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

How to prevent viremia rebound? Evidence from a PRRSv data-supported model of immune response Natacha Go1,2,3*, Suzanne Touzeau

International audienceBACKGROUNDUnderstanding what determines the between-host variability in infection dynamics is a key issue to better control the infection spread. In particular, pathogen clearance is desirable over rebounds for the health of the infected individual and its contact group. In this context, the Porcine Respiratory and Reproductive Syndrome virus (PRRSv) is of particular interest. Numerous studies have shown that pigs similarly infected with this highly ubiquitous virus elicit diverse response profiles. Whilst some manage to clear the virus within a few weeks, others experience prolonged infection with a rebound. Despite much speculation, the underlying mechanisms responsible for this undesirable rebound phenomenon remain unclear.RESULTSWe aimed at identifying immune mechanisms that can reproduce and explain the rebound patterns observed in PRRSv infection using a mathematical modelling approach of the within-host dynamics. As diverse mechanisms were found to influence PRRSv infection, we established a model that details the major mechanisms and their regulations at the between-cell scale. We developed an ABC-like optimisation method to fit our model to an extensive set of experimental data, consisting of non-rebounder and rebounder viremia profiles. We compared, between both profiles, the estimated parameter values, the resulting immune dynamics and the efficacies of the underlying immune mechanisms. Exploring the influence of these mechanisms, we showed that rebound was promoted by high apoptosis, high cell infection and low cytolysis by Cytotoxic T Lymphocytes, while increasing neutralisation was very efficient to prevent rebounds.CONCLUSIONSOur paper provides an original model of the immune response and an appropriate systematic fitting method, whose interest extends beyond PRRS infection. It gives the first mechanistic explanation for emergence of rebounds during PRRSv infection. Moreover, results suggest that vaccines or genetic selection promoting strong neutralising and cytolytic responses, ideally associated with low apoptotic activity and cell permissiveness, would prevent rebound

Potential alternatives to antimicrobials in pig production based on perceived effectiveness, feasibility and return on investment

Based on a questionnaire returned by 111 pig health experts from six European countries a list of potential alternatives to antimicrobials was ranked. These althernatives provide input for further studies to find strategies to reduce the widely discussed use of antimicrobials and the potential risk from antimicrobial resistance

Effectiveness of alternative measures to reduce antimicrobial usage in pig production in four European countries

Background The reduction of antimicrobial usage (AMU) is in the focus in modern pig production. The objective of this study was to assess the effectiveness of alternatives to reduce AMU at herd level. In a prospective study, 68 farrow-to-finish pig herds located in Belgium, France, Germany and Sweden were recruited on a voluntary basis to implement tailor-made intervention plans to reduce their AMU. Alternative measures included improvement of biosecurity (n = 29 herds), vaccination (n = 30), changes of feeding schemes or drinking water quality (n = 45), improved pig health and welfare care (n = 21) as well as changes in stable climate and zootechnical measures (n = 14). Herds were followed for 1 year after implementation of measures. Annual antimicrobial expenditures or treatment records, as well as disease incidence scores were collected and compared to those of the year before intervention. AMU was measured as the treatment incidence and calculated by age category, antimicrobial class and administration route. Results Compliance with the intervention plans was high (median 93%). AMU was significantly reduced following the implementation of alternative measures: in the median herd of the four countries, pigs were treated before intervention 25% of their expected lifespan (200 days from birth to slaughter) and after intervention 16%. AMU of suckling and weaned pigs were significantly reduced by 37 and 54%, respectively. The usage of polymyxins and tetracyclines was significantly reduced by 69 and 49%, respectively. AMU via feed and water, as well as parenteral AMU were significantly reduced by 46 and 36%, respectively. Herds with a higher AMU level before intervention achieved a bigger reduction. The majority of disease incidence were similar before and after intervention, with a few exceptions of disorders related to the gastro-intestinal tract in suckling pigs (decreased) and in breeding pigs (increased). Conclusion Following tailor-made implementation of alternative measures, a substantial reduction of AMU in pig production was achievable without jeopardizing animal health. The AMU reduction in the youngest age categories (suckling and weaned pigs) and the reduction of group treatments via feed and water was in line with the recent European Guidelines on the prudent use of antimicrobials in veterinary medicine

Review: Towards the agroecological management of ruminants, pigs and poultry through the development of sustainable breeding programmes. II. Breeding strategies

Agroecology uses ecological processes and local resources rather than chemical inputs to develop productive and resilient livestock and crop production systems. In this context, breeding innovations are necessary to obtain animals that are both productive and adapted to a broad range of local contexts and diversity of systems. Breeding strategies to promote agroecological systems are similar for different animal species. However, current practices differ regarding the breeding of ruminants, pigs and poultry. Ruminant breeding is still an open system where farmers continue to choose their own breeds and strategies. Conversely, pig and poultry breeding is more or less the exclusive domain of international breeding companies which supply farmers with hybrid animals. Innovations in breeding strategies must therefore be adapted to the different species. In developed countries, reorienting current breeding programmes seems to be more effective than developing programmes dedicated to agroecological systems that will struggle to be really effective because of the small size of the populations currently concerned by such systems. Particular attention needs to be paid to determining the respective usefulness of cross-breeding v. straight breeding strategies of well-adapted local breeds. While cross-breeding may offer some immediate benefits in terms of improving certain traits that enable the animals to adapt well to local environmental conditions, it may be difficult to sustain these benefits in the longer term and could also induce an important loss of genetic diversity if the initial pure-bred populations are no longer produced. As well as supporting the value of within-breed diversity, we must preserve between-breed diversity in order to maintain numerous options for adaptation to a variety of production environments and contexts. This may involve specific public policies to maintain and characterize local breeds (in terms of both phenotypes and genotypes), which could be used more effectively if they benefited from the scientific and technical resources currently available for more common breeds. Last but not least, public policies need to enable improved information concerning the genetic resources and breeding tools available for the agroecological management of livestock production systems, and facilitate its assimilation by farmers and farm technicians

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

Evaluation of the relationship between the biosecurity status, production parameters, herd characteristics and antimicrobial usage in farrow-to-finish pig production in four EU countries

Background: High antimicrobial usage and the threat of antimicrobial resistance highlighted the need for reduced antimicrobial usage in pig production. Prevention of disease however, is necessary to obtain a reduced need for antimicrobial treatment. This study aimed at assessing possible associations between the biosecurity level, antimicrobial usage and farm and production characteristics in order to advice on best practices for a low antimicrobial usage and maximum animal health and production. A cross-sectional study was conducted in 227 farrow-to-finish pig herds in Belgium, France, Germany and Sweden between December 2012 and December 2013. Associations between biosecurity status, antimicrobial usage, and production parameters were evaluated with multivariable general linear models, according to an assumed causal pathway. Results: The results showed that higher antimicrobial usage in sows tended to be associated with higher antimicrobial usage from birth until slaughter (p = 0.06). The antimicrobial usage from birth until slaughter was positively associated with the number of pathogens vaccinated against (p < 0.01). A shorter farrowing rhythm (p < 0.01) and a younger weaning age (p = 0.06) tended to be also associated with a higher antimicrobial usage from birth until slaughter whereas a better external biosecurity (p < 0.01) was related with a lower antimicrobial usage from birth until slaughter. Conclusion: Management practices such as weaning age and biosecurity measures may be important factors indirectly impacting on antimicrobial usage. We therefore promote a holistic approach when assessing the potential to reduce the need for antimicrobial treatments