Bovine tropical theileriosis, caused by Theileria annulata, is one of the economically important fatal tick borne haemoprotozoan diseases of dairy animals. The aim of present investigation was to map the distribution of T. annulata in bovines of Punjab state of India in relation to various risk factors including age, sex of animals, location and management of farms. In a cross sectional study, a total of 1278 blood samples were randomly collected from twenty districts falling in five major agro-climatic zones of Punjab. All the samples were screened by blood smear examination followed by polymerase chain reaction targeting SSU rRNA gene for Theileria spp. PCR positive samples (n = 386) for Theileria spp. were then analyzed for T. annulata by amplification of Tams1 gene. Overall prevalence of T. annulata was found to be 29.26% in Punjab, with highest in western Zone (40.49%, 95% CI = 35.57-45.41) and lowest in submountain zone (18.90%, 95% CI = 13.73-24.06). The propensity of incidence of T. annulata was found to be highest in cross bred cattle (32.40%, 95% CI = 29.87-34.94), followed by indigenous cattle (19.64%, 95% CI = 10.67-28.61) and buffaloes (19.2%, 95% CI = 14.99-23.41). Between the two sexes, incidence of T. annulata was higher in female animals. Calves less than 6 months of age were found to be more prone to theileriosis.

Specific duplex polymerase chain reaction (PCR) was employed on 411 (386 cattle and 25 buffaloes) blood samples of dairy animals from 9 districts of Punjab, India, for simultaneous detection of Babesia bigemina and Trypanosoma evansi. The results were compared and correlated with conventional Giemsa stained thin blood smear (GSTBS) examination and haematological alterations to know the clinical status and pathogenicity of infections. The Bg3/Bg4 and TR3/TR4 primers were used in duplex PCR for B. bigemina and T. evansi amplified products of 689 bp and 257 bp, respectively. The overall prevalence by duplex PCR was found to be 36.49, 2.43, and 3.41% for T. evansi, B. bigemina, and dual infection, respectively. A more significant difference was observed for dual infection status (P ≤ 0.005) as compared to T. evansi (P ≤ 0.05) and B. bigemina (P ≤ 0.01) among various districts under study. A very low prevalence of T. evansi (0.73%) and B. bigemina (0.48%) was seen by GSTBS. The highly sensitive, specific, and cost-effective duplex PCR was able to detect latent T. evansi and B. bigemina infection in cattle and buffaloes. Haematological evaluation revealed marked pathology in B. bigemina infected group and in dual infected group in contrast to that infected with T. evansi alone.

Duplex PCR consisting of two primer sets within a single mixture for the simultaneous detection of Anaplasma marginale and Trypanosoma evansi was standardized and employed on 219 blood samples collected from cattle (165) and buffaloes (54) from eastern Punjab to evaluate the status of concurrent infection and associated risk factors. The reaction produced 257- and 407-bp amplification products targeting repetitive nucleotide sequence of T. evansi and msp1β gene of A. marginale, respectively. The nucleotide sequence analysis of individual amplicons expressed the fidelity of the primer pairs used; duplex PCR was 100% sensitive and 92.66 % specific with conventional microscopy for the detection of mixed infections. Among the agro-climatic zones of interest, undulating zone was at higher risk of T. evansi infection (odds ratio (OR) = 1.75, 95% confidence interval (CI) = 0.94-3.27), and submountain zone (OR = 1.89, 95% CI = 1.11-3.33) for A. marginale. For the concurrent infection, the relative risk among the two zones was almost unity. The cross-bred cattle population was at the highest risk of infection, may it be solo infection of T. evansi (OR = ∞, 95% CI = 1.18-∞)/A. marginale (OR = 6.39, 95% CI = 1.14-125.3) or dual infection (OR = ∞, 95% CI = 0.39-∞) of both as the indigenous cattle are resistant to the infection. Cross-bred cattle were at approximately three times the risk than buffaloes. For the dual infection, the cattle calves were at about 2.5 times higher risk than buffalo calves. Results indicate the endemic status of these infections in the region and mark out the commodities at great risk and requiring better surveillance.