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.

Quantification of Pasteurella multocida in experimentally infected pigs using a real-time PCR assay.

The aim of the study was to quantify Pasteurella multocida in experimentally infected pigs using a new qPCR assay based on the sodA gene and validated with 35 P. multocida strains, including strains isolated from pigs with pneumonia, clinically healthy pigs (nasal cavities), and human infections. The specificity of the test was verified with a collection of 60 strains of bacterial species other than P. multocida. The estimated detection threshold was 10 genome equivalents per microliter. The amplification efficiency and value of the correlation coefficients were 95.5% (±3.5%) and 0.995 (±0.005), respectively. Analysis of P. multocida suspensions in Buffered Peptone Water Broth and of samples prepared from lungs experimentally spiked with P. multocida revealed detection thresholds of 1.4CFU/µl and 8.4CFU/µl, respectively. In live pigs, experimentally-infected, approximately 105, 107 and 108genomeequivalents/ml of P. multocida DNA was detected on Day 8 post-infection in the nasal cavities, tonsils and trachea samples, respectively. In dead pigs, approximatively 107genomeequivalents/ml of P. multocida DNA was detected in the lung tissue with pneumonia. The qPCR assay's diagnostic specificity and sensitivity were 100% and 96%, respectively. This new qPCR assay should be a very useful tool for controlling enzootic pneumonia and studying the dynamics of infections in pig herds.

Factors associated with herd-level PRRSV infection and age-time to seroconversion in farrow-to-finish herds.

Factors associated with porcine reproductive and respiratory syndrome virus (PRRSV) infection were investigated in 109 herds. Serums from four batches of pigs (4, 10, 16 and 22 weeks, 15 pigs/batch) were tested by ELISA for PRRSV antibodies. Infection by Mycoplasma hyopneumoniae (Mhp), Actinobacillus pleuropneumoniae, H1N1 and H1N2 swine influenza A viruses (swIAV) and PCV2 were detected by specific serological or PCR tests. Data related to herd characteristics, biosecurity, management housing and climatic conditions were collected during a herd visit. Factors associated with the herd's PRRSV seropositive status were identified by logistic regression. Large herd size, the lack of disinsectisation in the gestation facilities, on-farm semen collection, a short time-period for gilt quarantine and a low temperature setpoint for the ventilation controller in the fattening room significantly increased the odds of a herd being seropositive for PRRSV. Infection by Mhp and H1N2 swIAV were associated with a PRRSV seropositive status. A Cox proportional hazards model was used to identify the factors associated with the age-time to seroconversion in infected herds. Joint housing for the gilts and sows when lactating, a large nursery pen, a small number of pens per fattening room and lack of all-in all-out management in the fattening section significantly reduced the age-time to seroconversion. A small range of temperatures controlling ventilation rate in the nursery room was also associated with time to PRRSV seroconversion. Infection by Mhp and a high PCV2 infection pressure were associated with a shorter time to seroconversion. Biosecurity measures minimising the risk of introducing PRRSV into the herd, management practices reducing contacts between animals from different batches and within batches and favourable climatic conditions should be implemented to better control PRRSV infection.

Infectious agents associated with respiratory diseases in 125 farrow-to-finish pig herds: a cross-sectional study.

A study was carried out in 125 farrow-to-finish pig herds to assess the relationships between pathogens involved in respiratory disorders and to relate these findings to clinical signs of respiratory diseases and pneumonia and pleuritis at slaughter. Clinical examination and sampling were carried out on four different batches in each herd (pigs aged 4, 10, 16 and 22 weeks). Mycoplasma hyopneumoniae, Actinobacillus pleuropneumoniae, swine influenza viruses (SIV), porcine reproductive and respiratory syndrome virus (PRRSV) and porcine circovirus type 2 (PCV2) were detected by serological or PCR tests. Pneumonia-like gross lesions and pleuritis were scored at the slaughterhouse. The results indicate that the percentage of pigs PCR-positive for PCV2 at 4, 10 and 16 weeks old was associated with the percentage of pigs PCR-positive for M. hyopneumoniae at these ages. On the other hand, the percentage of pigs with antibodies against PRRSV at 10, 16 and 22 weeks was positively correlated with the percentage of pigs seropositive for M. hyopneumoniae at 22 weeks, with the percentage of pigs with antibodies against SIV H1N1 and SIV H1N2 and the percentage of pigs sero-positive for A. pleuropneumoniae serotype 2. The findings also indicate that, within the five studied pathogens, M. hyopneumoniae, PRRSV and SIV H1N1 are the major pathogens involved in pneumonia-like gross lesions even though PCV2 may play a role. A. pleuropneumoniae serotype 2, in association with PRRSV, is significantly associated with extensive pleuritis. Respiratory diseases could be significantly reduced by implementing measures including appropriate management practices to control these pathogens.