Blouin E F, Barbet A F, Yi J, Kocan K M, Saliki J T
Department of Anatomy, Pathology and Pharmacology, College of Veterinary Medicine, Oklahoma State University, Stillwater 74078, USA.
Vet Parasitol. 2000 Feb 1;87(4):301-13. doi: 10.1016/s0304-4017(99)00183-1.
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.
边缘无形体是一种全球范围内通过蜱传播的牛血液寄生虫。弗吉尼亚州的边缘无形体分离株先前在源自肩突硬蜱胚胎的细胞系中进行繁殖。培养的无形体（VA-tc）连续传代超过4年，并保持其对牛的感染力和抗原稳定性。我们在此报告了从俄克拉荷马州一头自然感染的母牛分离得到的第二种边缘无形体分离株（OK-tc）的连续体外培养情况。将感染母牛的血液接种到一头脾切除的小牛体内，在寄生虫血症高峰期采集血液，冷冻、解冻后用作接种物接种到长满单层蜱细胞的培养皿中。暴露后9天（PE）可见少量无形体菌落，到4 - 5周PE时单层细胞感染率达到100%。通过将上清液以1:5或1:10的稀释度接种到新鲜的蜱细胞单层上进行传代培养。到第三次传代时，OK-tc的生长情况与VA-tc相似，1:5的稀释度在10 - 12天内导致100%感染。将OK-tc接种到脾切除的小牛体内会引发临床无形体病，以这头小牛为食的革蜱将该病原体传播给了第二头易感小牛。将OK-tc的主要表面蛋白（MSP）1 - 5与VA-tc以及俄克拉荷马分离株红细胞阶段的同源蛋白进行了比较。OK-tc上的MSP 1、2、4、5是保守的，但有证据表明无形体培养阶段和红细胞阶段的MSP3存在结构差异。MSP2和MSP3是感染牛血清识别的主要蛋白。二维凝胶电泳还鉴定出VA-tc和OK-tc在MSP2和MSP3上存在位置差异。OK-tc可能有潜力用作抗原，用于开发美国中南部地区改进型无形体病疫苗。
