S. Rouxel

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 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.

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

Abstract 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.

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.

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.

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

ABSTRACT 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.

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.

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.

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.