Identification of Anaplasma ovis appendage-associated protein (AAAP) for development of an indirect ELISA and its application

Zhenguo Wang, Jifei Yang, Qingli Niu, Kelly A. Brayton, Jianxun Luo, Guangyuan Liu, Hong Yin, Zhijie Liu

Ovine anaplasmosis is a tick-borne disease that is caused by Anaplasma ovis in sheep and goats. The pathogen is widely distributed in tropical and subtropical regions of the world. At present, diagnosis of the disease mainly depends on microscopy or nucleic acid based molecular tests, although a few serological tests have been applied for the detection of A. ovis infection. Here we describe the identification of an A. ovis protein that is homologous to the A. marginale appendage-associated protein (AAAP). We expressed a recombinant fragment of this protein for the development of an indirect enzyme-linked immunosorbent assay (ELISA) for the detection of A. ovis. Anaplasma ovis-positive serum showed specific reactivity to recombinantly expressed AAAP (rAAAP), which was further confirmed by the rAAAP ELISA, which also demonstrated no cross-reactivity with sera from animals infected with A. bovis or other related pathogens in sheep and goats. Testing antibody kinetics of five experimentally infected sheep for 1 year demonstrated that the rAAAP ELISA is suitable for the detection of early and persistent infection of A. ovis infections. Investigation of 3138 field-collected serum samples from 54 regions in 23 provinces in China demonstrated that the seroprevalence varied from 9.4% to 65.3%, which is in agreement with previous reports of A. ovis infection. An A. ovis derived antigenic protein, AAAP, was identified and the antigenicity of the recombinant AAAP was confirmed. Using rAAAP an indirect ELISA assay was established, and the assay has been proven to be an alternative serological diagnostic tool for investigating the prevalence of ovine anaplasmosis of sheep and goats.