Martine Denis

Risk factors for Campylobacter spp. contamination in French broiler-chicken flocks at the end of the rearing period

Our objectives were to identify risk factors for contamination of French broiler flocks by Campylobacter. We used 75 broiler farms in western France. A questionnaire was administered to the farmers and samples of fresh droppings were taken to assess the Campylobacter status of the broiler flocks. 42.7% of the flocks were positive for Campylobacter spp. The risk of contamination of the broiler flocks by Campylobacter was increased in summer/autumn, in houses with static air distribution, when two or more people took care of the flock, in poultry farms with three or more houses and when the drinking water for the chickens was acidified. The presence of litter-beetles in the change room also increased the risk of contamination. The administration of an antibiotic treatment following a disease decreased the risk of a flock being contaminated by Campylobacter.

Rapid identification and quantification of Campylobacter coli and Campylobacter jejuni by real-time PCR in pure cultures and in complex samples

BackgroundCampylobacter spp., especially Campylobacter jejuni (C. jejuni) and Campylobacter coli (C. coli), are recognized as the leading human foodborne pathogens in developed countries. Livestock animals carrying Campylobacter pose an important risk for human contamination. Pigs are known to be frequently colonized with Campylobacter, especially C. coli, and to excrete high numbers of this pathogen in their faeces. Molecular tools, notably real-time PCR, provide an effective, rapid, and sensitive alternative to culture-based methods for the detection of C. coli and C. jejuni in various substrates. In order to serve as a diagnostic tool supporting Campylobacter epidemiology, we developed a quantitative real-time PCR method for species-specific detection and quantification of C. coli and C. jejuni directly in faecal, feed, and environmental samples.ResultsWith a sensitivity of 10 genome copies and a linear range of seven to eight orders of magnitude, the C. coli and C. jejuni real-time PCR assays allowed a precise quantification of purified DNA from C. coli and C. jejuni. The assays were highly specific and showed a 6-log-linear dynamic range of quantification with a quantitative detection limit of approximately 2.5 × 102 CFU/g of faeces, 1.3 × 102 CFU/g of feed, and 1.0 × 103 CFU/m2 for the environmental samples. Compared to the results obtained by culture, both C. coli and C. jejuni real-time PCR assays exhibited a specificity of 96.2% with a kappa of 0.94 and 0.89 respectively. For faecal samples of experimentally infected pigs, the coefficients of correlation between the C. coli or C. jejuni real-time PCR assay and culture enumeration were R2 = 0.90 and R2 = 0.93 respectively.ConclusionThe C. coli and C. jejuni real-time quantitative PCR assays developed in this study provide a method capable of directly detecting and quantifying C. coli and C. jejuni in faeces, feed, and environmental samples. These assays represent a new diagnostic tool for studying the epidemiology of Campylobacter by, for instance, investigating the carriage and excretion of C. coli and C. jejuni by pigs from conventional herds.

Diversity of Pulsed-Field Gel Electrophoresis Profiles of Campylobacter jejuni and Campylobacter coli from Broiler Chickens in France

In 2003 to 2004, 26 free-range broilers flocks excreting Campylobacter were studied for identification of Campylobacter species and genotype diversity. Seventeen flocks were sampled at the end of the indoor rearing period and 9 before departure to the slaughterhouse after access to an open area. Out of 513 isolates, 315 were identified as Campylobacter jejuni and 198 as Campylobacter coli. Pulsed-field gel electrophoresis analysis revealed 35 genotypes for C. jejuni and 43 genotypes for C. coli; 38.4% of the isolates were clustered into 16 genetic groups. This kind of poultry production system is characterized by a large number of Campylobacter coli isolates. Flocks sampled during the indoor phase were predominantly contaminated by C. jejuni, whereas those sampled during warm months were predominantly contaminated by C. coli. The Campylobacter population was genetically highly diverse: multiple genotypes were detected in a single flock. Six flocks were each found to harbor a mixture of genotypes; these isolates were genetically closely related and were grouped into clusters of at least 80% genetic similarity. Isolates with genotypes found in different flocks and strains, but sharing the same genetic clusters, were detected in different farms and at different times in the year. Flocks sampled during the indoor rearing period and when farmers use fresh farm-made litter were associated with a small number of clusters. In conclusion, Campylobacter species were genetically highly diverse. Our findings suggest the presence of genomic rearrangements in Campylobacter colonizing the chick intestine and that some Campylobacter strains are adapted to poultry. In addition, the species diversity in the flocks was affected by season and cloistering measures. Litter and exposure to an open area were likely sources of flock Campylobacter contamination.

A simplified method for detecting pathogenic Yersinia enterocolitica in slaughtered pig tonsils

The aim of this study was to collect preliminary data on the carriage of pathogenic Yersinia enterocolitica in slaughtered pigs in France and to test a simplified method for detecting these strains from tonsils. From January to March 2009, 900 tonsil swabs were taken from pigs at one slaughterhouse in Brittany, France. The swabs were vortexed in 10 ml PSB broth, then 1 ml was added to 9 ml ITC broth. The media were incubated for 48 h at 25°C. The PSB enrichment broth was streaked on CIN plates and the ITC enrichment broth on SSDC plates. In addition to the ISO 10273 method, we also streaked ITC enrichment broth on CIN plates. The plates were incubated for 24h at 30°C, and we then streaked a maximum of four typical colonies per plate onto a plate containing chromogenic medium (YeCM), for the isolation of pathogenic Y. enterocolitica isolates. In parallel, biochemical assays were carried out to confirm the identification of the isolates as Yersinia and to determine biotype. After passage on a YeCM plate and biochemical tests, 380 strains were confirmed to be pathogenic Y. enterocolitica. Finally, with the ISO 10273 method, 9.1% (CI(95%) [5.8-12.4]) of tonsil swabs and 60% (CI(95%) [45.4-74.6]) of the batches were positive. With the ITC-CIN method, 14.0% (CI95% [10.7-17.3]) of the tonsil swabs and 68.9% (CI(95%) [54.3-83.5]) of the batches were positive. Identification as pathogenic Y. enterocolitica was confirmed for 97.0% of the typical colonies obtained on the chromogenic medium, YeCM. The most prevalent biotype was biotype 4 (80.5% of the isolates), followed by biotype 3. This study demonstrates that the ITC-CIN method, followed by streaking on YeCM, may be an effective approach to the isolation of pathogenic Y. enterocolitica from tonsil swabs and the recovery of positive samples. This method is less time-consuming than the ISO 10273 method and reduces the number of biochemical tests required for the confirmation of Yersinia identification, through the use of YeCM.

Quantification of Campylobacter spp. in pig feces by direct real-time PCR with an internal control of extraction and amplification

The rapid and direct quantification of Campylobacter spp. in complex substrates like feces or environmental samples is crucial to facilitate epidemiological studies on Campylobacter in pig production systems. We developed a real-time PCR assay for detecting and quantifying Campylobacter spp. directly in pig feces with the use of an internal control. Campylobacter spp. and Yersinia ruckeri primers-probes sets were designed and checked for specificity with diverse Campylobacter, related organisms, and other bacterial pathogens before being used in field samples. The quantification of Campylobacter spp. by the real-time PCR then was realized on 531 fecal samples obtained from experimentally and naturally infected pigs; the numeration of Campylobacter on Karmali plate was done in parallel. Yersinia ruckeri, used as bacterial internal control, was added to the samples before DNA extraction to control DNA-extraction and PCR-amplification. The sensitivity of the PCR assay was 10 genome copies. The established Campylobacter real-time PCR assay showed a 7-log-wide linear dynamic range of quantification (R²=0.99) with a detection limit of 200 Colony Forming Units of Campylobacter per gram of feces. A high correlation was found between the results obtained by real-time PCR and those by culture at both qualitative and quantitative levels. Moreover, DNA extraction followed by real-time PCR reduced the time needed for analysis to a few hours (within a working day). In conclusion, the real-time PCR developed in this study provides new tools for further epidemiological surveys to investigate the carriage and excretion of Campylobacter by pigs.

Experimental infection of Specific-Pathogen-Free pigs with Campylobacter: excretion in faeces and transmission to non-inoculated pigs

Campylobacter species are leading agents of human bacterial gastroenteritis and consumption of food of animal origin is a major source of infection. Although pigs are known to frequently exhibit high counts of Campylobacter in their faeces, more information is needed about the dynamics of this excretion. An experimental trial was conducted to evaluate the faecal excretion of Campylobacter by 7-week-old specific pathogen-free piglets inoculated per os with three Campylobacter strains (one C. coli isolated from a pig, one C. coli and one C. jejuni from chickens) alone or simultaneously (5x10(7)CFU/strain). Non-inoculated pigs were housed in adjacent pens. Pigs were monitored for 80 days for clinical signs and by bacteriological analysis of faeces. Pigs inoculated with porcine C. coli or with a mix of the three strains excreted from 10(3) to 10(6)CFU/g of faeces with a slight decrease at the end of the trial. Animals inoculated with poultry C. coli or C. jejuni strain excreted a lower quantity and some of them stopped excreting. At the end of the trial, only C. coli was detected in the faeces of pigs inoculated simultaneously with the three bacteria. Moreover, the transmission of Campylobacter was noticed between pens for the two C. coli strains and all the neighbouring animals became shedders with a level of excretion similar to the inoculated pigs. Intermittence in the Campylobacter excretion was also observed. Finally, our study highlighted a host preference of Campylobacter, namely C. coli seems to have a higher colonization potential for pigs than C. jejuni.

Attribution of the French human Salmonellosis cases to the main food-sources according to the type of surveillance data.

Salmonella are the most common bacterial cause of foodborne infections in France and ubiquitous pathogens present in many animal productions. Assessing the relative contribution of the different food-animal sources to the burden of human cases is a key step towards the conception, prioritization and assessment of efficient control policy measures. For this purpose, we considered a Bayesian microbial subtyping attribution approach based on a previous published model (Hald et al., 2004). It requires quality integrated data on human cases and on the contamination of their food sources, per serotype and microbial subtype, which were retrieved from the French integrated surveillance system for Salmonella. The quality of the data available for such an approach is an issue for many countries in which the surveillance system has not been designed for this purpose. In France, the sources are monitored simultaneously by an active, regulation-based surveillance system that produces representative prevalence data (as ideally required for the approach) and a passive system relying on voluntary laboratories that produces data not meeting the standards set by Hald et al. (2004) but covering a broader range of sources. These data allowed us to study the impact of data quality on the attribution results, globally and focusing on specific features of the data (number of sources and contamination indicator). The microbial subtyping attribution model was run using an adapted parameterization previously proposed (David et al., 2012). A total of 9076 domestic sporadic cases were included in the analyses as well as 9 sources among which 5 were common to the active and the passive datasets. The greatest impact on the attribution results was observed for the number of sources. Thus, especially in the absence of data on imported products, the attribution estimates presented here should be considered with caution. The results were comparable for both types of surveillance, leading to the conclusion that passive data constitute a potential cost-effective complement to active data collection, especially interesting because the former encompass a greater number of sources. The model appeared robust to the type of surveillance, and provided that some methodological aspects of the model can be enhanced, it could also serve as a risk-based guidance tool for active surveillance systems.

Genetic Diversity and Antimicrobial Resistance Profiles of Salmonella enterica Serotype Derby Isolated from Pigs, Pork, and Humans in France

In France, Salmonella enterica serotypes Typhimurium and Derby are the most often isolated serotypes in pigs. Moreover, serotype Derby usually ranks between third and fourth in prevalence among human isolates in France. The aim of this study was to evaluate the genetic relationships between human and pig Salmonella Derby isolates based on their pulsed-field gel electrophoresis (PFGE) patterns after XbaI, BlnI, and SpeI restriction and on their antimicrobial resistance profiles. The 196 studied isolates were isolated in 2006 and 2007: 73 from fattening pigs, 27 from pork, and 96 from humans. Forty-four PFGE XbaI patterns were identified. A major pattern (SDX01) was identified for 96 isolates (49%). This pattern was common to pig, pork, and human isolates. Among the 146 isolates tested for their antimicrobial resistance, 84.2% (n=123) showed resistance to at least one antibiotic and 69.2% (n=101) were simultaneously resistant to at least streptomycin, sulfonamides, and tetracycline. Most of the isolates that are resistant to these three antibiotics also displayed the major SDX01 pattern. The use of two other restriction enzymes on a part of the panel (155 isolates) brought a significant increase in the discriminatory index, in particular for SDX01 strains. As Salmonella Derby is essentially isolated from pigs, and major resistance and PFGE patterns of isolates from pigs and pork were very similar to human isolates, human salmonellosis due to Salmonella Derby may be related to pigs.

Prevalence of pathogenic Yersinia enterocolitica in slaughter-aged pigs during a one-year survey, 2010–2011, France

The prevalence of pathogenic Yersinia enterocolitica in French slaughter-aged pigs was estimated by sampling 3120 pigs from 96 batches in 16 slaughterhouses from January 2010 to February 2011. Respectively, 36 batches (20 pigs/batch) and 60 batches (40 pigs/batch) were considered during the cold period and the warm period. Tonsils were swabbed before the chilling step. Pathogenic Y. enterocolitica was detected after enrichment in ITC and streaking on CIN and YeCM media. Typical isolates were confirmed as Y. enterocolitica and biotyped by biochemical tests as described in the ISO 10273:2003 method. Of the tested pigs, 13.7% (CI95% [10.1-17.3]) were found positive for pathogenic Y. enterocolitica and 74.3% (CI95% [64.8-83.8]) of the pig batches contained at least one positive pig. The percentage of positive pigs per batch was generally low; 60.3% of positive batches contained fewer than 5 positive pigs. The prevalence of the pathogen at the batch level remained unchanged throughout this one-year study, but the prevalence in pigs was significantly higher during the warm period than during the cold period. Biotype 4 was the most prevalent biotype among the 827 isolated strains (91.9% of the isolates), followed by biotype 3 (7.25% of the isolates). Six isolates were of biotype 5 and one of biotype 2. Biotype 4 was found in all the 16 participating slaughterhouses, biotype 3 in ten slaughterhouses and biotype 5 in four. This study provides valuable recent figures for the prevalence of pathogenic Y. enterocolitica in French pigs. It also highlights the seasonal aspect of the carriage of this pathogen by pigs, a pattern which differs from those in other countries.

Antibiotic Resistance in Escherichia coli from Pigs in Organic and Conventional Farming in Four European Countries

Organic pig production differs in many ways from conventional production of pigs, e.g., in antibiotic use, herd structure, feeding regimes, access to outdoor areas and space allowance per pig. This study investigated if these differences result in a lower occurrence of antibiotic resistance in organic slaughter pigs in Denmark, France, Italy and Sweden. Samples were taken from the colon content and/or faeces and minimum inhibitory concentrations (MIC) of ten antibiotics were determined in isolates of Escherichia coli. In addition, the proportion of tetracycline (TET) resistant E. coli in colon content and/or faeces from individual pigs was determined. In all four countries the percentage resistance to ampicillin, streptomycin, sulphonamides or trimethoprim was significantly lower in E. coli from organic pigs. In France and Italy, the percentage of isolates resistant to chloramphenicol, ciprofloxacin, nalidixic acid or gentamicin was also significantly lower in the E. coli from organic pigs. Resistance to cefotaxime, was not found in any country. The percentage of E. coli isolates resistant to TET as well as the proportion of TET-resistant E. coli was significantly lower in organic than in conventional pigs, except in Sweden where TET-resistance was equally low in both production types. There were also differences between countries within production type in the percentage resistance to individual antibiotics as well as the proportion of TET-resistant E. coli with lower median proportions in Sweden and Denmark compared to France and Italy. The study shows that in each of the four countries resistance in intestinal E. coli was less common in organic than in conventional pigs, but that there were also large differences in resistance between countries within each production type, indicating that both country- and production-specific factors influence the occurrence of resistance.