End Point Multiplex PCR for Diagnosis of Haemoprotozoan Diseases in Cattle

Background Theileriosis, trypanosomosis and babesiosis are the three major haemoprotozoan diseases causing huge economic losses worldwide. Difficulty in diagnosis of these diseases lies with the detection of carrier state with low parasitemia and concurrent infection. Purpose The present study was conducted to standardize and evaluate multiplex PCR assay for specific, fast and simultaneous detection of Theileria annulata , Trypanosoma evansi and Babesia bovis in bovines. Methods Positive parasitic DNA was obtained from microscopically positive samples. Simplex PCR assay was developed targeting repetitive nucleotide sequences for Trypanosoma evansi and gene coding enzyme carbamoyl phosphate synthetase II for Babesia bovis . For theileriosis conditions already standardized targeting cytochrome b gene was used. Gradient PCR assay was used to determine common amplification conditions and develop multiplex PCR assay. Limit of detection was determined using tenfold serial dilution of parasitic DNA. Blood samples collected from 117 bovines suspected for haemoparasite infection was tested by simplex and multiplex PCR assay. Results Simplex PCR assay was able to detect Theileria annulata , Trypanosoma evansi and Babesia bovis at dilution 10 −9 , 10 −8 and 10 −8 which corresponds to copy number 1, 10 and 10, respectively, whereas of multiplex PCR assay was found to be 10 −7 dilution corresponding to 100 copy number. PCR products bands obtained in multiplex PCR assay at 257, 312 and 446 bp were easily distinguishable. Results of simplex PCR assay for detection of individual parasites revealed 48 (41.02%), 27 (23.07%) and 5 (4.27%) samples positive for T. annulata , T. evansi and B. bovis , respectively. Sixty-three (53.8%) samples were found positive by multiplex PCR assay with 15 samples (23.8%) showing mixed infection. Conclusion Multiplex PCR assay was found to be highly specific and can be used for easy, early, sensitive, specific and simultaneous diagnosis of haemoprotozoan diseases in epidemiological survey as a robust tool.