Bingqian Zhou,Guangpu Yang,Zhe Hu,Kewei Chen,Wei Guo,Xiaojun Wang,Cheng Du

Microorganisms.2023 Oct 26;11(11):2633.doi: 10.3390/microorganisms11112633.

Abstract

Equine piroplasmosis (EP) is a parasitic disease caused by  Theileria equi  ( T. equi ),  Babesia caballi  ( B. caballi ) and  Theileria haneyi  ( T. haneyi ). This disease is considered to be reportable by the World Organization for Animal Health (WOAH). Real-time quantitative PCR (qPCR) is regarded as a straightforward, rapid and sensitive diagnostic method to detect pathogens. However, qPCR has not been employed in the various epidemiological investigations of  T. haneyi . In this study, we developed a new qPCR method to detect  T. haneyi  based on the chr1sco (chromosome 1 single-copy open reading frame (ORF)) gene, which has no detectable orthologs in  T. equi  or  B. caballi.  A TaqMan MGB probe was used in the development of the qPCR assay. A plasmid containing the chr1sco gene was constructed and used to establish the standard curves. The novel qPCR technique demonstrated great specificity for detecting additional frequent equine infectious pathogens and sensitivity for detecting diluted standard plasmids. This qPCR was further validated by comparison with an optimized nested PCR (nPCR) assay in the analysis of 96 clinical samples. The agreement between the nPCR assay and the established qPCR assay was 85.42%. The newly established method could contribute to the accurate diagnosis of  T. haneyi  infections in horses.

Keywords: Theileria haneyi; nested PCR; real-time quantitative PCR.