Development and validation of direct PCR-RFLP assay for rapid detection and differentiation of Anaplasma species in naturally infected goats

Abstract

This study was carried out with the objective to develop a novel, rapid and cost effective direct PCR-RFLP assay for detection and differentiation of two Anaplasma species (A. ovis and A. marginale). Blood samples were collected randomly from 112 goats. DNA was extracted from blood samples and a direct blood polymerase chain reaction (DT-PCR) for amplifying a fragment of the major surface protein 5 (msp5) gene of A. ovis/A. marginale from whole blood was developed and standardized. Additionally, the 16srRNA and msp4 genes were analysed by nested PCR, semi nested PCR and PCR-RFLP methods. Blood smear examination revealed 33 samples (29.46%) to be positive for Anaplasma inclusion bodies. On the contrary, 54 (48.21%) samples were positive by DT-PCR. The results revealed that DT-PCR was 100% sensitive and 73.41% specific when compared with microscopy based detection (k =0.63). All DT-PCR positive samples were confirmed as A. ovis by RFLP analysis. DT-PCR showed 94.44% sensitivity and 100% specificity compared to conventional PCR results with suspected blood samples (k=0.94). The phylogenetic tree and comparative sequence analysis revealed msp5 gene of Anaplasma species of Indian isolate had maximum distance from A. phagocytophilum followed by A. centrale and A. marginale and 100% sequence identity with A. ovis isolates of Chinese origin. The in-house standardized DT-PCR-RFLP method based on msp5 gene is an easy and reliable diagnostic tool to identify and discriminate between these closely related pathogens in ruminants. This type of study is the first of its kind as it depicts the molecular detection, differentiation and phylogenetic characterization of Anaplasma ovis among goat flocks in India.