Molecular prevalence of Babesia bigemina and Trypanosoma evansi in dairy animals from Punjab, India, by duplex PCR: a step forward to the detection and management of concurrent latent infections

Abstract

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.

Figure 1

Agarose gel (1.5%) eletrophoresis showing amplified DNA from B. bigemina (689 bp) and T. evansi (257 bp). Lane M molecular size marker 100 bp plus. Lane A showing no amplification for Theileria annulata genomic DNA. Lane B showing amplification for Trypanosoma evansi genomic DNA. Lane C showing no amplification for host leucocyte DNA. Lane D showing amplification for Babesia bigemina genomic DNA. Lane E showing no amplification for Anaplasma marginale genomic DNA. Lane F showing amplification for Babesia bigemina and Trypanosoma evansi genomic DNA.

Figure 2

Agarose gel (1.5%) eletrophoresis showing amplified DNA from B. bigemina (689 bp) and T. evansi (257 bp). Lane M molecular size marker 100 bp plus. Lane P positive control. Lane N negative controls (genomic DNA from host leucocytes). Lanes A and B showing simultaneous amplified B. bigemina and T. evansi genomic DNA from the blood of animals positive for infection. Lane C showing amplified B. bigemina genomic DNA from the blood of animal infected for B. bigemina alone. Lane D showing amplified T. evansi genomic DNA from the blood of animal infected for T. evansi alone. Lane E showing no amplification indicating being negative for dual infection by PCR.

Figure 3

Haematological parameters of animals infected with T. evansi (group a), B. bigemina (group b), both T. evansi and B. bigemina (group c), and non infected control (group d). Values of TLC, TEC, Hb, PCV, MCV, MCH, MCHC, and Plt with different letters a–d differ significantly at P ≤ 0.05.