The establishment and application of a semi-nested PCR assay for the detection of Mycoplasma ovis

Ovine eperythrozoonosis is a zoonosis caused by Mycoplasma ovis (M. ovis), which parasitizes the surfaces of erythrocytes and plasma. However, the methods available for the diagnosis of eperythrozoonosis have revealed a conspicuous lack of specificity and low sensitivity with the result that certain infected carriers cannot be detected. The objective of this study was to establish a highly sensitive and specific method for the detection of M. ovis and to investigate the epidemiology of this pathogen in Hubei Province of China. According to the published 16S rRNA gene sequence of M. ovis in GenBank (EU165509.1), primers were designed, and a semi-nested PCR assay was developed. The sensitivity of the newly developed assay was as high as 10−10 fg. The specificity analysis revealed no cross reaction with Mycoplasma suis, Mycoplasma wenyonii, Mycoplasma ovipneumoniae, Theileria sergenti as well as Babesia orientalis. A total of 371 blood samples collected from seven different regions in Hubei Province of China were analyzed for the presence of M. ovis using both semi-nested PCR and universal PCR. The prevalence of M. ovis was determined to be 41% (151) and 26% (97) by the newly developed semi-nested PCR method and universal PCR, respectively. These results indicated that the semi-nested PCR was much more sensitive than the universal PCR. The occurrence of M. ovis in female and male goats was found to be 50% (84/167) and 54% (13/24) by semi-nested PCR. The statistical analysis indicated significant differences in the positive rates between the samples obtained from areas north and south of the Yangtse River (*P < 0.05), whereas no differences were observed between samples obtained from different genders (P > 0.05). The present study demonstrates that the semi-nested PCR assay is more suitable for the detection of M. ovis and the investigation of epidemiology.