A pan-Theileria FRET-qPCR survey for Theileria spp. in ruminants from nine provinces of China

Abstract

BackgroundTheileria spp. are tick transmitted protozoa that can infect large and small ruminants causing disease and economic losses. Diagnosis of infections is often challenging, as parasites can be difficult to detect and identify microscopically and serology is unreliable. While there are PCR assays which can identify certain Theileria spp., there is no one PCR that has been designed to identify all recognized species that occur in ruminants and which will greatly simplify the laboratory diagnoses of infections.MethodsPrimers and probes for a genus-specific pan-Theileria FRET-qPCR were selected by comparing sequences of recognized Theileria spp. in GenBank and the test validated using reference organisms. The assay was also tested on whole blood samples from large and small ruminants from nine provinces in China.ResultsThe pan-Theileria FRET-qPCR detected all recognized species but none of the closely related protozoa. In whole blood samples from animals in China, Theileria spp. DNA was detected in 53.2% of the sheep tested (59/111), 44.4% of the goats (120/270) and 30.8% of the cattle (380/1,235). Water buffaloes (n = 29) were negative. Sequencing of some of the PCR products showed cattle in China were infected with T. orientalis/T. sergenti/T. buffeli group while T. ovis and T. luwenshuni were found in sheep and T. luwenshuni in goats. The prevalence of Theileria DNA was significantly higher in Bos p. indicus than in Bos p. taurus (77.7% vs. 18.3%) and copy numbers were also significantly higher (104.88 vs. 103.00Theileria 18S rRNA gene copies/per ml whole blood).ConclusionsThe pan-Theileria FRET-qPCR can detect all recognized Theileria spp. of ruminants in a single reaction. Large and small ruminants in China are commonly infected with a variety of Theileria spp.

Identification of 12 Piroplasms Infecting Ten Tick Species in China Using Reverse Line Blot Hybridization

“…A previous study reported a high infection rate of T. luwenshuni in ticks by RLB, or in small ruminants via PCR and FRET-qPCR (Niu et al, 2012;Li et al, 2014a;Yang et al, 2014). In our study, T. luwenshuni was mostly prevalent in Linzhou, Henan province, corresponding with a previous study, which reported T. luwenshuni infecting sheep in the same province (Chen et al, 2014b).…”

Temporal dynamics and subpopulation analysis of Theileria orientalis genotypes in cattle

“…Although stress (transport, pregnancy, lactation) has been widely reported as a disease trigger (Eamens et al, 2013c;Izzo et al, 2010), the inter-and intra-breed susceptibility of cattle to T. orientalis infection has not been extensively investigated. There is however, some suggestion that there may be differential susceptibility of Bos taurus versus Bos indicus breeds (Yang et al, 2014) and that Japanese black cattle (Wagyu) may be less susceptible to disease (Terada et al, 1995). The animals tested in our temporal study were all of a single breed (Ayrshire) and the patterns of genotype switching and disease progression were remarkably similar amongst the majority of animals.…”

Development and Validation of a Quantitative PCR Assay Using Multiplexed Hydrolysis Probes for Detection and Quantification of Theileria orientalis Isolates and Differentiation of Clinically Relevant Subtypes

“…While real-time assays that detect T. orientalis have been developed, most have focused on species-level detection (not discrimination of genotypes) (34), discrimination of different Theileria spp. (43,44), or are only semiquantitative (28). The UIC multiplex qPCR assay developed here was both sensitive and specific for T. orientalis detection compared to cPCR and reliably identified the clinically relevant Ikeda and Chitose genotypes.…”