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 oligonucleo tide primers were designed from the sequence of a specific DNA probe (I 141 ; accession number U02537), The primer combination was Hp1/Hp3 for the firs t step PCR while the nested primers (Hp4/Hp6) allowed amplification of a 70 6 bp fragment. All strains of nl. hyopneumoniae tested in this study could be detected by the nested PCR. DNA from other bacterial species isolated fr om the respiratory tract of pigs or from other mycoplasmal species were not amplified. The detection limit was estimated to be 1 fg, corresponding app roximately to one organism, while in the one step PCR previously described 4x10(2) organisms were required. The nested PCR was evaluated on 362 trache obronchiolar lavages collected from pigs at 2, 4 and 6 months of age in eig ht herds chronically infected with M. hyopneumoniae. The nested PCR was com pared 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 lung s from 6-month old pigs. The comparison indicated that the nested PCR was s ignificantly (p<0.05) more sensitive (157 positive results of 362 samples) than ELISA (118 positive results of 362 samples) for detection of M. hyopne umoniae infection. Nested PCR was also significantly more sensitive (54 pos itive results of 65 samples) than immunofluorescence (29 positive results o f 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 i n a pig herd without any history of M. hyopneumoniae infection. Thus, neste d PCR appears to be a useful test to assess M. hyopneumoniae infection on p ig farms. (C) 2000 Elsevier Science B.V. All rights reserved.