AUTHOR=Lv Kunying , Zhang Yiwei , Yang Yixin , Liu Zheng , Deng Liang 

TITLE=Development of Nested PCR and Duplex Real-Time Fluorescence Quantitative PCR Assay for the Simultaneous Detection of Theileria equi and Babesia caballi

JOURNAL=Frontiers in Veterinary Science

VOLUME=9

YEAR=2022

URL=https://www.frontiersin.org/journals/veterinary-science/articles/10.3389/fvets.2022.873190

DOI=10.3389/fvets.2022.873190

ISSN=2297-1769

ABSTRACT=<p>Equine piroplasmosis (EP) is a type of blood protozoan disease caused by tick-borne parasites, <italic>Theileria equi</italic> (<italic>T. equi</italic>), <italic>Babesia caballi</italic> (<italic>B. caballi</italic>) and <italic>Theileria haneyi</italic>. While many studies have been conducted on EP diagnosis, diagnostic methods exhibiting high sensitivity and specificity remain lacking. Therefore, nested PCR (nPCR) and duplex real-time fluorescence quantitative PCR (qPCR) that can simultaneously detect both <italic>T. equi</italic> and <italic>B. caballi</italic> causing agents were established and compared. The two techniques were used to analyze 36 horse blood samples for EP. This set of samples was also detected by a multinested PCR (mnPCR) targeting the <italic>EMA-1</italic> gene of <italic>T. equi</italic> and the <italic>RAP-1</italic> gene of <italic>B. caballi</italic>. By nPCR, duplex real-time fluorescence qPCR and mnPCR, infections with <italic>B. caballi</italic> were detected in 16.67% (6/36), 2.78% (1/36), 19.44% (7/36) of the horses, respectively. The <italic>T. equi</italic> prevalence was 58.33% (21/36) by the nPCR, 33.33% (12/36) by the duplex real-time fluorescence qPCR and 2.78% (1/36) by the mnPCR. The overall prevalence of infection with mixed parasites by nPCR was 5.56% (2/36), by duplex real-time fluorescence qPCR was 2.78% (1/36) and by mnPCR 0% (0/36). Results suggest that nPCR can detect <italic>T. equi</italic> and <italic>B. caballi</italic> positive samples with good specificity and sensitivity, although distinguishing between the two parasites requires an electrophoresis with 4% agarose gels. The duplex real-time fluorescence qPCR can readily distinguish between <italic>T. equi</italic> and <italic>B. caballi</italic> infection, but with low sensitivity.</p>