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Detection and assessment of risk factors associated with natural concurrent infection of Trypanosoma evansi and Anaplasma marginale in dairy animals by duplex PCR in eastern Punjab


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.

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Polymerase chain reaction


Deoxyribonucleic acid


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Thanks are due to the Director of Research for providing financial assistance under RKVY project, ‘Employment of Immuno-molecular Diagnostic Tools for Haemoprotozoan Diseases of livestock in Punjab State’.

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Correspondence to Lachhman Das Singla.

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Sharma, A., Singla, L.D., Tuli, A. et al. Detection and assessment of risk factors associated with natural concurrent infection of Trypanosoma evansi and Anaplasma marginale in dairy animals by duplex PCR in eastern Punjab. Trop Anim Health Prod 47, 251–257 (2015).

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  • Anaplasma marginale
  • Dairy animals
  • Duplex PCR
  • Prevalence
  • Punjab risk factors
  • Trypanosoma evansi