Development of a real-time PCR assay for detection and quantification of Anaplasma ovis infection

Abstract

Anaplasma ovis is a tick-borne intra-erythrocytic rickettsial pathogen of small ruminants. Real-time PCR possesses merits of rapidity, accuracy, reliability, automation and ease of standardization, but has not been used for detection of A. ovis, to the best of our knowledge. In this study, a real-time PCR assay was developed for detection and quantification of A. ovis. Species-specific primers and TaqMan probe were designed based on the gltA gene. No cross-reactions were observed with Anaplasma marginale, Anaplasma bovis, Anaplasma phagocytophilum, Borrelia burgdorferi s. l., Chlamydia psittaci, Mycoplasma mycoides, Theileria luwenshuni and Babesia sp. Xinjiang isolate. Analytic sensitivity results revealed that real-time PCR could detect as few as 10 copies of the gltA gene. The performance of real-time PCR was assessed by testing 254 blood samples from goats and comparing with the results from conventional PCR. This demonstrated that the real-time PCR assay was significantly more sensitive than conventional PCR. Our results indicated that real-time PCR is a useful approach for detecting A. ovis infections and has potential as an alternative tool for ecological and epidemiological surveillance of ovine anaplasmosis.

Keywords: Anaplasma ovis; real-time PCR; small ruminant.