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Background  Oriental theileriosis is a tick-borne disease of bovines caused by the members of the  Theileria orientalis  complex. Recently, we developed a multiplexed tandem (MT) PCR to detect, differentiate and quantitate four genotypes (i.e.,  buffeli ,  chitose ,  ikeda  and  type  5) of  T. orientalis . In this study, we used MT PCR to assess the prevalence and infection intensity of four  T. orientalis  genotypes in selected cattle herds that experienced oriental theileriosis outbreaks in New Zealand, and compared the sensitivities and specificities of MT PCR, PCR-high resolution melting (PCR-HRM) and a TaqMan® qPCR.    Methods  MT PCR, PCR-HRM analysis for  T. orientalis  and a TaqMan® qPCR assay for  ikeda  genotype were employed to test 154 and 88 cattle blood samples from North (where oriental theileriosis outbreaks had occurred; designated as Group 1) and South (where no outbreaks had been reported; Group 2) Islands of New Zealand, respectively. Quantitative data from MT PCR assay were analyzed using generalized linear model and paired-sample  t -test. The diagnostic specificity and sensitivity of the assays were estimated using a Bayesian latent class modeling approach.    Results  In Group 1, 99.4% (153/154) of cattle were test-positive for  T. orientalis  in both the MT PCR and PCR-HRM assays. The apparent prevalences of genotype  ikeda  in Group 1 were 87.6% (134/153) and 87.7% (135/154) using the MT PCR and Ikeda TaqMan® qPCR assays, respectively. Using the MT PCR test, all four genotypes of  T. orientalis  were detected. The infection intensity estimated for genotype  ikeda  was significantly higher ( P  = 0.009) in severely anaemic cattle than in those without anaemia, and this intensity was significantly higher than that of  buffeli  ( P  < 0.001) in the former cattle. Bayesian latent class analysis showed that the diagnostic sensitivities (97.1-98.9%) and specificities (96.5-98.9%) of the three PCR assays were very comparable.    Conclusion  The present findings show the advantages of using the MT PCR assay as a useful tool for in-depth epidemiological and transmission studies of  T. orientalis  worldwide.

Comparison of the performance of three PCR assays for the detection and differentiation of Theileria orientalis genotypes