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

<p>Abstract</p> <p>Background</p> <p><it>Campylobacter </it>spp., especially <it>Campylobacter jejuni </it>(<it>C. jejuni</it>) and <it>Campylobacter coli </it>(<it>C. coli</it>), are recognized as the leading human foodborne pathogens in developed countries. Livestock animals carrying <it>Campylobacter </it>pose an important risk for human contamination. Pigs are known to be frequently colonized with <it>Campylobacter</it>, especially <it>C. coli</it>, 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 <it>C. coli </it>and <it>C. jejuni </it>in various substrates. In order to serve as a diagnostic tool supporting <it>Campylobacter </it>epidemiology, we developed a quantitative real-time PCR method for species-specific detection and quantification of <it>C. coli </it>and <it>C. jejuni </it>directly in faecal, feed, and environmental samples.</p> <p>Results</p> <p>With a sensitivity of 10 genome copies and a linear range of seven to eight orders of magnitude, the <it>C. coli </it>and <it>C. jejuni </it>real-time PCR assays allowed a precise quantification of purified DNA from <it>C. coli </it>and <it>C. jejuni</it>. The assays were highly specific and showed a 6-log-linear dynamic range of quantification with a quantitative detection limit of approximately 2.5 × 10²CFU/g of faeces, 1.3 × 10²CFU/g of feed, and 1.0 × 10³CFU/m²for the environmental samples. Compared to the results obtained by culture, both <it>C. coli </it>and <it>C. jejuni </it>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 <it>C. coli </it>or <it>C. jejuni </it>real-time PCR assay and culture enumeration were R²= 0.90 and R²= 0.93 respectively.</p> <p>Conclusion</p> <p>The <it>C. coli </it>and <it>C. jejuni </it>real-time quantitative PCR assays developed in this study provide a method capable of directly detecting and quantifying <it>C. coli </it>and <it>C. jejuni </it>in faeces, feed, and environmental samples. These assays represent a new diagnostic tool for studying the epidemiology of <it>Campylobacter </it>by, for instance, investigating the carriage and excretion of <it>C. coli </it>and <it>C. jejuni </it>by pigs from conventional herds.</p

Modelling Salmonella transmission among pigs from farm to slaughterhouse: Interplay between management variability and epidemiological uncertainty

International audienceSalmonella carriage and cutaneous contamination of pigs at slaughter are a major risk for carcass contamination. They depend on Salmonella prevalence at farm, but also on transmission and skin soiling among pigs during their journey from farm to slaughterhouse. To better understand and potentially control what influences Salmonella transmission within a pig batch during this transport and lairage step, we proposed a compartmental, discrete-time and stochastic model. We calibrated the model using pork chain data from Brittany. We carried out a sensitivity analysis to evaluate the impact of the variability in management protocols and of the uncertainty in epidemiological parameters on three model outcomes: prevalence of infection, average cutaneous contamination and number of new infections at slaughter. Each outcome is mainly influenced by a single management factor: prevalence at slaughter mainly depends on the prevalence at farm, cutaneous contamination on the contamination of lairage pens and new infections on the total duration of transport and lairage. However, these results are strongly affected by the uncertainty in epidemiological parameters. Re-excretion of carriers due to stress does not have a major impact on the number of new infections

Control of Salmonella environmental contamination during

This study aims at investigating Salmonella environmental contamination of trucks and lairage pens and evaluating the efficiency of an improved cleaning and disinfection protocol to reduce Salmonella environmental contamination. During four days, the lairage of two French pig slaughterhouses were sampled twice a day when pigs were present and once at the end of the week after the cleaning protocol. In parallel, six trucks per day were randomly selected and sampled at their arrival and after the cleaning procedure. The samples consisted of floor surface swabbing. Salmonella occurrence, level of contamination and serotypes were determined. The efficiency of different cleaning and disinfection procedures on the presence of Salmonella was also estimated. Salmonella was isolated in 97.7% of the lairage samples when pigs were present (contamination levels \u3e104UFC/m2) and in 65% of the truck samples (contamination levels from \u3c10 to \u3e104UFC/m2). An improved cleaning and disinfection procedure reduced efficiently the occurrence and the level of contamination in the trucks (almost 100%) compared to a simple wash with cold water (no effect), more partially in the lairage. This study showed the importance of a good cleaning and disinfection protocol to decrease the level of contamination or eliminate the bacteria in the trucks used for the transport of pigs

Evaluation of the potential benefits of iron supplementation in organic pig farming

Background: Iron from the stock acquired during foetal life and the ingestion of milk is not sufficient to cover the needs of the piglets during their first weeks of life. In organic farming, systematic supplementation with iron is problematic due to a strong limitation in pharmaceutic treatments. Methods: Erythroid parameters around weaning were measured in piglets from organic outdoor and indoor farms, and related to indicators of the inflammatory status. Blood samples were collected from 28.9±2.6 piglets/herd at 42.0±3.2 days of age and 11.9±3.0 kg live weight (mean ± SD) in 21 farms from the west part of France. Among the 11 outdoor farms, only one had supplemented piglets with 200 mg iron while among the 10 indoor farms, only one had not supplemented piglets, one had supplemented them with 100 mg, 8 with 200 mg and one with 400 mg. Results: Compared to outdoor piglets without supplementation, piglets kept indoors and receiving 200 mg iron had lower haemoglobin concentration (105 vs 118±2 g/l, mean ± SE) and red blood cell volume (56 vs 60±1 fl) (P<0.005). The reduction in haemoglobin concentration and red blood cell volume was more pronounced in indoor piglets supplemented with 100 mg of iron and even more when they had not received iron. The plasma concentration of haptoglobin was lower in outdoor than in indoor piglets (0.51±0.06 vs 0.78±0.09 g/l) whereas no effect of housing was observed for markers of oxidative stress (dROM, BAP). In the 14 farms where sow parity was known, the haemoglobin concentration was lower in piglets from primiparous than from multiparous sows (109 versus 114±2 g/l, P < 0.001). Conclusion: With the exception of soils where the content of bioavailable iron is very low, piglets from outdoor farms do not require iron supplementation, unlike those raised indoors

Hair cortisol concentration in finishing pigs on commercial farms: variability between pigs, batches, and farms

Hair cortisol is a stress indicator and could be used to assess the pigs’ exposure to stressors in the weeks/months prior to non-invasive hair sampling. The main aim of this study was to describe the hair cortisol concentration (HCC) variability between individuals within a batch, between farms and between batches within a farm. The secondary aim was to determine how the number of sampled pigs influences the characterization of HCC within a batch. Twenty farrow-to-finish pig farms were recruited considering the diversity of their management practices and health status (data collected). Hair was sampled in two separate batches, 8 months apart. The necks of 24 finishing pigs were clipped per batch the week prior to slaughter. To describe the variability in HCC, an analysis of the variance model was run with three explanatory variables (batch, farm and their interaction). To identify farm clusters, a principal component analysis followed by a hierarchical clustering was carried out with four active variables (means and standard deviations of the two batches per farm) and 17 supplementary variables (management practices, herd health data). We determined how the number of sampled pigs influenced the characterization of HCC within a batch by selecting subsamples of the results. HCC ranged from 0.4 to 121.6 pg/mg, with a mean of 25.9 ± 16.2 pg/mg. The variability in HCC was mainly explained by differences between pigs (57%), then between farms (24%), between batches within the same farm (16%) and between batches (3%). Three clusters of farms were identified: low homogeneous concentrations (n = 3 farms), heterogeneous concentrations with either higher (n = 7) or lower (n = 10) HCC in batch 2 than in batch 1. The diversity of management practices and health statuses allowed to discuss hypotheses explaining the HCC variations observed. We highlighted the need to sample more than 24 pigs to characterize HCC in a pig batch. HCC differences between batches on six farms suggest sampling pigs in more than one batch to describe the HCC at the farm level. HCC variations described here confirm the need to study its links with exposure of pigs to stressors

Coinfections and their molecular consequences in the porcine respiratory tract

Understudied, coinfections are more frequent in pig farms than single infections. In pigs, the term “Porcine Respiratory Disease Complex” (PRDC) is often used to describe coinfections involving viruses such as swine Influenza A Virus (swIAV), Porcine Reproductive and Respiratory Syndrome Virus (PRRSV), and Porcine CircoVirus type 2 (PCV2) as well as bacteria like Actinobacillus pleuropneumoniae, Mycoplasma hyopneumoniae and Bordetella bronchiseptica. The clinical outcome of the various coinfection or superinfection situations is usually assessed in the studies while in most of cases there is no clear elucidation of the fine mechanisms shaping the complex interactions occurring between microorganisms. In this comprehensive review, we aimed at identifying the studies dealing with coinfections or superinfections in the pig respiratory tract and at presenting the interactions between pathogens and, when possible, the mechanisms controlling them. Coinfections and superinfections involving viruses and bacteria were considered while research articles including protozoan and fungi were excluded. We discuss the main limitations complicating the interpretation of coinfection/superinfection studies, and the high potential perspectives in this fascinating research field, which is expecting to gain more and more interest in the next years for the obvious benefit of animal health

Campylobacter chez le porc (méthodes d identification quantitative et dynamique d infection)

L objectif de ces travaux est de produire des méthodes et des connaissances permettant d étudier le portage de Campylobacter en élevage porcin. Des techniques de PCR quantitative en temps réel rapides et fiables ont été mises au point pour la quantification de Campylobacter spp., C. coli et C. jejuni. dans les matières fécales ou les prélèvements environnementaux. Des porcs EOPS âgés de sept semaines ont été inoculés expérimentalement avec plusieurs souches de Campylobacter d origine différente, seules ou en association. Cette infection expérimentale a permis de souligner les grandes tendances décrites dans la littérature (portage asymptomatique, niveaux d excrétion élevés et légère baisse des quantités excrétées en fin d engraissement) et de mettre en évidence une intermittence de l excrétion, une une transmission rapide à distance aux animaux des cases adjacentes et l existence d une interaction spécifique hôte/espèce. Elle a également permis de calibrer deux méthodes de typage, la macrorestriction génomique en champ pulsé et la PCR-RFLP sur le gène flaA et d établir un seuil de similarité entre souches. Une variabilité génomique in vivo dans l animal a été mise en évidence avec un effet souche marqué (seul C. coli d origine porcine a varié). La dernière étape était l application de ces méthodes lors d enquêtes en élevage naisseur-engraisseur pour décrire la contamination de l environnement et la dynamique d infection des Campylobacter en élevage porcin. Notre étude souligne le rôle de la truie comme source de contamination mais l environnement est un élément possible de transmission des Campylobacter entre les animaux à considérer.This work aimed at providing new methods and knowledge to study the carriage of Campylobacter in conventional pig herds. Campylobacter specific real-time PCR assays developed in this study allow a direct and reliable quantification of Campylobacter spp, Campylobacter coli and Campylobacter jejuni in complex substrates like faeces or environmental samples due to the presence of an internal control of DNA extraction and PCR amplification. To study excretion in controlled condition, we inoculated three different Campylobacter strains to specific pathogen-free pigs. Our results confirmed an asymptomatic carriage and a high excretion There is a transmission between inoculated and contact pigs, which are Campylobacter-free at the beginning of the study and which live in the same experimental unit. The results suggest a specific interaction between the Campylobacter species and the host. Moreover, this experimental infection allowed to calibrate two molecular typing methods, namely RFLP-PFGE and flaA PCR-RFLP. Variability was evidenced only in pigs inoculated with C. coli of porcine origin, either alone or in the mix, or in sham inoculated pigs neighbouring the latter. A discrimination threshold was established with the two methods. Finally, to describe Campylobacter infection in commercial pig herds, faecal shedding as well as contamination of pens (either empty or with animals), feed and water were monitored from birth to finishing for pigs, and during one production cycle for sows. Our study underlines the role of the sows as a Campylobacter contamination source for their piglets and the role of the environment in the transmission of Campylobacter between animalsRENNES1-BU Sciences Philo (352382102) / SudocSudocFranceF

Mise au point d'une PCR quantitative en temps réel pour la mise en évidence de campylobacter (études préliminaires)

Les campylobacter thermotolérants sont considérés comme la première cause de maladies infectieuses d'origine alimentaire dans les pays développés et les denrées d'origine animale ont été identifiées comme source de ces contaminations.NANTES-Ecole Nat.Vétérinaire (441092302) / SudocSudocFranceF