Standardization of a SYBR Green Based Real-Time PCR System for Detection and Molecular Quantification of Babesia bovis and B. bigemina in Water Buffaloes (Bubalus bubalis)

Abstract

Water buffalo (Bubalus bubalis) is a potential reservoir for Babesia bovis and B. bigemina in tropical regions, but the epidemiological evidence of their reservoir competence is limited, especially due to the lack of diagnostic tests capable of detecting and quantifying the low-level parasitemia present in the carrier animals. In this paper we present the standardization process of a SYBR Green based real-time PCR system (qPCR), consisting of two single qPCR assays, for the detection and quantification of B. bovis and/or B. bigemina. Both assays were optimized in similar protocols, including reagent concentrations and thermocycling parameters, so it is possible its use as a multiple qPCR in a single run. Both single assays showed a suitable analytical performance, especially by allowing detection of a greater number of carrier animals when compared with nested PCR assays (nPCR) against a reference panel of 60 DNA samples extracted from blood of both, infected-and non-infected buffaloes. Furthermore, a mathematical algorithm to convert the qPCR outcomes in percent of infected red blood cell was used, and was found that the estimated parasitemia in carrier buffaloes within the reference sample panels were close to those described in carrier cattle. This method could be a useful tool for epidemiological studies on the participation of the bubaline specie in the epidemic process of bovine babesiosis.

Efficient Transovarial Transmission of Babesia Spp. in Rhipicephalus microplus Ticks Fed on Water Buffalo (Bubalus bubalis)

“…The primers used were those described by Buling et al [46], which amplify 88 base pairs (bp) fragments of the mitochondrial cytochrome b gene (mt-Cytb). The standardized thermocycling conditions, as well as the quantification procedures, were as described previously by Obregon et al [47].…”

Ticks and Tick-Borne Diseases in Cuba, Half a Century of Scientific Research

“…However, epidemiological studies using molecular (i.e., Polymerase chain reaction PCR and nested (n) PCR) and serological (i.e., indirect (i) ELISA) diagnostic tests found a prevalence greater than 50% for B. bovis , B. bigemina and A. marginale in buffaloes of the western region of Cuba [ 21 , 99 ]. Prevalence and infection levels found in water buffaloes suggest that these animals are potential reservoirs of CTF pathogens in the ecoepidemiological conditions of Cuba, especially in areas where cattle and buffaloes coexist [ 100 , 101 , 102 ]. Remarkably, A. marginale strains infecting buffalo were genetically related to those infecting cattle in the same region [ 100 ], suggesting circulation of this pathogen between buffalo and cattle.…”

“…The multiplex qPCR described by Díaz-Sánchez et al [ 14 ] proved to be a rapid, specific and cost-effective means for the simultaneous detection of A. marginale and T. annulata , confirming a high prevalence of A. marginale infection of carrier cattle in Cuba. On the other hand, a SYBR Green-based real-time PCR system developed for the detection and quantification of B. bovis and B. bigemina [ 101 ] detected low parasitemia levels in carrier buffaloes, and R. microplus tick larvae [ 70 ]. Likewise, a TaqMan-based real-time PCR assay enabled exploration of the reservoir competence of water buffalo for A. marginale in endemic areas of Cuba [ 100 ].…”