Development of a multiplex polymerase chain reaction assay for the detection of Piroplasma (Babesia spp. and Theileria spp.), Anaplasma spp., and Trypanosoma evansi in cattle.

Abstract

Background: Blood parasites cause serious drawbacks in the livestock industry. The detection methods rely on microscopic examination and polymerase chain reaction (PCR), which requires an expert person, labor, time, and cost.

Aim: In this study, a multiplex PCR has been developed for hemoparasites consisting of Piroplasma (Babesia spp. and Theileria spp.), Anaplasma spp., and Trypanosoma evansi detections.

Methods: Blood parasite infections in cattle were investigated using three different methods: microscopic examination, single PCR, and multiplex PCR. The Anaplasma 16s rRNA gene, Babesia 18s rRNA gene, Theileria major piroplasm surface protein gene, and Trypanosome ITS1 gene have been used to detect Anaplasma spp., Babesia spp., Theileria spp., and T. evansi, respectively, using molecular methods. The sensitivity and specificity of multiplex PCR were evaluated. Multiplex PCR results were compared with microscopic examination and single PCR.

Results: Multiplex PCR assay revealed a limit of detection of 0.01-10 pg of parasite DNA. According to the evaluation of 60 bovine blood samples, blood smear, single PCR, and multiplex PCR revealed 50.0%, 18.3%, and 26.7% of single infections, and 40.0%, 81.7%, and 50.0% of co-infections, respectively. The comparative analysis between multiplex PCR with microscopic examination and single PCR revealed that triple infection, Babesia spp., Theileria spp., and T. evansi, showed 50.0% sensitivity, 100% specificity, and positive predictive value, 98% negative predictive value, and substantial agreement indicated by a Cohen's Kappa value of 0.659.

Conclusion: The multiplex PCR assay developed in this study may be helpful for improved hemoparasite prevention and control when combined with farmer education, proper hygiene practices, and effective environmental management.