title: Assessing the performance of multiplexed tandem PCR for the diagnosis of pathogenic genotypes of Theileria orientalis using pooled blood samples from cattle.
abstract: Oriental theileriosis caused by multiple genotypes of Theileria orientalis is an important tick-borne disease of bovines. Here, we assessed the performance of an established multiplexed tandem PCR (MT-PCR) for the diagnosis of the two recognized, pathogenic genotypes (chitose and ikeda) of T. orientalis in cattle using pooled blood samples. We used a total of 265 cattle blood samples, which were divided into two groups according to previous MT-PCR results for individual samples. Samples in group 1 (n = 155) were from a herd with a relatively high prevalence of T. orientalis infection; and those in group 2 (n = 110) were from four herds with a low prevalence. For group 1, 31 and 15 batches of five- and ten-pooled samples (selected at random), respectively, were formed. For group 2, 22 and 11 batches of five- and ten-pooled samples (selected at random), respectively, were formed. DNAs from individual pooled samples in each batch and group were then tested by MT-PCR. For group 1, the apparent prevalences estimated using the 31 batches of five-pooled samples (97%) and 15 batches of ten-pooled samples (100%) were significantly higher compared with individual samples (75%). For group 2, higher apparent prevalences (9% and 36%) were also recorded for the 22 and 11 batches of pooled samples, respectively, compared with individual samples (7%). Overall, the average infection intensity recorded for the genotypes of chitose and ikeda were considerably lower in pooled compared with individual samples. The diagnostic specificities of MT-PCR were estimated at 95% and 94%, respectively, when batches of five- and ten-pooled samples were tested, and 94% for individual samples. The diagnostic sensitivity of this assay was estimated at 98% same for all individual, five- and ten-pooled samples. This study shows that screening batches of five- and ten-pooled blood samples from cattle herds are similar to those obtained for individual samples, and, importantly, that the reduced cost for the testing of pooled samples represents a considerable saving to herd managers.
authors.affiliation: Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Werribee, Victoria, 3030, Australia.
authors.forename: Hagos
authors.initials: H
authors.lastname: Gebrekidan
authors.affiliation: Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Werribee, Victoria, 3030, Australia.
authors.forename: Robin B
authors.initials: RB
authors.lastname: Gasser
authors.affiliation: Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Werribee, Victoria, 3030, Australia.
authors.forename: Mark A
authors.initials: MA
authors.lastname: Stevenson
authors.affiliation: Millicent Veterinary Clinic, Millicent, South Australia, Australia.
authors.forename: Sean
authors.initials: S
authors.lastname: McGrath
authors.affiliation: Faculty of Veterinary and Agricultural Sciences, The University of Melbourne, Werribee, Victoria, 3030, Australia. Electronic address: jabbara@unimelb.edu.au.
authors.forename: Abdul
authors.initials: A
authors.lastname: Jabbar
mesh_terms: D000818:Animals; D002417:Cattle / Q000097:blood* / Q000469:parasitology*; D003365:Costs and Cost Analysis; D016054:DNA, Protozoan / Q000235:genetics; D005838:Genotype; D060885:Multiplex Polymerase Chain Reaction / Q000191:economics / Q000379:methods*; D015995:Prevalence; D012680:Sensitivity and Specificity; D016794:Theileria / Q000235:genetics* / Q000302:isolation & purification*; D013801:Theileriasis / Q000097:blood* / Q000175:diagnosis* / Q000453:epidemiology / Q000469:parasitology
chemical_list: D016054:DNA, Protozoan
keywords: Cattle; Diagnosis; Multiplexed-tandem PCR; Pooled blood sample; Theileria orientalis