First report on comparative assessment of real-time polymerase chain reaction to standard PCR and microscopy for detection of Babesia bigemina in cattle and buffaloes of North India.

Abstract

Abstract Purpose: Detection efficacy of real-time PCR targeting 18s SSU ribosomal RNA (18S rRNA) gene of Babesia bigemina in comparison to standard PCR and classical microscopy was evaluated from the samples suspected to be positive for bovine babesiosis. Materials & methods: Blood samples (n =95) suspected to be positive B. bigemina based on clinical signs and history of tick infestation were analyzed comparatively by three techniques. Real-time PCR was standardized targeting the 18s rRNA gene with the 10 fold serial dilutions in duplication of the given positive control (2x106 copy number) ranging from 106- 100 copy number/µL and mean Ct value of each dilution was taken to extrapolate the curve. Results: Blood samples (n=95) analysis revealed 5.26, 22.10 and 51.58% to be positive by microscopy, standard PCR and real-time PCR, respectively. Samples positive by microscopy, PCR and real-time PCR were cut down in range of >106-104, 104-103, and 103-<10 copy number/ µL, respectively of 18s rRNA gene. The concordance of real-time PCR with conventional PCR and microscopy was moderate (Kappa= 0.523) and mild (Kappa= 0.09), respectively. Real-time PCR assay indicated cows to be at four times more at risk than buffaloes (Odds ratio:3.85, CI:1.4255 - 10.4370) for getting B. bigemina infection and also indicated as most important risk factor associated with the prevalence of babesiosis. Conclusion: Results of this first report on comparative analysis indicated real-time PCR to be more perceptive than conventional PCR and microscopy. This needs further investigations on large random sample size to evaluate the factual depiction.