云南松鼠鼠疫噬菌体的分离鉴定及其流行病学意义

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目的:了解云南松鼠是否携带鼠疫噬菌体,并探讨其流行病学意义。方法:2015 - 2018年,在云南鼠疫疫源地和非疫源地的5个调查点进行鼠疫宿主动物调查。对调查中捕获的松鼠,取其脾、肝、肠道标本,低温保存备用。取肠道标本磷酸盐缓冲液(PBS)增菌液,经0.22 μm滤膜滤过后加入到含有100 μl鼠疫疫苗株(EV76)菌悬液的LB液体培养基中,28 ℃、220 r/min恒温气浴振荡培养18~24 h,采用双层平板法观察噬菌斑的生长情况,电镜下观察鼠疫噬菌体的形态结构;同时取脾、肝、肠道标本进行鼠疫菌特异标志基因caf1检测。结果:共捕获到10只松鼠,其中赤腹松鼠8只,珀氏长吻松鼠2只。共分离出4株鼠疫噬菌体,其中在赤腹松鼠分离出2株,在珀氏长吻松鼠分离出2株;家鼠鼠疫疫源地弥勒县和新平县各分离出1株,野鼠鼠疫疫源地剑川县和洱源县未分离出,非鼠疫疫源地永善县分离出2株。肉眼下观察,家鼠鼠疫疫源地分离出的2株噬菌体产生的噬斑为透明状,生长状态良好;非鼠疫疫源地分离出的2株噬菌体产生的噬斑为半透明状,生长状态欠佳。电镜下观察,噬菌体为较典型的肌尾噬菌体,噬菌体头部直径约40 nm,肌尾约120 nm,肌尾末梢可见尾丝簇。10份松鼠脾、肝、肠道标本caf1检测均为阴性。结论:云南松鼠中携带鼠疫噬菌体的比例较高,虽然鼠疫菌特异标志基因caf1检测均为阴性,但由于鼠疫噬菌体的存在,松鼠有可能成为鼠疫菌传播的一种载体。“,”Objective:To investigate whether the squirrels in Yunnan Province carried n Yersinia pestis phages and their epidemiological significance.n Methods:From 2015 to 2018, plague host animals were investigated in five of Yunnan plague foci and non-plague foci. The spleen, liver and intestinal specimens of the squirrels captured in the investigation were taken and stored at low temperature for later use. Intestinal specimens with PBS solution, were filtered by 0.22 μm and added to LB liquid medium containing 100 μl suspension of plague vaccine strain (EV76) and then oscillated in a constant temperature gas bath at 28 ℃ and 220 r/min for 18 to 24 h. The double-layer plate method was used to isolate and observe the growth of plaque. The morphology and structure of n Yersinia pestis phages were observed under electron microscope. Meanwhile, spleen, liver and intestinal specimens were taken for detection of n Yersinia pestis specific marker gene n caf1.n Results:A total of 10 squirrels were captured (8 n Callosciurus erythraeus and 2 n Dremomys pernyi), and four n Yersinia pestis phages were isolated (2 in n Callosciurus erythraeus and 2 in n Dremomys pernyi). Two were isolated from non-plague foci (Yongshan County), two from house rats plague foci (Mile County and Xinping County), and none was isolated from wild radents plague foci (Jianchuan County and Eryuan County). By naked eye observation, two bacteriophages from the plague foci produced transparent plaques and grew well, while two bacteriophages from non-plague foci produced translucent plaques and with poor growth. By electron microscopy, these n Yersinia pestis phages were of typical n Myoviridae family, their head diameter was about 40 nm, muscle tail was about 120 nm, and tail filament cluster was slightly visible at the end of muscle tail. And all the 10 samples of squirrels were negative of plague-specific n caf1 gene.n Conclusions:The proportion of plague phages carried by Yunnan squirrels is relatively high. Although the detection of n caf1 is negative. Squirrels may be a carrier of plague transmission due to the existence of n Yersinia pestis phages.n
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