Determination of Anaplasma species variety in cattle from suburb of Isfahan city, Iran.

Anaplasmosis is an arthropod born disease of cattle and other ruminants caused by intracellular rickettsia of the genus Anaplasma. The disease caused considerable economical loss to cattle industries worldwide. The genus Anaplasma includes A. marginale, A. centrale, A. bovis and A. phagocytophilium, from which A. marginale belong to the most spread agent worldwide. The most common used method for the diagnosis of anaplasma infected cattle is the microscopic examination of Giemsa stained blood smears. But due to the low amount of parasitemia in carrier cattle, this method is not recommended for the characterization of persistently infected cattle. The golden standard method for the differential diagnosis of anaplasmosis is the method of polymerase chain reaction. In this study hundred and fifty blood samples and corresponding blood smears of cattle without any signs of diseases were prepared from a region in Isfahan / Iran with the previous history of tick-borne diseases. The blood smears were first analyzed by Giemsa staining and the extracted DNA from blood cells were amplified by Anaplasma-all primers, which amplify an approximately 781bp DNA fragment from region of 16S rRNA gene from various members of the genus Anaplasma. Anaplasma like structures could be identified in the limited amount of erythrocytes of 75 blood smears. In these samples, the percentage of erythrocytes harboring Anaplasma like structures varied from 10-3% to 10-2%. In 150 total blood samples, 58 samples were Anaplasma spp. positive by first PCR. All positive samples were further analysed for the presence of A. centrale (Amori strain), A. bovis and A. phagocytophilum by specific nested PCR. A. centrale (Amori strain), A. bovis and A.phagocytophilum were identified by specific nested PCR in 1.33%, 2.66% and 1.33% of blood samples, respectively. Because of highly sequence similarity among hyper variable region (V1) of 16S rRNA of A. marginale, A. centrale (South Africa strain) and A. ovis designing of species-specific primers was impossible. Therefore a new primer was designed for amplification of these specieses. The resulted semi-nested PCR products were analyzed by PCR-RFLP using restriction endonuclease Bst1107I and MvnI (FunD II). Restriction endonucleasis Bst1107I can cut only the semi-nested PCR product derived from A. marginale genome and can not cut the corresponding PCR product of A. centrale (south Africa strain) and A. ovis, whereas restriction endonucleasis MvnI can cut the PCR product of A. centrale (south Africa strain) and A. ovis but had no effect on PCR product of A. marginale. The results of PCR-RFLP showed that 38.76% of blood samples were A. marginale positive. A. centrale (south Africa strain) and A. ovis could not found in any samples. In this study, the molecular determination of A. centrale (Amori strain), A. bovis and A. phagocytophilum was for the first time performed in Iran using molecular techniques. Our results suggest that PCR-RFLP based on the chosen primers and restriction endonucleasis used in this work is an innovation method for differentiation anaplasma spp. and can be recommended for epidemiological studies dealing with the characterization of anaplasma in persistently infected cattle. Furthermore, our results revealed that microscopic examination is not a suitable method for diagnosis of carrier animals because of the very low amount of the Anaplasma infected erythrocytes, and the difficulty to differentiate between Anaplasma organism and other structures. Keywords: Iran, Isfahan, Anaplasma species, Cattle, A. marginale, A. centrale, A. bovis, A. phagocytophilum, PCR-RFLP, 16S rRNA.