of 959 cattle intestinal samples, from one of the largest French slaughterhouses, were analyzed. Isolated strains were genotyped by pulsed field gel electrophoresis (PFGE), and a sub-selection was taken by whole genome sequencing (WGS). Twenty-nine samples were positive for Salmonella spp., yielding an estimated prevalence of 3% in cattle production. Eight different Salmonella serotypes were found: Montevideo was the most prevalent (34%), followed by Mbandaka (24%) and Anatum (14%). PFGE genotyping allowed the clustering of Salmonella isolates according to their serotype. Within the clusters, some isolates presented 100% similarity. To investigate potential epidemiological links between them, WGS and core genome multilocus sequence typing (cgMLST) were used, revealing identical profiles between isolates originating from different areas and/or different animal breeds. This investigation provides new insights on Salmonella serotype epidemiology in cattle production in France.

Epidemiological and Bacteriological Investigations Using Whole-Genome Sequencing in a Recurrent Outbreak of Pullorum Disease on a Quail Farm in France.

An outbreak of pullorum disease causing septicemia and high mortality was diagnosed in 2019 on a quail farm in western France. An initial episode had been detected in another building at the same site eight months earlier. Given the exceptional nature and the extent of the potential economic consequences of pullorum disease, epidemiological and bacteriological investigations using molecular sequencing tools were carried out. Salmonella Gallinarum and Salmonella Infantis were isolated (using the NF U 47-101 reference method) from samples taken from birds at the infected site. A resurgence of the initial episode by horizontal transmission of S. Gallinarum is the most likely hypothesis, supported by whole-genome sequencing (WGS) of the strains isolated during the two episodes. Risk health practices have been identified, including the rearing of animals of different ages and species on the same site. Recurrence is explained by the probable persistence of reservoirs of the pathogen on the site (manure, lesser mealworm beetles). The article also highlights the importance of decontamination measures, including pest control, as a key element in the success of the disease control protocol.

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.

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.

Detection of Clostridium botulinum group III in environmental samples from farms by real-time PCR using four commercial DNA extraction kits.

OBJECTIVES: Few studies have tested DNA extraction methods to optimize the detection of Clostridium botulinum in environmental samples that can be collected during animal botulism outbreaks. In this study, we evaluated four commercial DNA extraction kits for the detection of C. botulinum group III in 82 various environmental samples (9 manure, 53 swabs, 3 insects, 8 water, 1 silage and 8 soil samples) collected in a context of animal botulism outbreaks. RESULTS: The PowerSoil® kit was the most efficient for almost all matrices (83.6% of the 73 tested samples), except manure for which the NucleoSpin® Soil kit was the most efficient. The NucleoSpin® Soil kit enabled detection in 75.3%, the QIAamp® DNA Mini Kit in 68.5%, and the QIAamp® Fast DNA Stool Mini Kit in 45.2%. However, the NucleoSpin® Soil kit detected C. botulinum in 9 of the 9 manure samples tested, while the PowerSoil® kit found C. botulinum in only two samples, and the other two kits in none of the samples. This study showed that PowerSoil® can be recommended for DNA extraction from environmental samples except for manure, for which the NucleoSpin® Soil kit appeared to be far more appropriate.

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.

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.

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.

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.

Quantitative and qualitative evaluation of Campylobacter spp. contamination of turkey cecal contents and carcasses during and following the slaughtering process.

The present study aimed to document quantitatively and qualitatively the contamination by thermotolerant Campylobacter spp. of turkey samples during slaughtering. Four Campylobacter-positive turkey flocks were investigated at the slaughterhouse at three different stages: evisceration (cecal content), after carcass rinses but before chilling (neck skin), and after breast meat cut (meat). In each case, the studied flock was slaughtered first thing in the morning any given day of the week. The efficiency of cleaning and disinfecting operations was examined in the facility prior to processing the studied flock. For each flock, 90 samples were collected from cecal contents, neck skins, and meat pieces and checked quantitatively and qualitatively for Campylobacter. Identification of Campylobacter species was determined by PCR, and genetic patterns were determined by pulsed-field gel electrophoresis. Campylobacter contamination levels of ceca range from 2 to more than 7 Log CFU/g, while those of neck skin range from 0.5 to 3.5 Log CFU/g and those of meat range from 0.1 to 1.9 Log CFU/g. These differences in Campylobacter counts were not associated with a modification of Campylobacter species ratio; however, in the Campylobacter jejuni population, four genetic groups identified from the ceca were not recovered during slaughtering operations and two other genetic groups were only detected after chilling at the cutting stage of the breast meat. The present study suggests that the slaughtering process did not affect Campylobacter species populations; however, it might have influenced the strain population. Finally, the Campylobacter populations found on breast meat were similar to those isolated from the digestive tract of the birds.

Prevalence and risk factors for Salmonella enterica subsp. enterica contamination in French breeding and fattening turkey flocks at the end of the rearing period.

An epidemiological study was conducted to estimate the prevalence of Salmonella spp. contamination in French commercial breeding and fattening turkey flocks at the end of the rearing period, as part of a European Union-wide baseline study. Two hundred and five breeding turkey flocks and three hundred and two fattening turkey flocks were included in the study, between October 2006 and September 2007. The Salmonella status of flocks was assessed by collecting five environmental faeces samples, analysed by classical bacteriological method. The prevalence of Salmonella positive flocks was 1.5% for breeding turkeys and 15.6% (95% CI: 11.5; 19.7) for fattening turkeys. Information on potential risk factors of the turkey flock being Salmonella positive was collected by questionnaire at the same time as sample collection. The association between management practices, general hygiene and Salmonella status in French turkey flocks was assessed by logistic regression. The risk of Salmonella contamination in fattening turkey flocks was decreased when floors were disinfected during decontamination procedures, when Salmonella detection was carried out during rearing and when there was a metering pump in the house. However in this study, the risk was increased when the farmer used a footbath at the turkey house entrance. Risk factors for Salmonella in breeding turkey flocks could not be subjected to formal statistical analysis since only three flocks were contaminated.