Assessment of a novel multiplex real-time PCR assay for the detection of the CBPP agent Mycoplasma mycoides subsp. mycoides SC through experimental infection in cattle

Background: Mycoplasma mycoides subsp. mycoides SC is the pathogenic agent of contagious bovine pleuropneumonia (CBPP), the most important disease of cattle in Africa causing significant economic losses. The re-emergence of CBPP in Europe in the 1980s and 1990s illustrates that it is still a threat also to countries that have successfully eradicated the disease in the past. Nowadays, probe-based real-time PCR techniques are among the most advanced tools for a reliable identification and a sensitive detection of many pathogens, but only few protocols have been published so far for CBPP diagnosis. Therefore we developed a novel TaqMan®-based real-time PCR assay comprising the amplification of two independent targets (MSC_0136 and MSC_1046) and an internal exogenous amplification control in a multiplex reaction and evaluated its diagnostic performance with clinical samples. Results: The assays detected 49 MmmSC strains from diverse temporal and geographical origin, but did not amplify DNA from 82 isolates of 20 non-target species confirming a specificity of 100%. The detection limit was determined to be 10 fg DNA per reaction for the MSC_0136 assay and 100 fg per reaction for the MSC_1046 assay corresponding to 8 and 80 genome equivalents, respectively. The diagnostic performance of the assay was evaluated with clinical samples from 19 experimentally infected cattle and from 20 cattle without CBPP and compared to those of cultivation and a conventional PCR protocol. The two rt-PCR tests proved to be the most sensitive methods and identified all 19 infected animals. The different sample types used were not equally suitable for MmmSC detection. While 94.7% of lung samples from the infected cohort were positively tested in the MSC_0136 assay, only 81% of pulmonal lymph nodes, 31% of mediastinal lymph nodes and 25% of pleural fluid samples gave a positive result. Conclusions: The developed multiplex rt-PCR assay is recommended as an efficient tool for rapid confirmation of a presumptive CBPP diagnosis in a well-equipped laboratory environment.