A new multiplex real-time TaqMan® PCR for quantification of Mycoplasma hyopneumoniae, M. hyorhinis and M. flocculare: exploratory epidemiological investigations to research mycoplasmal association in enzootic pneumonia-like lesions in slaughtered pigs.

AIMS: A new multiplex qPCR, targeting Mycoplasma (M.) hyopneumoniae, M. hyorhinis and M. flocculare, was developed and the relationship between detection of those mycoplasma species and the extent of gross pneumonia-like lesions in slaughtered pigs lungs were investigated. METHODS AND RESULTS: The multiplex qPCR method targets the p102, p37 and fruA genes and has detection limits of 14, 146 and 16 genome equivalents µl-1 for M. hyopneumoniae, M. hyorhinis and M. flocculare, respectively. In all, 671 lungs were collected and analysed, among them 666 were scored for macroscopic pneumonia and categorized according to the extent of the lesions (no or minor lesions, moderate lesions and extensive lesions). According to results of multiplex qPCR, 59·5% were positive for M. hyopneumoniae, 3·4% for M. hyorhinis and 34·7% for M. flocculare, with on average, 3·1 × 107 , 9·7 × 106 and 5·7 × 106 genome equivalents of mycoplasma ml-1 , respectively. More results showed that no or minor lesions were associated with multiplex qPCR-negative results or qPCR-positive results for M. flocculare. Moderate to extensive lesions were positively correlated with qPCR-positive results for M. hyopneumoniae. Extensive lesions were associated with qPCR-positive results for at least two mycoplasma species (M. hyopneumoniae and M. hyorhinis). CONCLUSION: The findings also indicated that M. hyopneumoniae and M. hyorhinis significantly increased the odds for a lung to have macroscopic pneumonia. No relationship was found between the extent of lesions and the mycoplasma genome load. SIGNIFICANCE AND IMPACT OF THE STUDY: This new multiplex qPCR appears to be specific, sufficiently sensitive and repeatable. The validation of this method with field samples guarantees its use for field epidemiological investigations, particularly to gain more insight into the aetiology of the porcine respiratory disease complex.

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.

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.

Impact of two different colistin dosing strategies on healthy piglet fecal microbiota.

Colistin is often used in piglets but underdosing and overdosing are frequent. The impact of such administrations on fecal microbiota was studied. Piglets were given either underdoses of colistin by oral gavage for five days or overdoses by in-feed medication for 14days. The composition of fecal microbiota was studied by quantitative PCR, 16S rRNA sequencing, culture of Enterobacteriaceae, and quantification of short-chain fatty acids (SCFAs). The mean colistin concentrations during the treatment for underdosed and overdosed groups were 14.4µg/g and 64.9µg/g of feces respectively. Whatever the piglet and the sampling day, the two main phyla were Firmicutes and Bacteroidetes, The main families were Lactobacillaceae, Clostridiales, Lachnospiraceae and Ruminococcaceae. The main perturbation was the significant but transitory decrease in the Escherichia coli population during treatment, yet all the E. coli isolates were susceptible to colistin. Moreover, colistin did not affect the production of SCFAs. These results show that under- or overdoses of colistin do not result in any major disturbance of piglet fecal microbiota and rarely select for chromosomal resistance in the dominant E. coli population.

Impact of ceftiofur injection on gut microbiota and Escherichia coli resistance in pigs.

Resistance to extended-spectrum cephalosporins (ESCs) is an important health concern. Here, we studied the impact of the administration of a long-acting form of ceftiofur on the pig gut microbiota and ESC resistance in Escherichia coli. Pigs were orally inoculated with an ESC-resistant E. coli M63 strain harboring a conjugative plasmid carrying a gene conferring resistance, bla CTX-M-1. On the same day, they were given or not a unique injection of ceftiofur. Fecal microbiota were studied using quantitative PCR analysis of the main bacterial groups and quantification of short-chain fatty acids. E. coli and ESC-resistant E. coli were determined by culture methods, and the ESC-resistant E. coli isolates were characterized. The copies of the bla CTX-M-1 gene were quantified. After ceftiofur injection, the main change in gut microbiota was the significant but transitory decrease in the E. coli population. Acetate and butyrate levels were significantly lower in the treated group. In all inoculated groups, E. coli M63 persisted in most pigs, and the bla CTX-M-1 gene was transferred to other E. coli. Culture and PCR results showed that the ceftiofur-treated group shed significantly more resistant strains 1 and 3 days after ESC injection. Thereafter, on most dates, there were no differences between the groups, but notably, one pig in the nontreated group regularly excreted very high numbers of ESC-resistant E. coli, probably leading to a higher contamination level in its pen. In conclusion, the use of ESCs, and also the presence of high-shedding animals, are important features in the spread of ESC resistance.

Risk factors associated with the presence of hepatitis E virus in livers and seroprevalence in slaughter-age pigs: a retrospective study of 90 swine farms in France.

The frequency of sporadic cases of hepatitis E in humans in developed countries has increased in recent years. The consumption of raw or undercooked pig liver-based products has been identified as an important source of human infection. The question of possible massive human exposure to this zoonotic agent has been raised by the high prevalence of hepatitis E virus (HEV) in swine herds. However, little is known about the epidemiology of HEV on pig farms. A retrospective study, based on a previous prevalence study of 185 farms, was conducted on 90 farms located in Western France, randomly selected from this database, to identify factors associated with the presence of HEV in pig livers and HEV seroprevalence in slaughter-age pigs. At least one HEV RNA-positive liver was found in 30% of the sampled farms while seroprevalence in slaughter-age pigs at the farm level reached almost 75%. Different factors were associated with the two conditions. The risk of having HEV-positive livers was increased by early slaughter, genetic background, lack of hygiene measures and surface origin of drinking water. High HEV seroprevalence was associated with mingling practices at the nursery stage and hygiene conditions. These results can be used to determine on-farm measures to reduce within-farm HEV spread and infection of slaughter-age pigs.

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.

Bacterial pathogens associated with lung lesions in slaughter pigs from 125 herds.

Relationships between macroscopic lesions and Polymerase Chain Reaction (PCR) detection of Mycoplasma hyopneumoniae (Mhp), Pasteurella multocida (Pm), Actinobacillus pleuropneumoniae (App), Haemophilus parasuis (Hps) and Streptococcus suis (Ssuis) of the lungs of 3731 slaughter pigs from 125 herds were assessed in France. Pneumonia and pleuritis were the most frequent lesions (69.3% and 15% of the lungs, respectively). Mhp, Pm, App, Ssuis and Hps were detected in 69.3%, 36.9%, 20.7%, 6.4% and 0.99% of the lungs, respectively. Mhp and Pm were associated with pneumonia at both the pig and herd levels. Pleuritis was not associated with any pathogen at the pig level, but was associated with a high percentage of pigs PCR-positive for App at the herd level. Measures focused on control of Mhp, Pm and App should significantly reduce the occurrence of both pneumonia and pleuritis.

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.

Longitudinal study of respiratory infection patterns of breeding sows in five farrow-to-finish herds.

A longitudinal study was carried out in five French farrow-to-finish herds differently affected by respiratory diseases to describe the carrying and infection patterns of batches of sows to various respiratory pathogens during gestation and lactation. An entire batch of sows was followed during two successive reproduction cycles. Nasal, tonsillar and oro-pharyngeal swabs and blood samples were taken from each sow 9 and 4 weeks before farrowing and 1 and 4 weeks after farrowing. Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, Pasteurella multocida, Haemophilus parasuis and Streptococcus suis were detected from swab samples using PCR assays. Blood samples were tested for antibodies against M. hyopneumoniae, A. pleuropneumoniae serotypes 1-9-11 and 2, Porcine Circovirus type-2 (PCV-2) and Porcine Reproductive and Respiratory Syndrome virus (PRRSV) by ELISA tests. Antibodies against H(1)N(1), H(1)N(2) and H(3)N(2) Swine Influenza Viruses (SIV) of European lineages were tested by hemagglutination inhibition assay. The results indicated that S. suis is widespread among sows (67.1% of PCR-positive sows). A. pleuropneumoniae, P. multocida, and H. parasuis were detected by PCR in 30.9%, 24.6% and 23.4% of the sows, respectively. Antibodies against M. hyopneumoniae were recovered from more than 55% of the sows in all herds whereas the micro-organism was detected in 2.4% of the sows. Although PCV-2 and SIV infections were highly prevalent, the PRRSV infection patterns ranged from no infection in farms mildly affected by respiratory diseases to active circulation in more severely affected herds. The sow population thus constitutes a reservoir for a continuous circulation of respiratory pathogens and needs to be properly considered in control strategies.

Evaluation of common cleaning and disinfection programmes in battery cage and on-floor layer houses in France.

1. The aim in this study was to evaluate cleaning and disinfection programmes in battery cage and on-floor layer houses in France. 2. Cleaning and disinfection efficiency was assessed by a visual evaluation of cleaning and a bacteriological monitoring of surface contamination from counts of thermotolerant streptococci on contact agar plates. 3. In battery cage houses, dropping belts, manure conveyors, and house floors remained highly contaminated due to poor cleaning in half of the buildings examined. 4. In on-floor houses, a high standard of cleaning was achieved but errors in the planning of cleaning and disinfection operations sometimes led to a high residual contamination of nest boxes and egg sorting tables.