Abstract
Mycoplasma ovipneumoniae infects both sheep and goats causing pneumonia resulting in considerable economic losses worldwide. Current diagnosis methods such as bacteriological culture, serology, and PCR are time consuming and require sophisticated laboratory setups. Here we report the development of two rapid, specific and sensitive assays; an isothermal DNA amplification using recombinase polymerase amplification (RPA) and a real-time PCR for the detection of M. ovipneumoniae. The target for both assays is a specific region of gene WP_069098309.1, which encodes a hypothetical protein and is conserved in the genome sequences of ten publicly available M. ovipneumoniae strains. The RPA assay performed well at 39°C for 20 min and was combined with a lateral flow dipstick (RPA-LFD) for easy visualization of the amplicons. The detection limit of the RPA-LFD assay was nine genome copies of M. ovipneumoniae per reaction and was comparable to sensitivity of the real-time PCR assay. Both assays showed no cross-reaction with 38 other ovine and caprine pathogenic microorganisms and two parasites of ruminants, demonstrating a high degree of specificity. The assays were validated using bronchoalveolar lavage fluid and nasal swab samples collected from sheep. The positive rate of RPA-LFD (97.4%) was higher than the real-time PCR (95.8%) with DNA as a template purified from the clinical samples. The RPA assay was significantly better at detecting M. ovipneumoniae in clinical samples compared to the real-time PCR when DNA extraction was omitted (50% and 34.4% positive rate for RPA-LFD and real-time PCR respectively). The RPA-LFD developed here allows easy and rapid detection of M. ovipneumoniae infection without DNA extraction, suggesting its potential as a point-of-care test for field settings.
Highlights
Bronchopneumonia is a multifactorial disease that involves interactions between different bacterial and viral pathogens as well as predisposing factors such as immunocompromised hosts, environmental factors and stress [1,2,3]
We developed new highly sensitive and specific recombinase polymerase amplification (RPA)-LFD and real-time PCR assays for diagnosis of M. ovipneumoniae with a detection limit of 10 fg (9 genome equivalent)
A realtime PCR based method has been described for M. ovipneumoniae, which amplifies p113 gene with a detection limit of 220 genome copies [14]
Summary
Bronchopneumonia is a multifactorial disease that involves interactions between different bacterial and viral pathogens as well as predisposing factors such as immunocompromised hosts, environmental factors and stress [1,2,3] Because of this complexity and multifactorial nature, sheep pneumonia is commonly known as ovine respiratory disease complex and includes Mannheimia haemolytica, Mycoplasma ovipneumoniae and Parainfluenza virus type 3 [4, 5]. Mycoplasmas primarily infect animals that are under stress due to environmental factors such as cold, heat or dense housing This results in subclinical interstitial bronchopneumonia that often predisposes the lower respiratory tract to other secondary infections with pathogens such as M. haemolytica and Parainfluenza virus type 3 leading to chronic pneumonia [12]. These technical challenges have hindered the use of these methods as field diagnostic tools
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