Anaplasma marginale is a tick-borne hemoparasite of cattle worldwide. The Virginia isolate of A. marginale was propagated previously in a cell line derived from embryos of the tick, Ixodes scapularis. The cultured Anaplasma (VA-tc) was passaged continuously for over 4 years and retained its infectivity for cattle and antigenic stability. We report herein the continuous in vitro cultivation of a second isolate of A. marginale derived from a naturally infected cow in Oklahoma (OK-tc). Blood from the infected cow was subinoculated 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 were passaged by placing supernatant onto fresh tick cell monolayers at a dilution 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 clinical
anaplasmosis and Dermacentor ticks that fed on this calf transmitted the 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 the erythrocytic stage of the Oklahoma isolate. The MSPs 1, 2, 4, 5 were conserved on the OK-tc but there was evidence for structural variation in MSP3 between the cultured and erythrocytic stage of Anaplasma. MSP2 and MSP3 were the major
proteins recognized by serum from infected cattle. Two-dimensional
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 development of an improved
vaccine for
anaplasmosis in the South Central United States.