四川省德格县2株疑似喜马拉雅旱獭鼠疫菌株的鉴定

Identification of 2 strains of suspected Yersinia pestis isolated from Marmota himalayana in Dege County, Sichuan Province

目的通过对四川省德格县自毙喜马拉雅旱獭体内分离出的2株疑似鼠疫菌株进行生化特征、毒力鉴定，确定四川省是否存在早獭鼠疫自然疫源地。方法菌株鉴定采用细菌形态染色、培养特性、鼠疫噬菌体裂解试验、生化及糖醇类酵解试验、毒力因子、毒力、营养需求、质粒、基因检测及基因分型方法进行。结果光镜下，2株被检菌株均为革兰染色阴性，呈球杆状，两极浓染、两端钝圆；在赫氏普通琼脂上24～48h．被检菌株形态呈中心发暗，有黄褐色粗糙颗粒，边缘不整呈锯齿状、薄而透明的花边；存赫氏血琼脂t，菌落隆起，色泽深，花边消失或残留锯齿状痕迹。2株被检菌株均能酵解阿胶糖、麦芽糖、甘油，不酵解鼠李糖、密二糖，脱氮阳性；毒力因子均为F1’、VW’、Pgm’、PstI’；携带鼠疫菌常见的质粒，相对分子质量（胁）为6．0×10^6、45．0×10^6、65．0×10^6，具有鼠疫菌的毒力相关质粒基因Pla、calfl、krG；对小鼠的毒力试验，绝对致死量（LD100）均为50个菌；营养需求上，对苯丙氨酸、甲硫氨酸依赖；基因型是Genomovar5；与假结核菌（FFB）在鉴别培养基尿素、奥腾、枸橼酸盐上有明显区别，含鼠疫菌特有的PstI和F1抗原，22℃下能被鼠疫噬菌体完全裂解，含有鼠疫菌特有的3a片段。结论四川省德格县喜马拉雅旱獭体内分离的2株疑似菌株均为鼠疫耶尔森菌，具有青藏高原喜马拉雅旱獭型鼠疫菌株的特性，四川省存在喜马拉雅旱獭鼠疫自然疫源地。

Objective Through identify biochemical characteristics and virulence factors of 2 strains suspected Yersinia pestis （Y. pestis ） isolated from the dead Marmota himolayana（M, himalayana） to confirm the nature epidemic focus in Dege County, Sichuan Province. Methods Y. pestis was analyzed by specific staining and shape, culturing characteristics, splitting-test by bacteriophage, test of biochemical characteristics and glycolysis ability, virulence factors, virulence, nutritional requirement, plasmid, genetic test and genetic type. Results The tested strains were Grain staining baeilus. The main biochemical characteristics were Arabinose （＋）, Rhamnose （-）, Maltose （＋）, Melibiose （-）, Glycerol （＋）, Denitrifieation （＋）. The virulence factors with F l~, VW＋, Pgm＋, Pst I ＋; and with the common 6.0 ×10^6,45.0 ×10^6,65.0 ×10^6 plasmids, also with the virulence-relative plasmid gene. Both their absolutely lethal dose（LDl00） in mice were 50 bacteria. The nutritional requirement appeared which were depended on Phenylalanine and Methionine. With the Genomovar 5 genotype characteristics of M. himalayana plague loci of Qinghai-Tibet plateau. The difference between tested strains and Yersinia pseudotubercuosi. on the 3 different culture medium was obvious. The tested strains had a Y. pestis＇ specific 3a fragment, Pst I and F1-Ag, at 22 ℃, the strains could be split by bacteriophage completely. Conclusions According to the diagnostic criteria of plague in China, the 2 suspected strains isolated from Dege County, Sichuan Province are confirmed as Y. pestis, both with powerful virulenceand with the characteristics of the Y. pestis of M. himalayana in Qinghai-Tibet plateau plague natural focus.