A nested PCR assay for the detection of Mycoplasma hyopneumoniae in tracheobronchiolar washings from pigs

A nested polymerase chain reaction (PCR) was developed for the detection of Mycoplasma hyopneumoniae, the etiological agent of enzootic pneumonia, in tracheobronchiolar washings from live pigs. Two nested pairs of oligonucleotide primers were designed from the sequence of a specific DNA probe (I 141; accession number U02537). The primer combination was Hp1/Hp3 for the first step PCR while the nested primers (Hp4/Hp6) allowed amplification of a 706 bp fragment. All strains of M. hyopneumoniae tested in this study could be detected by the nested PCR. DNA from other bacterial species isolated from the respiratory tract of pigs or from other mycoplasmal species were not amplified. The detection limit was estimated to be 1 fg, corresponding approximately to one organism, while in the one step PCR previously described 4 x 10(2) organisms were required. The nested PCR was evaluated on 362 tracheobronchiolar lavages collected from pigs at 2, 4 and 6 months of age in eight herds chronically infected with M. hyopneumoniae. The nested PCR was compared to a blocking ELISA performed with sera collected from the same pigs at the same ages, and to an immunofluorescence test at slaughter on 65 lungs from 6-month old pigs. The comparison indicated that the nested PCR was significantly (p<0.05) more sensitive (157 positive results of 362 samples) than ELISA (118 positive results of 362 samples) for detection of M. hyopneumoniae infection. Nested PCR was also significantly more sensitive (54 positive results of 65 samples) than immunofluorescence (29 positive results of 65 samples) for detection of M. hyopneumoniae in pig lungs at slaughter. Moreover, the nested PCR was used to confirm the absence of the mollicute in a pig herd without any history of M. hyopneumoniae infection. Thus, nested PCR appears to be a useful test to assess M. hyopneumoniae infection on pig farms.