Application of a nested polymerase chain reaction assay to detect Mycoplasma hyopneumoniae from nasal swabs

The porcine respiratory disease complex (PRDC) is an increasingly important cause of decreased swine productivity and is characterized by slow growth, decreased feed efficiency, anorexia, cough, and dyspnea. Mycoplasma hyopne umoniae is among the most prevalent and important infectious agents associa ted with PRDC. Understanding of mycoplasmal pneumonia has been hindered by inadequate diagnostic methods. Many of the currently available tests are re latively insensitive or nonspecific when used in a diagnostic laboratory se tting or are too costly or difficult for routine diagnostic use. Several po lymerase chain reaction (PCR) assays have been described, but they are not sensitive enough to detect the microorganisms in live pigs, from either nas al or tracheal swabs. A nested PCR using 2 species-specific sets of primers from the 16S ribosomal DNA gave positive results with as little as 80 micr oorganisms and did not cross-react with other mycoplasma species or with ot her microorganisms commonly found in the respiratory tract of pigs. This as say was better suited for detection of M. hyopneumoniae from nasal swabs th an was conventional PCR. Nasal swab samples were taken at different time pe riods following experimental challenge of 10 susceptible pigs. Only 2 of th e 55 swabs examined gave a positive result with conventional PCR, whereas 3 0 of the 55 swabs gave a positive result using the nested PCR. Twenty of 40 (50%) nasal swabs from pigs experiencing a respiratory disease outbreak wh ere M. hyopneumoniae had been diagnosed also gave a positive result with th e nested PCR. To confirm that the amplified product was specific, 4 nested PCR products were purified, sequences were determined and aligned, and they were confirmed to be from M. hyopneumoniae.