Diagnosis and differentiation of Mycoplasma hyopneumoniae and Mycoplasma hyorhinis infections in pigs by PCR amplification of the p36 and p46 genes

The genome of Mycoplasma hyopneumoniae encodes several immunodominant prote ins, including a cytosolic protein (p36), three membranous proteins (p46, p 65, and p74), and an adhesin (p97), Cross-reactions with M. flocculare and Bt hyorhinis reduce the specificity of conventional serological detection m ethods. However, certain antigenic determinants of the p36 and p36 proteins have been shown to be specific for ill. hyopneumoniae. Ln the present stud y, pairs of oligonucleotide primers were designed to permit PCR amplificati on of entire p36 and p46 genes and of internal fragments of these genes. Sp ecific amplicons could be obtained with as low as 0.5 to 50 pg of extracted chromosomal DNA. No amplification product was obtained when testing p36 an d p46 primer pairs with genomic DNA or RNA from other mycoplasma species, b acteria, and viruses commonly associated,vith respiratory diseases in pigs, By using the single p36-PCR method, a positive reaction was demonstrated i n 100% (30 of 30) of lungs from pigs that developed typical lesions associa ted with an M. hyopneumoniae infection, and no false-positive results mere detected when 62 apparently normal lungs were tested. On the other hand, wi th the single p46-PCR method a sensitivity of 86.6% (26 of 30) and a specif icity of 96.7% (60 of 62) were obtained in comparison with the necropsy fin dings. A mixed infection with M. hyorhinis was diagnosed in 13.3% (4 of 30) of the cases by using species-specific primers for the heterologous p37 ge ne. The sensitivity of the single p36-PCR method for the detection of M; hy opneumoniae, when tested on tracheobronchial swabs, was 100% (20 positive s amples),with a specificity of 93.3% (14 of 15 negative samples), compared t o the necropsy findings. Both expected amplicons were obtained with 86.6% ( 26 of 30) positive lungs when p36 and p 16 primers were used simultaneously (multiplex PCR) to further increase the specificity of the PCR assay.