Neitz A W, Yunker C E
Department of Biochemistry, University of Pretoria, Republic of South Africa.
Ann N Y Acad Sci. 1996 Jul 23;791:24-34. doi: 10.1111/j.1749-6632.1996.tb53508.x.
Cowdria ruminantium (Rickettsiales) causes heartwater in ruminants of Africa, and some islands off Africa and in the Caribbean Sea. The in vitro culture method for the organism devised in 1985, which provided for the first time a means for production of adequate quantities of live organisms and their products, is erratic and requires improvement. We studied depletion of amino acids (AAs) and major proteins in culture medium taken daily from infected and uninfected ovine and bovine vascular endothelial cell cultures. AAs of these samples were analyzed by Pico Tag reversed phase HPLC precolumn derivatization, and major proteins determined by capillary electrophoresis using a 57 cm x 75 microns fused silica tube at high pH. In both ovine and bovine cell cultures, significant depletion of arginine and glutamine occurred over a 5-day observation period regardless of whether they were infected or uninfected. This indicates that supplementation of nutrient media with these AAs might improve conditions for growth of the organism. Both AAs are essential for survival of cultured cells, and probably for the rickettsia (although the metabolism of C. ruminantium is poorly understood). Concentrations of several AAs increased in infected cultures, implying de novo synthesis and/or proteolysis on the part of the organism. In fact, several protein fractions did decrease in culture medium throughout the course of infection, while increasing or remaining unchanged in uninfected control cultures. Proteolytic activity by C. ruminantium may be essential for nitrogen metabolism by the organism. It is suggested that studies such as these will facilitate the development of a specific medium for optimal in vitro growth of the heartwater organism, and may also lead to an understanding of the metabolic stratagem of C. ruminantium. This knowledge, in turn, could reveal the mechanism for pathogenesis of heartwater, with implications for control.
反刍兽立克次氏体（立克次氏体目）可引发非洲、非洲附近一些岛屿以及加勒比海地区反刍动物的牛心水病。1985年设计出的该病原体体外培养方法不稳定且需要改进，不过它首次提供了一种生产足量活病原体及其产物的手段。我们研究了每天从感染和未感染的绵羊及牛血管内皮细胞培养物中获取的培养基中氨基酸（AAs）和主要蛋白质的消耗情况。这些样品的氨基酸通过Pico Tag反相高效液相色谱柱前衍生化进行分析，主要蛋白质则使用57 cm×75微米熔融石英管在高pH值下通过毛细管电泳进行测定。在绵羊和牛细胞培养物中，无论是否感染，在5天的观察期内精氨酸和谷氨酰胺均出现显著消耗。这表明在营养培养基中补充这些氨基酸可能会改善该病原体的生长条件。这两种氨基酸对于培养细胞的存活至关重要，可能对立克次氏体也同样重要（尽管反刍兽立克次氏体的代谢情况了解甚少）。在感染的培养物中，几种氨基酸的浓度有所增加，这意味着该病原体进行了从头合成和/或蛋白水解。事实上，在整个感染过程中，培养基中的几个蛋白质组分确实减少了，而在未感染的对照培养物中则增加或保持不变。反刍兽立克次氏体的蛋白水解活性可能对该病原体的氮代谢至关重要。建议开展此类研究将有助于开发一种特定培养基，以实现牛心水病原体的最佳体外生长，还可能有助于了解反刍兽立克次氏体的代谢策略。反过来，这些知识可能揭示牛心水病的发病机制，对疾病控制具有重要意义。
