Comparative Evaluation of Microscopy and PCR Assay for Detection of Theileria annulata Infection in Ruminants

Bovine tropical theileriosis, a tick borne haemoprotozoan disease, is one of the major threats to the health and production of cattle in the tropics and sub tropics. Diagnosis of the disease mainly relies upon clinical signs and microscopic examination of blood and lymphnode aspirate smears, which suffers with low diagnostic sensitivity, especially in carrier animals. Hence, the microscopy (gold standard test) and a highly sensitive polymerase chain reaction assay (PCR) were compared in terms of sensitivity, in the prsesnt study. For this, a total of 250 blood samples (cattle-215 and buffaloe-35) suspected for theileriosis were collected and initially screened by microscopic examination of Giemsa stained thin blood smear followed by Tams1 gene based PCR assay. A total of 55 (22.0%) and 95 (38.0%) samples were found positive for Theileria annulata infection by microscopy and PCR assay, respectively. In order to analyze the detection limit of the given PCR assay, the amplifed product was cloned in pTZ57R/T cloning vector with DH5α (E. coli) as host cells. The recombinant plasmid was isolated from the bacterial cells and concentration of the same was measured and then, a 10 fold serial dilution of the same was used as template in PCR assay. Present study concludes that Tams1 based PCR has very high level of sensitivity (with respect to microscopy) and detection limit of the test is 10-⁷ng/μl parasite DNA.