Development of two real-time PCR assays for the detection of Mycoplasma hyopneumoniae in clinical samples

In order to improve the diagnosis of enzootic pneumonia (EP) in pigs two real-time polymerase chain reaction (rtPCR) assays for the detection of Mycoplasma hyopneumoniae in bronchial swabs from lung necropsies were established and validated in parallel. As a gold standard, the current "mosaic diagnosis" was taken, including epidemiological tracing, clinical signs, macro- and histopathological lesions of the lungs and immunofluorescence. One rtPCR is targeting a repeated DNA element of the M. hyopneumoniae genome (REP assay), the other a putative ABC transporter gene (ABC assay). Both assays were shown to be specific for M. hyopneumoniae and did not cross react with other bacteria and mollicutes from pig. With material from pigs of defined EP-negative farms the two assays showed to be 100% specific. When testing lungs from pig farms with EP, the REP assay detected 50% and the ABC assay 90% of the farms as positive. Both tests together detected all positive farms. Within a positive herd the two assays tested similarly with on average over 90% of the lung samples analysed from a single farm showing positive scores. A series of samples with suspicion of EP and samples from pigs with diseases other than respiratory taken from current routine diagnostic was assayed. None of the assays showed false positive results. The sensitivities in this sample group were 50% for the REP and 70% for the ABC assays and for both assays together 85%. The two assays run in parallel are therefore a valuable tool for the improvement of the current diagnosis of EP.