Abstract
BackgroundMycoplasma 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.ResultsThe 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.ConclusionsThe 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.
Highlights
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
We developed a new multiplex rt-PCR assay using TaqMan®-labelled locked nucleic acid (LNA) probes for the detection of Mycoplasma mycoides subsp. mycoides SC (MmmSC) and validated it using samples from experimentally infected Boran cattle taken at necropsy
All non-MmmSC strains belonging to the M. mycoides cluster (46 strains) and 36 isolates of 16 other mollicute and bacterial species yielded a signal below a Ct value of 39 in any of the two amplification reactions
Summary
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. Macropathological examinations show gross infection or by re- introduction, rapid action such as setting up exclusion zones and culling of affected live stock is essential to efficiently prevent transmission and spread of disease. All these measures depend on an early identification of the pathogen. Mycoplasmas including MmmSC are isolated by culture and identified by biochemical and antigenic techniques [6,7,8] Drawbacks of these methods include low sensitivity caused by bacterial contamination, low specificity due to cross-reactivity of antigenic determinants of closely related species and time- and labour-intensive laboratory procedures. Real time PCR (rtPCR) formats using SYBR green detection of PCR products compensated some of the disadvantages connected to conventional PCR [11,12], but are less specific than real-time PCR assays that include specific probes [13,14]
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