E. Eveno

An ORF2 protein-based ELISA for porcine circovirus type 2 antibodies in post-weaning multisystemic wasting syndrome

Abstract Porcine circovirus type 2 (PCV2) plays a crucial role in the pathogenesis of post-weaning multisystemic wasting syndrome (PMWS) in swine. As PCV2 displays significant homology with PCV1 (a non-pathogenic virus) at the nucleotide and amino-acid level, a discriminative antigen is needed for specific serological diagnosis. The ORF2-encoded capsid protein from PCV2 was used to develop an indirect enzyme-linked immunosorbent assay (ELISA). GST-fused capsid protein from PCV2 and GST alone (both expressed in recombinant baculovirus-infected cells) were used as antigens for serodiagnosis. The specificity of the ELISA for detection of PCV2 antibodies was demonstrated in sera from pigs experimentally infected with PCV1, PCV2 and other swine viruses. The semi-quantitative nature of the test was evaluated versus an immunoperoxidase monolayer assay (IPMA). The ELISA was performed on 322 sera from pigs in eight Brittany herds and compared with IPMA. The sensitivity (98.2%) and specificity (94.5%) of this test were considered suitable for individual serological detection. High PCV2 seroprevalence was found in sows and pigs at the end of the growth phase (18–19 weeks) in all eight herds. The seroprevalence in piglets (11–17 weeks) was statistically correlated with clinical symptoms of PMWS (93% in affected versus 54%, in non-affected farms). A cohort study performed in PMWS-free farms showed that 57% of piglets exhibited active seroconversion after 13 weeks, indicating that PCV2 infection occurred earlier in PMWS-affected piglets.

Longitudinal serological responses to Salmonella enterica of growing pigs in a subclinically infected herd

A longitudinal survey was conducted in France in a subclinically Salmonella-infected farrow-to-finish pig farm to describe the time-course of the serological response to Salmonella enterica in growing pigs. We used three batches of sows and their corresponding litters (n = 31 litters). Among these, 256 pigs randomly selected and individually identified were followed from the first week of age until slaughter. Serial individual blood samples were submitted to indirect Salmonella-ELISA testing. Salmonella shedding was investigated by bacteriological testing of faecal material regularly collected from the sows and pigs and by environment swabs taken from the pens. Caecal contamination was checked at the slaughterhouse. Information about litter composition (filiation), location of the pigs in post-weaning and fattening pens, sanitary events, sex and body weights was recorded. 11.6% of the pigs shed S. enterica; 52% of pigs seroconverted before slaughter. The age-related variation of the natural logarithm of calibrated optical densities (COD) of pigs was described with two linear mixed models. From 8 to 61 days of age, the decrease in COD with age was fitted with a model including random effects of the animal and the dam on the intercept and slope, a batch random effect on the intercept and an individual birth-weight fixed effect on the intercept. The dam random effect was explained by the parity of the sow, Salmonella shedding by the sow during the farrowing phase and the value of the optical density of colostrum collected at parturition. A second model fitting the increase in COD from 61 days of age until slaughter included the random effect on intercept of the batch and the random effects on slope and intercept of the animal, the dam and the pen in which the followed animals were located during the fattening phase and the environmental contamination as fixed effect. In this second model, no relation was found between individual slaughter-bacteriological results and increasing COD values. Considering seroconversion time between 61 days of age and slaughter, survival function were constructed using the Cox proportional-hazards model. Both methods suggested that seroconversions generally occurred during the last third of the fattening phase (from 140 days of age to slaughter), while shedding was observed during the first half of the fattening period. The fitted models suggest the existence of clusters (such as pen and litter of origin).

Individual risk factors for Post-weaning Multisystemic Wasting Syndrome (PMWS) in pigs: A hierarchical Bayesian survival analysis

Risk factors for Post-weaning Multisystemic Wasting Syndrome (PMWS) at the pig level were identified using data from a longitudinal study in seven PMWS-affected farms in France. In each farm, a representative sample of 120 pigs (180 in one farm) was randomly selected after farrowing and followed from birth to slaughter. Individual information included serological status for Porcine Circovirus type 2 (PCV-2), Porcine Reproductive and Respiratory Syndrome (PRRS) virus, and Porcine Parvovirus (PPV), individual weight, rearing conditions, and clinical observations recorded at 7, 13, 16 and 21 weeks of age and at slaughter. Two different Bayesian frailty models were used to identify variables related to time-to-PMWS: (i) a logistic-survival model and (ii) an accelerated failure time model (different survival time distributions) both with two levels of clustering (litter and farm). Similar results in terms of variables related to time-to-PMWS were obtained with both models. However, information provided by the different approaches were complementary. Piglets were more likely to exhibit PMWS after early infection by PCV-2 (i.e. before 7 weeks old) and if they were weaned early (before 21 days). Piglets born to PCV-2 seronegative sows and/or to sows with neck injuries due to poorly performed injections were also more at risk. With the accelerated failure time model, time ratios were obtained giving an estimation of the expected survival time (increased or decreased) after exposure to the factor. The logistic-survival model showed that the majority of the risk factors were mostly related to the odds of PMWS whereas the PCV-2 passive immunity derived from the dam also tended to postpone PMWS appearance later.

Dynamics of influenza A virus infections in permanently infected pig farms: evidence of recurrent infections, circulation of several swine influenza viruses and reassortment events

Concomitant infections by different influenza A virus subtypes within pig farms increase the risk of new reassortant virus emergence. The aims of this study were to characterize the epidemiology of recurrent swine influenza virus infections and identify their main determinants. A follow-up study was carried out in 3 selected farms known to be affected by repeated influenza infections. Three batches of pigs were followed within each farm from birth to slaughter through a representative sample of 40 piglets per batch. Piglets were monitored individually on a monthly basis for serology and clinical parameters. When a flu outbreak occurred, daily virological and clinical investigations were carried out for two weeks. Influenza outbreaks, confirmed by influenza A virus detection, were reported at least once in each batch. These outbreaks occurred at a constant age within farms and were correlated with an increased frequency of sneezing and coughing fits. H1N1 and H1N2 viruses from European enzootic subtypes and reassortants between viruses from these lineages were consecutively and sometimes simultaneously identified depending on the batch, suggesting virus co-circulations at the farm, batch and sometimes individual levels. The estimated reproduction ratio R of influenza outbreaks ranged between 2.5 [1.9-2.9] and 6.9 [4.1-10.5] according to the age at infection-time and serological status of infected piglets. Duration of shedding was influenced by the age at infection time, the serological status of the dam and mingling practices. An impaired humoral response was identified in piglets infected at a time when they still presented maternally-derived antibodies.

Noninfectious factors associated with pneumonia and pleuritis in slaughtered pigs from 143 farrow-to-finish pig farms.

A cross-sectional study involving 143 farrow-to-finish herds was carried out to identify herd-level noninfectious factors associated with pneumonia and pleuritis in slaughter pigs. Data related to herd characteristics, biosecurity, management and housing conditions were collected by questionnaire during a farm visit. Climatic conditions were measured over 20 h in the post-weaning and finishing rooms where the slaughter pigs were kept. After these on-farm investigations, the finishing pigs were examined at slaughter for lung lesions. A sample of 30 randomly selected pigs per herd was scored for pneumonia and pleuritis. Herds were grouped into three categories according to their pneumonia median score (class 1: ≤ 0.5; class 2: 0.53.75). For pleuritis, a herd was deemed affected if at least one pig had a high pleuritis score (≥ 3). A multinomial logistic regression model was used to identify factors associated with pneumonia classes 2 and 3. A logistic regression for binary outcome was used to identify risk factors for severe pleuritis. An interval of less than four weeks between successive batches (OR=4.5, 95% confidence interval (95% CI): 1.5-13.6, p<0.01), large finishing room size (OR=4.3, 95% CI: 1.6-11.6, p<0.01) and high mean CO(2) concentration in the finishing room (OR=4.2, 95%CI: 1.6-11.3, p<0.01), significantly increased the odds for a herd to be in class 2 for pneumonia. The same risk factors were found for class 3 and, in addition, a direct fresh air inlet from outside or from the corridor in the post-weaning room vs an appropriate ceiling above the pigs (OR=5.1, 95% CI: 1.4-18.8, p=0.01). The risk for a herd to have at least one pig with a high pleuritis score was increased when the farrowing facilities were not disinsected (OR=2.7, 95% CI: 1.2-5.8, p=0.01), when tail docking was performed later than 1.5 days after birth (OR=2.6, 95% CI: 1.2-5.7, p=0.01) and if the piglets were castrated when more than 14 days old (OR=2.7, 95%CI: 1.1-6.8, p=0.03). A temperature range of less than 5°C for the ventilation control rate in the farrowing room (OR=2.7, 95% CI: 1.2-5.9, p=0.01), a mean temperature in the finishing room below 23°C (OR=3.0, 95% CI: 1.3-6.8, p<0.01) and large herd size (OR=3.1, 95% CI: 1.4-6.9, p<0.01) were also associated with increased risk of pleuritis. The factors affecting pneumonia and pleuritis seemed to be different. All rearing steps from farrowing to finishing must be taken into account in any health programme aimed at controlling pneumonia and pleuritis and lung health may be improved through several pathways, i.e. correcting managerial and hygienic factors, implementing an appropriate and well-functioning ventilation in order to offer favorable climatic conditions.

Risk factors associated with leg disorders of gestating sows in different group-housing systems: A cross-sectional study in 108 farrow-to-finish farms in France

Group-housing, rather than individual-housing systems, is mandatory for gestating sows in the European Union (2008/120/EEC). However, leg problems occur more frequently in group-housing than in individual-housing systems and are a welfare and health concern. A cross-sectional study involving 108 farms in western France was carried out to see whether the type of the 4 main group-housing systems (i.e. large groups with electronic feeder station in stable or in dynamic groups, small groups in walk-in lock-in stalls or partial feeding stalls), and other husbandry practices, were associated with leg disorders. In each farm, the sows were examined visually for claw lesions, scored for lameness and their breeding characteristics were recorded. Lameness was positively correlated with heel lesions and dewclaw lesions. A concrete slatted floor, as compared to straw, was a major risk factor (unadjusted relative risk (RR)=9.9; 95% confidence interval (95% CI): 4.4-34.5). Walk-in lock-in stalls were found to be the most protective system. A logistic regression model was used to identify those factors which significantly increased the risk of leg problems. These factors were: housing in large groups (RR=1.5; 95% CI: 1.1-2.4), dirty floors (RR=1.6; 95% CI: 1.0-2.9), high level of ammonia (RR=1.5; 95% CI: 1.1-2.1), severely restricted feeding particularly during the last stage of pregnancy (RR=1.5; 95% CI: 1.0-2.1) and a high number of sows per stockman (RR=1.5; 95% CI: 1.0-2.4).

Helminth control practices and infections in growing pigs in France

Abstract Internal parasite control practices and helminth infestations were investigated in 78 pig farms in France. Pooled faecal samples were taken from pens housing 16-week-old pigs. Samples were examined by coproscopy. Farm practices were checked for the risk factors of infestation previously described in literature. Information was obtained during a visit of the facilities and an interview with the farm owner/manager. Anthelmintics were used in most herds (97%). Treatments were routinely prescribed, such as flubendazole in the diet of the piglets and ivermectin for sows. Finisher pig infestation may occur despite these treatments: five samples in our study contained helminth eggs, four samples contained strongylid eggs ( Hyostrongylus rubidus or Oesophagostomum spp.), and one sample contained Trichuris suis eggs. We can conclude that helminths are controlled, but that parasites can still be present in indoor intensive pig operations. Hygiene efforts must be continued.

Different herd level factors associated with H1N1 or H1N2 influenza virus infections in fattening pigs.

Herd-level factors associated with European H1N1 or H1N2 swine influenza virus (SIV) infections were assessed by mean of a cross-sectional study carried out in 125 herds in France. Serum samples from 15 fattening pigs in each herd were tested by haemagglutination inhibition. Data related to herd characteristics, biosecurity, management and housing conditions were collected by questionnaire during the farm visit. Climatic conditions in the post-weaning and fattening rooms, where the sampled pigs were housed, were measured over 20 h. Factors associated with H1N1 or H1N2 sero-positive status of the herd were identified by logistic regressions for binary outcome. For both subtypes, the odds for a herd to be SIV sero-positive increased if there were more than two pig herds in the vicinity (OR=3.2, 95% confidence interval (95% CI): 1.4-7.6, p<0.01 and OR=3.5, 95% CI: 1.5-8.1 p<0.01 for H1N1 and H1N2 respectively). Different factors were specifically associated with either H1N1 or H1N2 SIV infections. The odds for a herd to be H1N1 sero-positive were significantly increased by having a large number of pigs per pen in the post-weaning room (OR=3.2, 95% CI: 1.2-8.6, p=0.02), temperature setpoints below 25 °C (OR=2.6, 95% CI: 1.1-6.4, p=0.03) and below 24 °C (OR=2.6, 95% CI: 1.1-6.1, p=0.03) for the heating device in the farrowing room and the ventilation controller, respectively, and moving the pigs to the fattening facility via a room housing older pigs (OR=3.3, 95% CI: 1.1-9.6, p=0.03). A H1N2 sero-positive status was associated with a brief down period in the farrowing room (OR=2.6, 95% CI: 1.1-6.3, p=0.03), small floor area per pig in the post-weaning pen (OR=2.9, 95% CI: 1.2-7.0, p=0.02), large-sized fattening room (OR=2.5, 95% CI: 1.1-5.9, p=0.03), lack of all-in all-out management in the fattening room (OR=2.4, 95% CI: 1.0-5.8, p=0.04) and a temperature range of less than 5 °C controlling ventilation in the fattening facilities (OR=3.2, 95% CI: 1.4-7.4, p<0.01). Factors related to external and internal biosecurity and to the control of inside climatic conditions should be considered together when implementing programmes to better control SIV infections.

Maternally-derived antibodies (MDAs) impair piglets’ humoral and cellular immune responses to vaccination against porcine reproductive and respiratory syndrome (PRRS)

The influence of maternally-derived antibodies (MDAs) on the post-vaccination humoral and cellular immune responses in piglets vaccinated against PRRS was studied. The piglets came from a vaccinated breeding herd. Thirty piglets with a low (A-) or high level (A+) of PRRSV-neutralizing MDAs were vaccinated (V+) with a modified live vaccine at 3 weeks of age. Blood samples were collected before vaccination and then at 2, 4, 8 and 14 weeks post-vaccination (WPV). The samples were analysed to detect the vaccine viraemia (RT-PCR) and quantify the post-vaccination humoral (ELISA and virus neutralisation test) and cellular (ELISPOT IFNγ) immune responses. PRRSV vaccine strain was detected in 60%, 64%, 36% and 0% of A-V+ piglets 2, 4, 8 and 14 WPV respectively. No virus was detected in A+V+ piglets during the first four WPV but 32% and 6% of A+V+ piglets were PCR-positive at 8 and 14 WPV. Eighty-five percent of A-V+ piglets and 0% of A+V+ piglets seroconverted (ELISA) between 2 and 4 WPV. Neutralising antibodies appeared 4 WPV in the A-V+ piglets and 14 WPV in the A+V+ piglets. The number of PRRSV-specific IFNγ-secreting cells was significantly higher in A-V+ piglets at 2 and 4 WPV than in A+V+ piglets. These results show that MDAs can affect both post-vaccination humoral and cellular immune responses in piglets. Further studies are required to assess the impact of MDAs on vaccine efficacy following a PRRSV challenge and its ability to reduce viral transmission.

Oral fluid versus blood sampling in group-housed sows and finishing pigs: Feasibility and performance of antibody detection for porcine reproductive and respiratory syndrome virus (PRRSV)

The feasibility of using individual and pen-based oral fluid samples to detect PRRSV antibodies in growing-finishing pigs and group-housed sows was investigated. The diagnostic performances of a commercial oral fluid ELISA (OF-ELISA) and a serum ELISA (SER-ELISA) performed on individual or pooled samples from 5 or 10 pigs and sows was evaluated. The performance of the OF-ELISA was also assessed for pen-based oral fluids. Eight hundred and thirty-four pigs and 1598 sows from 42 PRRSV-infected and 3 PRRSV-negative herds were oral fluid sampled and bled. PRRSV antibodies were detected by an OF-ELISA performed at individual, pool (5 or 10 samples) and pen levels. Serum samples were tested by a SER-ELISA at individual and pool levels. The sensitivity and specificity of ELISAs for individual samples were assessed by Bayesian analysis. The relative diagnostic performance for the pools was calculated by taking individual samples as the gold standard. SER-ELISA and individual OF-ELISA results were used as references for estimating OF-ELISA performance for pen-based samples. Individual oral fluid collection was feasible in all kinds of pigs, whereas pen-based samples were unsuccessful in 40% of the group-housed sow pens. High levels of sensitivity comparable to those of the SER-ELISA were found for the OF-ELISA when performed on individual, 5-sample pool or pen-based samples from pigs or sows. The OF-ELISA lacked specificity for individual samples from sows. Pooling 5 individual oral fluid samples or using pen-based samples increased test specificity.