Catherine Belloc

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.

Serologic evidence for bovine immunodeficiency virus infection in France

We report herein on the first serologic detection of antibodies to bovine immunodeficiency virus (BIV) in France. Serum samples from dairy and beef cattle from southwestern and western France (Landes and Vendée) were tested using a western blot assay with a recombinant 53 kDa gag precursor derived from the Louisiana BIV R29 isolate. We performed our study on the oldest animals from 37 different herds that were under serologic follow up for previous bovine leukemia virus infection. Overall, 398 selected bovine sera were assayed and 15 serum samples from 8 herds reacted with the recombinant 53 kDa BIV R29 gag. Interestingly, reactions obtained with French sera were weaker than with positive Louisiana sera, a finding that may indicate the occurrence of distinct French and Louisiana BIV variants.

Alternatives to the use of antimicrobial agents in pig production: A multi-country expert-ranking of perceived effectiveness, feasibility and return on investment

Nineteen alternatives to antimicrobial agents were ranked on perceived effectiveness, feasibility and return on investment (ROI) from 0 (not effective, not feasible, no ROI) to 10 (fully effective, completely feasible, maximum ROI) by 111 pig health experts from Belgium, Denmark, France, Germany, Sweden and Switzerland. The top 5 measures in terms of perceived effectiveness were (1) improved internal biosecurity, (2) improved external biosecurity, (3) improved climate/environmental conditions, (4) high health/Specific Pathogen Free/disease eradication and (5) increased vaccination. The top 5 measures in terms of perceived feasibility were (1) increased vaccination, (2) increased use of anti-inflammatory products, (3) improved water quality, (4) feed quality/optimization and (5) use of zinc/metals. The top 5 measures in terms of perceived ROI were (1) improved internal biosecurity, (2) zinc/metals, (3) diagnostics/action plan, (4) feed quality/optimization and (5) climate/environmental improvements. Univariate linear regression showed that veterinary practitioners rank internal biosecurity, vaccination, use of zinc/metals, feed quality optimization and climate/environmental on average highest, while researchers and professors focused more on increased use of diagnostics and action plans. Financial incentives/penalties ranked low in all countries. Belgian respondents ranked feed quality significantly lower compared to the German respondents while reduction of stocking density was ranked higher in Belgium compared to Denmark. Categorical Principal Component Analysis applied to the average ranking supported the finding that veterinary practitioners had a preference for more practical, common and already known alternatives. The results showed that improvements in biosecurity, increased use of vaccination, use of zinc/metals, feed quality improvement and regular diagnostic testing combined with a clear action plan were perceived to be the most promising alternatives to antimicrobials in industrial pig production based on combined effectiveness, feasibility and ROI.

The biosecurity status and its associations with production and management characteristics in farrow-to-finish pig herds

Disease prevention through biosecurity measures is believed to be an important factor for improvement of the overall health status in animal production. This study aimed at assessing the levels of implementation of biosecurity measures in pig production in four European Union (EU) countries and to describe possible associations between the biosecurity level and farm and production characteristics. A cross-sectional study was conducted in 232 farrow-to-finish pig herds in Belgium, France, Germany and Sweden between December 2012 and December 2013. The biosecurity status in each of these herds was described and quantified by using the risk-based scoring tool Biocheck.UGentTM (www.biocheck.ugent.be). Production and management characteristics, obtained from the herd management system and by interviewing the farmer, were analysed for their association with the biosecurity level. A causal path was designed to study statistical associations. The results showed that there was substantial room for improvement in the biosecurity status on many pig farms. Significant differences (P<0.01) both in internal and external biosecurity levels were observed between countries. The external biosecurity status, combining all measures taken to prevent disease introduction into the herd, was highest in Germany and lowest in France. The internal biosecurity status, combining all measures taken to prevent within herd disease transmission, was highest in Sweden and lowest, with a large variation, in Belgium. External biosecurity scores were in general higher compared to internal biosecurity scores. The number of pathogens vaccinated against was significantly associated with internal biosecurity status, suggesting an overall more preventive approach towards the risk of disease transmission. A higher external biosecurity was associated with more weaned piglets per sow per year. Furthermore also the weaning age and the mortality till weaning were highly associated with the number of weaned piglets per sow per year. The negative association observed between the biosecurity level and the estimated frequency of treatment against certain clinical signs of disease as a proxy for disease incidence is consistent with the hypothesis that a higher biosecurity level results in healthier animals. These findings promote an improved biosecurity status at pig farms and are of relevance in the discussion on alternative ways to keep animals healthy with a reduced necessity of antimicrobials; Prevention is better than cure!

Infectiousness of pigs infected by the Porcine Reproductive and Respiratory Syndrome virus (PRRSV) is time-dependent

The time-dependent transmission rate of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) and the correlation between infectiousness, virological parameters and antibody responses of the infected pigs were studied in experimental conditions. Seven successive transmission trials involving a total of 77 specific pathogen-free piglets were carried out from 7 to 63 days post-inoculation (dpi). A semi-quantitative real time RT-PCR was developed to assess the evolution of the viral genome load in blood and nasal swabs from inoculated and contact pigs, with time. Virus genome in blood was detectable in inoculated pigs from 7 to 77 dpi, whereas viral genome shedding was detectable from nasal swabs from 2 to 48 dpi. The infectiousness of inoculated pigs, assessed from the frequency of occurrence of infected pigs in susceptible groups in each contact trial, increased from 7 to 14 dpi and then decreased slowly until 42 dpi (3, 7, 2, 1 and 0 pigs infected at 7, 14, 21, 28 and 42 dpi, respectively). These data were used to model the time-dependent infectiousness by a lognormal-like function with a latency period of 1 day and led to an estimated basic reproduction ratio, R0 of 2.6 [1.8, 3.3]. The evolution of infectiousness was mainly correlated with the time-course of viral genome load in the blood whereas the decrease of infectiousness was strongly related to the increase in total antibodies.

Quantitative and qualitative antimicrobial usage patterns in farrow-to-finish pig herds in Belgium, France, Germany and Sweden

Data on sales of antimicrobials using a standardised methodology have shown that there are vast differences between countries in amounts of antimicrobials sold for food-producing animals, but these data do not provide insight on how sales are distributed by species and age groups. The aim of this study was to compare herd level antimicrobial usage for pigs by age category, antimicrobial class and administration route for farrow-to-finish herds in four EU countries. A cross-sectional study was conducted among 227 farrow-to-finish pig herds with at least 100 sows and 500 finishing pigs in Belgium (n=47), France (n=60), Germany (n=60) and Sweden (n=60). Detailed information about the antimicrobial consumption for breeding and growing pigs was collected. Antimicrobial usage was quantified as active substance expressed as mg and then converted to treatment incidence (TI) based on Defined Daily Doses Animal per 1000 pig-days at risk. TIs varied between and within countries, herds and age groups. The Swedish herds had the lowest and the German herds the highest overall use. Most treatments were applied to weaned piglets except in the Swedish herds where treatments of suckling piglets were most frequent. Antimicrobials were most often applied through feed or water except in the Swedish herds where parenteral treatments were most frequent. Aminopenicillins was the antimicrobial class most commonly used. Use of third and fourth generation cephalosporins constituted 11% of use for the Belgian herds, which was higher compared to the other countries. There was a significant (p<0.01) association between the within-herd antimicrobial use across different age categories. This study has shown that there were large differences in antimicrobial use for pigs between countries, herds and age groups in farrow-to-finish herds of similar size when actual consumption data were compared. Collecting detailed usage data can be used to efficiently target high users in order to reduce antimicrobial consumption.

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.

Guidance on the Selection of Appropriate Indicators for Quantification of Antimicrobial Usage in Humans and Animals

An increasing variety of indicators of antimicrobial usage has become available in human and veterinary medicine, with no consensus on the most appropriate indicators to be used. The objective of this review is therefore to provide guidance on the selection of indicators, intended for those aiming to quantify antimicrobial usage based on sales, deliveries or reimbursement data. Depending on the study objective, different requirements apply to antimicrobial usage quantification in terms of resolution, comprehensiveness, stability over time, ability to assess exposure and comparability. If the aim is to monitor antimicrobial usage trends, it is crucial to use a robust quantification system that allows stability over time in terms of required data and provided output; to compare usage between different species or countries, comparability must be ensured between the different populations. If data are used for benchmarking, the system comprehensiveness is particularly crucial, while data collected to study the association between usage and resistance should express the exposure level and duration as a measurement of the exerted selection pressure. Antimicrobial usage is generally described as the number of technical units consumed normalized by the population at risk of being treated in a defined period. The technical units vary from number of packages to number of individuals treated daily by adding different levels of complexity such as daily dose or weight at treatment. These technical units are then related to a description of the population at risk, based either on biomass or number of individuals. Conventions and assumptions are needed for all of these calculation steps. However, there is a clear lack of standardization, resulting in poor transparency and comparability. By combining study requirements with available approaches to quantify antimicrobial usage, we provide suggestions on the most appropriate indicators and data sources to be used for a given study objective.

Factors associated with carcass contamination by Campylobacter at slaughterhouse in cecal-carrier broilers

A study was conducted in 2009 to identify risk factors of Campylobacter spp. transmission from the digestive tract to the carcasses of standard broilers (slaughter age: 37 day, carcass weight: 1.3 kg on average). Counts of Campylobacter were performed on pools of 10 ceca and 10 neck-skins from 108 Campylobacter ceca-positive batches in three slaughterhouses. Technical and health data also was collected on the broilers: age, size, carcass weight (mean and standard deviation), condemnation rate, mortality rate and nature of treatment during the rearing period. Cecal counts varied from 4.8 to 10.2 log(10) cfu/g. In seventeen batches (15.7%), the skin count was below the detection limit. In the 91 batches with positive neck-skin test results, the counts varied from 2.0 to 5.2 log(10) cfu/g. Standard deviation of carcass weight, condemnation rate, slaughter rate and cecal count were significantly lower and growth rate higher in the 17 batches where neck-skin results were not detected positive. Multivariate analysis showed that batches with higher standard deviation of carcass weight were 5 to 9 fold more at risk of having detectable carcass contamination. Among the 91 positive neck-skin batches, only slaughter rate and cecal counts were found to have a significant but limited effect on the level of neck-skin contamination. As far as body weight homogeneity may be affected by disease, better health control can contribute to a reduction of the contamination of the broiler carcasses in Campylobacter carrier batches.