Use of a Mycoplasma Hyopneumoniae Nested Polymerase Chain Reaction Test to Determine the Optimal Sampling Sites in Swine

Abstract

A number of polymerase chain reaction (PCR)-based diagnostic tests have been developed for Mycoplasma hyopneumoniae, including one from this research group. This report presents further development, optimization, and standardization of a nested PCR test. Detection sensitivity was 1 fg of M. hyopneumoniae chromosomal DNA (approximately 1 organism). This exceeded the sensitivity of or compared favorably with other published PCR tests. Polymerase chain reaction primers to porcine 2-microglobulin were included as internal controls for amplifiable chromosomal DNA from porcine samples. To standardize the test, a number of samples from experimentally infected pigs, including nasal, tonsil, tracheobronchial swabs, lung tissue, bronchial alveolar lavage (BAL) fluid, and tracheobronchial brush samples, were examined by PCR. Samples obtained from BAL fluid and tracheobronchial sites were most predictive of infection, whereas nasal swabs and lung tissue were not reliable indicators of experimentally induced infection. In conclusion, the nested PCR developed for this study was found to be a highly sensitive and specific diagnostic tool for M. hyopneumoniae, but the enhanced sensitivity may be unnecessary if the proper sites are sampled. Porcine enzootic pneumonia caused by Mycoplasma hyopneumoniae continues to produce significant economic losses to swine producers. Mycoplasma hyop-neumoniae has recently gained new importance as an integral component of the porcine respiratory disease complex (PRDC). 24 Recent studies indicate that the incidence and severity of enzootic pneumonia have increased with the emergence of porcine reproductive and respiratory syndrome virus and the reemergence of swine influenza virus. Bacterial pathogens such as Pasteurella multocida also contribute to respiratory disease outbreaks. 17 Because of economic consideration , there is increased interest in the swine industry to improve diagnostic methods to gain a better understanding of PRDC in general and to provide farm-specific information for implementing effective prevention or control programs (or both) for M. hyopneu-moniae. Reliable diagnostic tests for M. hyopneumoniae are essential for developing cost-effective prevention and control programs. Visual analysis of lungs at the time of slaughter is commonly used to monitor the disease status of a herd. But it is not predictive of lifetime pneumonia. 21 The gold standard for detection of M. hyopneumoniae has been culture of the organism. But culture methods are rarely used because of several reasons: 1) prolonged time before a definitive diagnosis is obtained, 2) overgrowth with normal flora bacteria and non-M. hyopneumoniae mycoplasmas, and 3) lack of experience and capabilities to perform culture diagnosis of swine tissue samples because of the fastidious nature of M. hyopneumoniae. 18 Detection of serum antibodies is the most common diagnostic tool used to monitor M. hyopneumoniae infection. Both fluorescent antibody and enzyme-linked immunosor-bent assay (ELISA) are commonly used assays. 2,9,...

Array-Based Genomic Comparative Hybridization Analysis of Field Strains ofMycoplasma hyopneumoniae

“…This chromosomal region involved mhp024 and included both a deletion and sequence variation. The region was identified using a nested PCR test that failed to identify these two strains with the inner primer pair (11). It is significant that the present study confirmed the variation within mhp024 in one of these strains (00MP1502), and the data are just outside our q value cutoff for 00MP1301 (P Ͻ 0.0053, q Ͻ 0.234) since the regions containing this sequence variation are represented on the array.…”

Quantification of the spread of Mycoplasma hyopneumoniae in nursery pigs using transmission experiments

“…We used the positive results in the BAL fluid for the calculation of the R n because this is believed to be the most sensitive parameter to predict an infection with M. hyopneumoniae at individual level (Kurth et al, 2002). Nasal swabs were not used since results are less reliable.…”

Real-Time PCR Assays To Address Genetic Diversity among Strains ofMycoplasma hyopneumoniae

“…Also included were single isolates of several infrequently identified mycoplasmas and acholeplasmas of swine and of Mycoplasma bovis from cattle. Clinical samples were obtained from pigs experimentally inoculated with different isolates of M. hyopneumoniae as previously described (11,28) and from uninfected control pigs. Samples were obtained 28 days postinfection at necropsy (11).…”

“…In addition, seroconversion to M. hyopneumoniae is often slow within a herd and can vary considerably among pigs, making the use of enzyme-linked immunosorbent assays less effective. A number of PCR assays have been developed and reported to be both sensitive and specific for M. hyopneumoniae (4,6,11,19,20,22,24,26). As a result, this technique is now among the most widely used for detection of M. hyopneumoniae in pigs.…”