Investigation of a type C/D botulism outbreak in free-range laying hens in France.

In 2014, a botulism outbreak in a flock of laying hens was investigated in France. In the flock of 5020 hens, clinical signs of botulism occurred at 46 weeks of age. A type C/D botulism outbreak was confirmed using the mouse lethality assay for detection of botulinum toxin in serum and a real-time PCR test to detect Clostridium botulinum in intestinal contents. The disease lasted one week with a mortality rate of 2.6% without recurrence. Botulism in laying hens has rarely been reported. Five monthly visits were made to the farm between December 2014 and May 2015 for a longitudinal study of the persistence of C. botulinum in the poultry house after the outbreak, and to assess egg contamination by C. botulinum. Several samples were collected on each visit: in the house (from the ventilation circuit, the egg circuit, water and feed, droppings) and the surrounding area. Thirty clean and 30 dirty eggs were also swabbed at each visit. In addition, 12 dirty and 12 clean eggs were collected to analyse eggshell and egg content. The samples were analysed using real-time PCR to detect type C/D C. botulinum. The bacterium was still detected in the house more than 5 months after the outbreak, mostly on the walls and in the egg circuit. Regarding egg contamination, the bacteria were detected only on the shell but not in the content of the eggs. Control measures should therefore be implemented throughout the egg production period to avoid dissemination of the bacteria, particularly during egg collection.

Influence of enrichment and isolation media on the detection of Campylobacter spp. in naturally contaminated chicken samples.

Investigating Campylobacter epidemiology requires adequate technique and media to ensure optimal culturing and accurate detection and isolation of Campylobacter strains. In the present study, we investigated the performances of three enrichment durations in Bolton broth (0, 24 and 48h) and compared four isolation media (mCCDA, Karmali, Butzler no. 2 and CampyFood agar (CFA)) for the detection of Campylobacter positive samples and the identification of Campylobacter species, from naturally contaminated broiler chicken samples (caeca, neck skin from carcasses, and skin from thighs). We compared our local results to those we obtained with samples from a European survey (caeca and neck skin) and a national survey (neck skin, thigh skin, and breast). Direct plating favored the detection of positive samples highly contaminated by Campylobacter (caeca and neck skin from carcasses) whatever the media. A longer enrichment reduced the rates of Campylobacter recovery except when using Butzler no. 2, more particularly for neck skin which background microflora was less important than in caeca. As a matter of fact, enrichment allowed a higher detection rate of positive samples with low Campylobacter contamination levels (breast, thigh skin), this detection being enhanced when using Butzler no. 2. When comparing the 3 other selective media, CFA was the 2nd most efficient media prior to mCCDA and Karmali. Interestingly, enrichment promoted the growth of Campylobacter coli but this promotion was least with Butzler no. 2 agar. Our study has confirmed the need to adapt the method to the types of samples for improving the detection of Campylobacter and that the method may affect the prevalence of the species.

Occurrence of C. botulinum in healthy cattle and their environment following poultry botulism outbreaks in mixed farms.

Ten cattle farms located in an area with a recent history of poultry botulism outbreaks were investigated to evaluate the occurrence of toxigenic C. botulinum in healthy cattle. Environmental samples in the 10 cattle farms and bovine fecal contents in farms with a confirmed environmental contamination were collected. Detection of C. botulinum toxin genes C, D, C/D, D/C and E was performed using real-time PCR. 4.9% (7/143) of the environmental samples collected in the 10 investigated cattle farms were positive for C. botulinum type C/D. Theses samples (boot-swabs in stalls and on pasture and water of a stream) were collected in 3 different farms. One cow dung sample and 3 out of 64 fecal contents samples collected in a single farm were also positive for C. botulinum type C/D. This study demonstrates that cattle are probably indirectly contaminated via poultry botulism in the area and that they can be intermittent carrier of C. botulinum type C/D after poultry botulism outbreaks in mixed farms.

Prevalence of and risk factors for Campylobacter colonisation in broiler flocks at the end of the rearing period in France.

1. A study was conducted to estimate the prevalence and quantification by species of Campylobacter infection in broiler flocks at the end of the rearing period and to identify associated risk factors. 2. A questionnaire about the rearing period was completed and caecal samples were collected from 121 broiler flocks in Brittany, France, during 2008. 3. Campylobacter was isolated in 87 out of 121 flocks--a prevalence of 71.9% (95% CI, 63.7-80.1%), including 40.5% of Campylobacter jejuni and 29.8% of Campylobacter coli. 4. The average concentration, in positive flocks, was 7.96 log10 cfu/g and ranged from 3.15 to 10.32 log10 cfu/g. 5. The average concentration by species was: 7.57 log10 cfu/g for C. jejuni and 8.44 log10 cfu/g for C. coli. 6. There was a seasonal effect, with increased risk of Campylobacter colonisation in June, July and August (odds ratio (OR) = 9.59, 95% CI 1.15-79.75). 7. The other factors, associated with lower risk of Campylobacter colonisation, were the acidification of drinking water (OR = 0.33, 95% CI 0.13-0.86), antibiotic treatment at the beginning of the rearing period (OR = 0.20, 95% CI 0.07-0.55) and rodent control around the house (OR = 0.18, 95% CI 0.03-0.95). 8. The results show that hygiene practices and biosecurity measures could lead to a reduction in Campylobacter colonisation.

Distribution of serotypes and genotypes of Salmonella enterica species in French pig production.

The population of Salmonella found at various stages of pig production in France was characterised to analyse the distribution and spread of Salmonella in the pig production chain. We serotyped and genotyped by PFGE 174 isolates collected from breeding pigs from breeding farms, 163 collected from breeding pigs from production farms, and 325 collected from fattening pigs. Forty-seven serovars and 110 genotypes were identified. The major serovars were S Derby (263 isolates) and S Typhimurium (162 isolates). The percentage of S Derby isolates decreased slightly through the production system (44.3, 41.1 per cent and 36.5 per cent) and 79.1 per cent of the S Derby isolates were distributed in the five genotypes common to all three stages. The percentage of S Typhimurium isolates was high for slaughter pigs (40.8 per cent) and 43 of the 46 S Typhimurium genotypes were only identified at this stage. Distributions of S Derby and S Typhimurium between breeding and fattening pigs were different. S Derby was found throughout the pig production pyramid, suggesting that this serotype may be transmitted by the transfer of animals between herds. The presence of multiple S Typhimurium genotypes in fattening pigs suggests that there were many sources of contamination at this stage, with fattening pigs having higher levels of exposure and/or sensitivity to this serotype.

Prevalence and risk factors for Salmonella spp. contamination in French broiler-chicken flocks at the end of the rearing period.

A nation-wide survey was carried out in 370 randomly chosen French commercial broiler chicken flocks from October 2005 to September 2006 to determine Salmonella spp. prevalence and to identify risk factors for contamination, at the end of the rearing period. The Salmonella status of the flocks was assessed from five faecal samples (litter swabs) analysed by classical bacteriological methods. A flock with at least one contaminated sample was classified as a Salmonella-positive flock. The apparent prevalence of Salmonella was 8.6% (95% CI: 5.7, 11.5%). The most prevalent serovar was S. hadar followed by S. anatum and S. mbandaka. Logistic regression methods were used to analyse the associations between husbandry practices, farm characteristics, general hygiene and the Salmonella status of the sample. The risk for Salmonella contamination of the flock at the end of the rearing period increased when neighbours helped in the placement of day-old chicks. On the contrary, the risk decreased when mobile equipment was dismantled before cleaning and disinfection, when the farm had a specific container for dead-bird disposal and when acetic acid was added to the drinking water.

Bayesian estimation of flock-level sensitivity of detection of Salmonella spp., Enteritidis and Typhimurium according to the sampling procedure in French laying-hen houses.

A study was carried out to estimate the prevalence of flocks infected by Salmonella spp., S. Enteritidis and S. Typhimurium in 521 French laying-hen farms from October 1st 2004 to September 30th 2005 as part of a European Union-wide baseline study to define targets for Salmonella reduction in member states. The sampling scheme prescribed and financed by the European Commission to detect Salmonella in laying-hen flocks was based on 2 dust-samples and 5 faeces-samples per farm. A latent-class Bayesian approach for correlated tests was used to estimate the sensitivity of detection of reduced sampling schemes corresponding to the 16 combinations of 2 dust- and 5 faeces-samples. For each model the full sampling scheme (7 samples) and the reduced protocol were considered as two correlated tests, the biological principle being identical and the reduced protocol being a subset of the full sampling scheme. As the observed apparent prevalence in cage flocks was higher than in other systems (barns, outdoor, or organic) these two sub-populations were considered separately. Bayesian estimation of posterior medians with 95% probability intervals for true prevalence in cage flocks were 0.34 (0.29; 0.39) and 0.13 (0.10; 0.18) for Salmonella spp. and Salmonella Enteritidis+Typhimurium respectively. In alternative flocks posterior medians with 95% probability intervals for true prevalence were 0.09 (0.06; 0.13) and 0.05 (0.03; 0.08) for Salmonella spp. and Salmonella Enteritidis+Typhimurium, respectively. In cage flocks Bayesian estimation of posterior distributions for sensitivity indicated that at least 5 samples, including 2 dust samples were necessary to attain comparable sensitivity levels to the full sampling scheme. In alternative flocks and for Salmonella spp. 6 samples were required to ensure a comparable sensitivity level to the full sampling scheme. Detection sensitivity was improved by increasing the number of dust samples in cage farms and by increasing the total number of samples whatever their type in alternative farms.