Multiplex PCR for detection of Trypanosoma evansi and Theileria equi in equids of Punjab, India

- Multiplex PCR was standardized for the first time targeting haemoprotozoan in equines.
- Standardized PCR was applied on equine samples.
- Geographic trend of equine piroplasmosis in infection prone area.
- Association of disease preponderance with various related risk factors.

Multiplex PCR for simultaneous detection of Trypanosoma evansi and Theileria equi in single-step reaction was optimized and employed on 108 equids (99 horses and 9 donkeys/mules) blood samples collected from two agro-climatic zones (Sub-mountain undulating zone and Undulating plain zone) of Punjab to evaluate the status of concurrent infection and associated risk factors. The amplification products of 257 and 709 bp targeting repetitive nucleotide sequence of variable surface glycoproteins of T. evansi and 18S rRNA gene of T. equi, respectively expressed high fidelity of the primer pairs with sequence homology to neighboring geographic isolates. The overall prevalence of T. evansi and T. equi was 3.7 and 1.85%, with Undulating plain zone at higher infection risk for T. equi (OR = 3.24, 95% CI = 0.28-83.65); and Sub-mountain undulating zone (OR = ∞, 95% CI = 0.25-∞) for T. evansi. Multiplex PCR revealed higher risk of infection of both T. equi (OR = 6.75, 95% CI = 0.58-175.38) and T. evansi (OR = 2.11, 95% CI = 0.05-80.36) in the farms with inappropriate management system. The risk factor associated with the type of host species had an odds ratio of 12.35 (95% CI = 0.29-508.37) for donkeys/mules versus horses for T. evansi infection. This group was also at higher risk of infection with Odds ratio (OR) of 4 (95% CI = 0.14-53.99) for T. equi. The current investigation brings out various commodities at risk of infection pertaining to equid trypanosomosis and theileriosis evaluated by a rapid and sensitive multiplex PCR assay.