Françoise Lalande

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.

Miniaturized Most Probable Number and Enrichment Serology Technique for the Enumeration of Salmonella spp. on Poultry Carcasses

The ability of two 8-tube most probable number (MPN) techniques to quantitatively recover Salmonella spp. from 26 fresh, naturally contaminated chicken skin samples was compared. Individual macerated skin samples were tested in parallel using a traditional (tMPN) and a miniaturized (mMPN) analytical procedure. In the tMPN assay, replicate aqueous portions from each macerated sample were preenriched individually in buffered peptone water, selectively enriched in Müller-Kauffmann tetrathionate brilliant green broth (MKTBG), and plated on Rambach agar. Each MKTBG was also postenriched in M Broth, and the resulting postenrichment culture screened for the presence of Salmonella cells by enrichment serology (ES). Although a similar analytical approach was used in the mMPN assay, it differed from the tMPN in the use of smaller test volumes dispensed in microplates, and on a sedimented portion of skin macerate as test material. Of the 26 Salmonella -contaminated samples examined in this study, the tMPN coupled to Rambach agar or ES identified 23 and 24 positive samples, respectively. Under homologous conditions, the mMPN detected all 26 positive Salmonella contaminated samples. The most probable numbers in 100-g skin samples analyzed by the tMPN ranged from 18/100 g to 9,530,000/100 g with a median value of 570/100 g. Levels of contamination by the mMPN procedure ranged from 90/100 g to 556,000/100 g with a median value of 1,200/100 g. Statistical analysis of experimental data underlined the equivalence of the tMPN and the mMPN procedures and nonequivalence of the Rambach plating and ES conditions. It is suggested that the microplate mMPN coupled to ES offers a reliable and more cost-effective analytical approach for the quantitative recovery of Salmonella on broiler carcasses.

Heterogeneity of Persistence of Salmonella enterica Serotype Senftenberg Strains Could Explain the Emergence of this Serotype in Poultry Flocks

Salmonella enterica serotype Senftenberg (S. Senftenberg) has recently become more frequent in poultry flocks. Moreover some strains have been implicated in severe clinical cases. To explain the causes of this emergence in farm animals, 134 S. Senftenberg isolates from hatcheries, poultry farms and human clinical cases were analyzed. Persistent and non-persistent strains were identified in chicks. The non-persistent strains disappeared from ceca a few weeks post inoculation. This lack of persistence could be related to the disappearance of this serotype from poultry farms in the past. In contrast, persistent S. Senftenberg strains induced an intestinal asymptomatic carrier state in chicks similar to S. Enteritidis, but a weaker systemic infection than S. Enteritidis in chicks and mice. An in vitro analysis showed that the low infectivity of S. Senftenberg is in part related to its low capacity to invade enterocytes and thus to translocate the intestinal barrier. The higher capacity of persistent than non-persistent strains to colonize and persist in the ceca of chickens could explain the increased persistence of S. Senftenberg in poultry flocks. This trait might thus present a human health risk as these bacteria could be present in animals before slaughter and during food processing.

Effect of four antibiotic additives on the Salmonella contamination of chicks protected by an adult caecal flora.

Avoparcin (10 mg/kg feed), bacitracin (50 mg/kg), flavomycin (5 mg/kg) and virginiamycin (20 mg/kg) were tested for their synergy or antagonism on the protective effect of an adult caecal flora administered to 1-day-old chicks. The chicks were challenged experimentally per os with 10(4) to 10(5)Salmonella typhimurium (a rifampicin-resistant strain) when aged 2 days. Chicks receiving avoparcin continuously in the feed had significantly more Salmonella in their caeca than control birds given feed containing no antibiotics; those receiving flavomycin had similar numbers to the controls whereas the groups fed on a diet supplemented with bacitracin or virginiamycin exhibited the lowest level of Salmonella carriage.

Evaluation of a French ELISA for the detection of Salmonella Enteritidis and Salmonella Typhimurium in flocks of laying and breeding hens

Abstract In France, the regular and compulsory detection of Salmonella Enteritidis (SE) and Salmonella Typhimurium (ST) in flocks of breeding and laying hens is based on bacteriological examination of environmental swabs and faeces samples. The aim of this study was to compare this bacteriological examination with a serological method (ELISA) developed in our laboratory. This ELISA was first evaluated by use of artificially infected hens. During these experimental infection studies, several groups of hens were inoculated with SE, ST, different vaccines and different Salmonella serovars to calculate the experimental parameters of our ELISA. Then, in a field study, 43 flocks were followed monthly using two bacteriological samples (environmental swab and pool of faeces) and 20 serological samples (sera or yolks). Twenty-seven flocks without SE or ST gave a negative serological response throughout their surveillance. Among the 10 various serovars different from SE and ST isolated in this study, S. Heidelberg, S. Agona and S. Hadar gave seropositive results in seven flocks. Consequently, this ELISA was not specific of SE and ST as it detected serovars sharing or not common antigens with SE and ST. Seropositive results were also obtained each month for two flocks where no Salmonella could be isolated. Finally, in seven flocks found infected with SE or ST, the positive ELISA results appeared later than the bacteriological detection. Therefore, for the detection of chicken flocks recently infected with SE or ST, bacteriological examination currently used in France seems to be more appropriate than this ELISA.

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.

Quantification of Campylobacter carriage in pigs using a real-time PCR assay

Campylobacter species are the major agent of bacterial gastroenteritis. C. jejuni and C. coli together are responsible for more than 95% of all cases of Campylobacter induced diarrheal d1sease 1n developped countries R1sk analysis shows consumption of foods of animal origin to be a maJor source of human 1nfect1on Pigs are known to be frequently infected with Campylobacter and to exh1bit h1gh counts of th1s pathogen in their faeces. The study descnbes a rap1d, sens1tive. and spec1fic real-t1me polymerase cham react1on (PCR} assay capable of detecting and quantifying Campylobacter sp., C. JBJUnl and C. coli directly from faecal samples. The description of excretion of Campylobacter was carried out by inoculating pigs with three different strams of Campylobacter. one C. coli of porcine origin, one C. coli and one C jejuni of poultry ongm, alone or m a mix. The number of Campylobacter excreted in faeces was determ1ned by numeration on Karmali plates and by the real time PCR assay. The quantitative PCR results were cons1stent w1th data obtamed by the bactenolog1cal method Two days after the inoculation. the inoculated p1gs excreted from 104 to 106 CFU of Campylobacter per gramme of faeces. These levels of excret1on were s1m1lar to those observed 1n the fattening p1gs after spontaneous mfection Moreover, the real time PCR allowed species-specific detection of Campylobacter Indeed, when p1gs were Infected w1th a m1x of the three strams, the PCR showed that C. co/1 was predominant.