title: Quantitative PCR detection of Theileria equi using laboratory workflows to detect asymptomatic persistently infected horses.
abstract: Equine piroplasmosis is the most important tick-borne disease of horses. Regulations on movement of horses into disease-free countries are in place to preserve international trade. Introduction of infectious disease, such as equine piroplasmosis, into non-endemic countries remains a substantial risk owing to the wide-spread distribution of vectors. Identification and restriction of movement of Theileria equi persistently infected horses is an integral part of control strategies, because persistently infected horses with low parasitaemia are an important reservoir. We used real-time PCR for diagnosis of T. equi DNA in clinically healthy horses in an equine piroplasmosis endemic area. The sensitivity was assessed using a synthetic plasmid DNA and a laboratory workflow was developed to maximise detection of persistently infected horses. The detection limit was 10 rDNA copies of the plasmid DNA. Assuming that each red blood cell contains a single T. equi genome the detection limit corresponded to 2.5 T. equi/μl of total blood and parasitaemia as low as 2-3.8 × 10(-5)%. A laboratory workflow was developed and assessed on samples from Saudi Arabia. The laboratory workflow focused on samples returning no or single positive result in duplicate PCR. In total, we obtained 42% (59/141; 95% confidence interval: 33.85-50.15) T. equi positive samples, 26% (37/141) negative for T. equi samples. The remaining 45 samples were judged as suspect with no definitive diagnosis made. The Saudi Arabia's T. equi small subunit ribosomal DNA (SSU rDNA) sequencing (n=16) demonstrated A clade (n=15) as the dominant T. equi clade. Clade B was sequenced in a single case. We present an approach for diagnostic workflow to detect T. equi in clinically healthy but persistently infected horses. Results from Saudi Arabia confirm that T. equi is widespread in the Middle East region. High proportion of horses with low parasitaemia calls for caution with results based on a single blood sample. Understating of the fluctuation of the parasitaema in persistently infected horses in endemic areas is needed to establish the required sample numbers for reliable detection of T. equi.
authors.affiliation: Department of Biological Sciences, Faculty of Science and Humanities, Shaqra University, P.O. Box 1040, Ad-Dawadimi 11911, Saudi Arabia. Electronic address: aalanazi@su.edu.sa.
authors.forename: Abdullah D
authors.initials: AD
authors.lastname: Alanazi
authors.affiliation: Department of Biological Sciences, Faculty of Science and Humanities, Shaqra University, P.O. Box 1040, Ad-Dawadimi 11911, Saudi Arabia.
authors.forename: Ashraf E
authors.initials: AE
authors.lastname: Said
authors.affiliation: Faculty of Veterinary Science, University of Sydney, New South Wales 2006, Australia.
authors.forename: Victoria
authors.initials: V
authors.lastname: Morin-Adeline
authors.affiliation: Department of Zoology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia.
authors.forename: Mohammed S
authors.initials: MS
authors.lastname: Alyousif
authors.affiliation: Faculty of Veterinary Science, University of Sydney, New South Wales 2006, Australia. Electronic address: jan.slapeta@sydney.edu.au.
authors.forename: Jan
authors.initials: J
authors.lastname: Slapeta
mesh_terms: D000818:Animals; D001404:Babesiosis / Q000469:parasitology / Q000517:prevention & control*; D001483:Base Sequence; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics; D004275:DNA, Ribosomal / Q000737:chemistry / Q000235:genetics; D004197:Disease Reservoirs; D006734:Horse Diseases / Q000175:diagnosis* / Q000469:parasitology; D006736:Horses; D006801:Humans; D008969:Molecular Sequence Data; D018512:Parasitemia / Q000662:veterinary; D010802:Phylogeny; D060888:Real-Time Polymerase Chain Reaction / Q000662:veterinary; D012529:Saudi Arabia; D017422:Sequence Analysis, DNA / Q000662:veterinary; D016794:Theileria / Q000235:genetics / Q000302:isolation & purification*; D013801:Theileriasis / Q000175:diagnosis* / Q000469:parasitology; D057188:Workflow
chemical_list: D016054:DNA, Protozoan; D004275:DNA, Ribosomal
keywords: Diagnostics; Horse; Phylogeny; Piroplasmosis; Real time PCR; Saudi Arabia