Pentaplex PCR assay for rapid differential detection of Babesia bigemina, Theileria annulata, Anaplasma marginale and Trypanosoma evansi in cattle.

A multiplex PCR (mPCR) assay for simultaneous detection and differentiation of four major haemoparasites in crossbred cattle was established using parasite specific genomic DNA and four sets of primer pairs targeting AMA-1, Tams1, MSP5 and VSG genes of Babesia bigemina, Theileria annulata, Anaplasma marginale and Trypanosoma evansi generating precise amplicons of 448, 156, 382 and 110 bp, respectively. An internal amplification control, 202 bp bovine β-casein gene fragment, was simultaneously amplified with four target genes to avoid false-negative results. The sensitivity of mPCR was 3.44 × 10, 5.9 × 10, 2.88 × 10 and 3.3 × 10 copies for B. bigemina, T. annulata, A. marginale and T. evansi, respectively. mPCR of cattle clinical samples (n = 516), suspected for haemoparasites, revealed single haemoparasitic infection in 279 (54.06%) cases, whereas mixed infection was recorded in 54 (10.46%) samples. In clinical samples, coinfection with T. annulata and A. marginale was the most common. The findings of mPCR were consistent with uniplex PCR under field conditions except for subtle variations in A. marginale infection. Overall, the mPCR assay represents an economical, reproducible and robust diagnostic tool for concurrent detection of cattle haemoparasites and large scale epidemiological studies.