Establishment and characterization of an Oklahoma isolate of Anaplasma marginale in cultured Ixodes scapularis cells

Anaplasma marginale is a tick-borne hemoparasite of cattle worldwide. The V irginia isolate of A. marginale was propagated previously in a cell line de rived from embryos of the tick, Ixodes scapularis. The cultured Anaplasma ( VA-tc) was passaged continuously for over 4 years and retained its infectiv ity for cattle and antigenic stability. We report herein the continuous in vitro cultivation of a second isolate of A. marginale derived from a natura lly infected cow in Oklahoma (OK-tc). Blood from the infected cow was subin oculated into a splenectomized calf and blood collected at peak parasitemia was frozen, thawed and used as inoculum on confluent tick cell monolayers. Colonies of Anaplasma were apparent in low numbers at 9 days post exposure (PE) and infection in monolayers reached 100% by 4-5 weeks PE. Cultures we re passaged by placing supernatant onto fresh tick cell monolayers at a dil ution of 1 : 5 or 1 : 10. By the third passage development of the OK-tc was similar to that of the VA-tc and a 1:5 dilution resulted in 100% infection in 10-12 days, Inoculation of OK-tc into a splenectomized calf caused clin ical anaplasmosis and Dermacentor ticks that fed on this calf transmitted t he organism to a second susceptible calf. Major surface proteins (MSPs) 1-5 of the OK-tc were compared with homologous proteins present on VA-tc and t he erythrocytic stage of the Oklahoma isolate. The MSPs 1, 2, 4, 5 were con served on the OK-tc but there was evidence for structural variation in MSP3 between the cultured and erythrocytic stage of Anaplasma. MSP2 and MSP3 we re the major proteins recognized by serum from infected cattle. Two-dimensi onal gels also identified positional differences between VA-tc and OK-tc in MSP2 and MSP3. The OK-tc may have potential to be used as antigen for deve lopment of an improved vaccine for anaplasmosis in the South Central United States. (C) 2000 Elsevier Science B.V. All rights reserved.