Development and evaluation of specifc polymerase chain reaction assays for detecting Theileria equi genotypes
Date
2023
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Parasites & Vectors
Abstract
Background Theileria equi causes equine piroplasmosis, an economically signifcant disease that afects horses
and other equids worldwide. Based on 18S ribosomal RNA (18S rRNA sequences), T. equi can be classifed into fve
genotypes: A, B, C, D, and E. These genotypes have implications for disease management and control. However,
no conventional polymerase chain reaction (PCR) assays are available to diferentiate the genotypes of T. equi. To over come this limitation, we developed and evaluated PCR assays specifc for the detection of each T. equi genotype.
Methods A pair of forward and reverse primers, specifcally targeting the 18S rRNA sequence of each genotype,
was designed. The genotype-specifc PCR assays were evaluated for their specifcity using plasmids containing inserts
of the 18S rRNA sequence of each genotype. Subsequently, the assays were tested on 270 T. equi-positive equine
blood DNA samples (92 from donkeys in Sri Lanka and 178 from horses in Paraguay). 18S rRNA sequences derived
from the PCR amplicons were analyzed phylogenetically.
Results Each genotype-specifc PCR assay accurately targeted the intended genotype, and did not produce any
amplicons when 18S rRNA from other T. equi genotypes or genomic DNA of Babesia caballi or uninfected horse
blood was used as the template. Previous studies employing PCR sequencing methods identifed T. equi genotypes
C and D in the Sri Lankan samples, and genotypes A and C in the Paraguayan samples. In contrast, our PCR assay
demonstrated exceptional sensitivity by detecting four genotypes (A, C, D, and E) in the Sri Lankan samples and all
fve genotypes in the Paraguayan samples. All the Sri Lankan samples and 93.3% of the Paraguayan samples tested
positive for at least one genotype, further emphasizing the sensitivity of our assays. The PCR assays also had the abil ity to detect co-infections, where multiple genotypes in various combinations were detected in 90.2% and 22.5%
of the Sri Lankan and Paraguayan samples, respectively. Furthermore, the sequences obtained from PCR amplicons
clustered in the respective phylogenetic clades for each genotype, validating the specifcity of our genotype-specifc
PCR assays.
Conclusions The genotype-specifc PCR assays developed in the present study are reliable tools for the diferential
detection of T. equi genotypes.
Description
Research Article
Keywords
Equine piroplasmosis, Genotype, Polymerase chain reaction, Theileria equi