Detection of Anaplasma ovis based on 16S rRNA gene by PCR-RFLP in sheep from central part of Iran

One of the most specific methods for the differential diagnosis of Anaplasma spp. is the method of polymerase chain reaction. PCR-based methods on the 16S rRNA gene can differentiate the species in genus Anaplasma but the sequence of 16S rRNA in A. ovis is most similar to that of A. marginale (99.2%-99.9%) and designing of species-specific primers is impossible. Sheep are affected by both A. ovis and A. marginale, so the aim of the present study was to develop and apply species-specific PCR-RFLP test based on 16S rRNA gene in sheep and goats. Blood samples and corresponding blood smears of 150 sheep without any signs of diseases were prepared from a region with the previous history of anaplasmosis in Isfahan, Iran. The blood smears were first analyzed by Giemsa staining and DNA extraction was performed only on positive blood samples with Anaplasma spp. in marginal point of erythrocytes in their blood smears. The extracted DNA from blood cells were analyzed by A. ovis and A. marginale specific semi-nested PCR and PCR-RFLP using primers derived from 16S rRNA gene and restriction endonuclease MvnI (FunD II). The restriction endonuclease MvnI (FunD II) recognizes the sequence (CGCG) in corresponding PCR product of A. ovis and cut it in the position 70, whereas the used restriction enzyme leave un-cut the corresponding PCR product of A. marginale (TACG). This simple PCR method based on 16S rRNA gene flowing by RFLP give a rapid discrimination between A. ovis and A. marginale, which is useful for epidemiological survey and development of control strategies.