Discrimination Between Anaplasma marginale and Anaplasma ovis by PCR-RFLP

Several tick-borne diseases with different etiological agents such as rickettsia, protozoa, bacteria and viruses can infect domestic ruminants. The only common feature between these diseases is that they can all be transmitted by ticks. Of these, Anaplasma spp. can cause diseases in the livestock with high economical losses. The Giemsa staining of blood smear is the common traditional method for the identification and characterization of anaplasmosis in Iran. The most specific method for the differential diagnosis of anaplasmosis is the method of polymerase chain reaction. Three hundred blood samples and corresponding blood smears of 150 cattle and 150 sheep without any signs of diseases were prepared from a region in Isfahan / Iran with the previous history of anaplasmosis. The blood smears were first analyzed by Giemsa staining and DNA extraction was performed only on blood samples with presence of Anaplasma spp. in marginal point of erythrocytes in their blood smears. The extracted DNA from blood cells were analyzed by A. marginale A. ovis specific semi-nested PCR and PCR-RFLP using primers derived from 16S rRNA gene and restriction endonuclease Bst1107 I. The restriction endonuclease Bst1107I recognizes the sequence (GTATAC) in corresponding PCR product of A. marginale and cut it in the position 68, whereas the used restriction enzyme can not cut the corresponding PCR product of A. ovis (GTACGC).