Pathogen exposure influences immune parameters around weaning in pigs reared in commercial farms.

BACKGROUND: Multiple antigenic stimulations are crucial to immune system training during early post-natal life. These stimulations can be either due to commensals, which accounts for the acquisition and maintenance of tolerance, or to pathogens, which triggers immunity. In pig, only few works previously explored the influence of natural exposition to pathogens upon immune competence. We propose herein the results of a multicentric, field study, conducted on 265 piglets exposed to contrasted pathogen levels in their living environment. Piglets were housed in 15 different commercial farms, sorted in two groups, low (HSLOW)- and high (HSHIGH)-health status farms, depending on their recurrent exposition to five common swine pathogens. RESULTS: Using animal-based measures, we compared the immune competence and growth performances of HSLOW and HSHIGH pigs around weaning. As expected, we observed a rise in the number of circulating leucocytes with age, which affected different cell populations. Monocyte, antigen-experienced and cytotoxic lymphocyte subpopulation counts were higher in piglets reared in HSLOW farms as compared to their HSHIGH homologs. Also, the age-dependent evolution in γδ T cell and neutrophil counts was significantly affected by the health status. With age, circulating IFNα level decreased and IgM level increased while being greater in HSLOW piglets at any time. After weaning, LPS-stimulated blood cells derived from HSLOW piglets were more prone to secrete IL-8 than those derived from HSHIGH pigs did. Monocytes and granulocytes issued from HSLOW pigs also exhibited comparable phagocytosis capacity. Altogether our data emphasize the more robust immunophenotype of HSLOW piglets. Finally, piglets raised under higher pathogen pressure grew less than HSHIGH piglets did and exhibited a different metabolic profile. The higher cost of the immune responses associated with the low farm health status may account for lower HSLOW piglet performances. CONCLUSIONS: Altogether, our data, obtained in field conditions, provide evidence that early exposure to pathogens shapes the immune competence of piglets. They also document the negative impact of an overstimulation of the immune system on piglets' growth.

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.

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.

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.

Towards a Better Use of Antimicrobials on Farms: Insights from a Participatory Approach in the French Pig and Poultry Sectors.

Despite the strong decrease in antimicrobial use in the French poultry and pig sectors over the last decade, room for improvement remains. A participatory approach was set up in France, involving representatives of veterinarians, the pig and poultry industries, technical institutes, the French Ministry of Agriculture, and researchers, to further improve how antimicrobials are used on farms. By successively defining a shared, long-term vision of future antimicrobial use on farms, identifying lock-in mechanisms impeding this future vision from being realized, and articulating practical questions on how to move in the desired direction, the group rapidly reached a consensus. The results highlight the need for consensual standardized monitoring tools that would allow farmers and veterinarians to jointly monitor the health, welfare, antimicrobial resistance, and antimicrobial use on farms. Other results relate to better communication and training for citizens regarding animal health, animal welfare, and proper antimicrobial use; some benefits but also counterproductive effects of antibiotic-free labels that imperil animal health and welfare; the economic competitiveness of farms on international markets; and the economic sustainability of farm animal veterinary practices. These results call for a concerted way to produce tools for farmers and veterinarians and the broader involvement of other food sector actors.

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

BACKGROUND: Campylobacter 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. RESULTS: With 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. CONCLUSION: The 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.

Feasibility of pooled oral fluid collection from pre-weaning piglets using cotton ropes.

Collection of pooled oral fluid (OF) by allowing pigs to chew on a cotton rope is an alternative to blood sampling. However, little is known about the applicability of this method to suckling piglets. The objectives of the present study were to describe the spontaneous interaction of suckling piglets with a rope and to investigate the influence of a rope pre-exposure on the success rate of sampling. We studied the interaction dynamics of 21 and 28 days-old suckling piglets with a cotton rope presented for 30 min. Ropes were manually wrung out inside plastic bags to release the oral fluid. A total of 49 litters were included. Percentages of success of pooled OF collection for 28-day-old, 21-day-old and 21-day-old pre-exposed litters were 82%, 62% and 100%, respectively. The mean volume collected did not differ between groups. Without pre-exposure, 84.7% and 95% of piglets interacted spontaneously with the rope at 21 and 28 days of age, respectively. The latency between rope presentation and interaction was highly variable between piglets within litters: from < 10 s to 30 min. Among piglets having interacted with the rope, the interaction lasted for at least 60 s for 90% and 91.4% of 21 and 28-day-old piglets, respectively. Pooled OF collection is achievable prior to weaning in piglets of at least 21 days of age. Pooled OF sampling is representative at litter level if collection is successful. In order to improve the success rate of collection, pre-exposing the piglets with a rope one day prior to sampling is effective.

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.

Effects of age and weaning conditions on blood indicators of oxidative status in pigs.

Weaning is a source of social, nutritional and environmental disorders that challenge piglet health. This study assesses the relevance of using plasma indicators of oxidative status as biomarkers of health around weaning in pigs. Blood antioxidant potential (BAP), hydroperoxides (HPO), oxidative stress index (OSI, e.g. HPO/BAP), vitamin A and E concentrations were investigated in two different trials. Trial A was carried out in an experimental unit to investigate the effects of age (from 12 to 147 days of age), weaning (at 21 or 28 days of age) and management at weaning (in optimal (OC) or deteriorated (DC) conditions) on those parameters. Trial B was performed in 16 commercial pig farms to describe the variability of these indicators on field between 26 and 75 days of age. In trial A, between 12 and 147 days of age, HPO globally increased (P < 0.001), vitamin E concentration decreased (P < 0.001) whereas BAP and vitamin A concentration remained relatively stable (P > 0.1). Vitamins E and A concentrations dropped 5 days after weaning independently of weaning age, weaning conditions and expression of diarrhea (P < 0.001). Twelve days after weaning, whatever the weaning age, HPO and OSI increased in DC compared to OC piglets (P = 0.05 and P < 0.01) and in piglets exhibiting diarrhea compared to those without diarrhea (P < 0.01 and P < 0.001). In DC pigs, BAP was also decreased (P < 0.05) 12 days after weaning. On trial B, plasma concentrations of vitamins A and E decreased and HPO increased 5 and 19 days respectively after weaning (P < 0,001). Contrarily to trial A, BAP values did not drop after weaning. Piglets which had the lowest ADG (Average Daily Gain) after weaning had greater HPO and OSI and lower vitamin A and E concentrations after weaning but also lower vitamin E concentration before weaning (P < 0.05). In conclusion, HPO or OSI seem to be good indicators of health disorders around weaning and plasma concentration of vitamin E before weaning is associated to growth after weaning.

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

Salmonella 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.

Metagenomic detection of viruses in aerosol samples from workers in animal slaughterhouses.

Published studies have shown that workers in animal slaughterhouses are at a higher risk of lung cancers as compared to the general population. No specific causal agents have been identified, and exposures to several chemicals have been examined and found to be unrelated. Evidence suggests a biological aetiology as the risk is highest for workers who are exposed to live animals or to biological material containing animal faeces, urine or blood. To investigate possible biological exposures in animal slaughterhouses, we used a metagenomic approach to characterise the profile of organisms present within an aerosol sample. An assessment of aerosol exposures for individual workers was achieved by the collection of personal samples that represent the inhalable fraction of dust/bioaerosol in workplace air in both cattle and sheep slaughterhouses. Two sets of nine personal aerosol samples were pooled for the cattle processing and sheep processing areas respectively, with a total of 332,677,346 sequence reads and 250,144,492 sequence reads of 85 bp in length produced for each. Eukaryotic genome sequence was found in both sampling locations, and bovine, ovine and human sequences were common. Sequences from WU polyomavirus and human papillomavirus 120 were detected in the metagenomic dataset from the cattle processing area, and these sequences were confirmed as being present in the original personal aerosol samples. This study presents the first metagenomic description of personal aerosol exposure and this methodology could be applied to a variety of environments. Also, the detection of two candidate viruses warrants further investigation in the setting of occupational exposures in animal slaughterhouses.

Genetic instability of Campylobacter coli in the digestive tract of experimentally infected pigs.

Campylobacter, a leading cause of food-borne illness worldwide, has a widespread distribution with a broad range of animal hosts and environmental reservoirs. The genetic description of bacterial strains is a powerful tool for epidemiological studies but can be impaired by the high genomic variability of Campylobacter. Our study aimed (i) at investigating the genotypic instability of Campylobacter generated either in vitro by subculturing or after in vivo passage on specific pathogen-free pigs and (ii) at evaluating the suitability of typing methods to detect such variation. Pigs were inoculated per os with three Campylobacter strains (one C. coli originating from pig faeces, one C. jejuni and one C. coli originating from poultry faeces) alone or in mixture and non-inoculated pigs were housed in adjacent pens. Genotypic instability was investigated using both macrorestriction combined with pulsed-field gel electrophoresis analysis (PFGE) and PCR restriction fragment length polymorphism analysis of the flaA gene (flaA PCR-RFLP). No variability in the genetic profile was observed for the three strains maintained through twenty times subculturing events in vitro. Genotypic variability was evidenced in vivo only in pigs inoculated with C. coli of porcine origin, either alone or in a mix, with both genotyping methods. In our study, for one porcine C. coli strain, 13% and 21% of variability were generated in the digestive tract of pigs by PFGE and flaA PCR-RFLP typing methods, respectively. This study is a first approach for a better understanding of the genomic instability of Campylobacter in pig under field conditions.

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.