| Plague is a zoonotic infection that spreads to humans from natural rodent reservoirs, commonly via the bite of an infected flea. Yersinia pestis, the causative agent of plague, has killed hundreds of millions of people in the three major historical plague pandemics. Parasitic relationship exists between Y. pestis and Y. pestis phages. Some atypical Y. pestis and/or Y. pestis phages were frequently isolated from body of rodents and natural environment during animal plague epidemic phase or silence intervals phase in natural plague focus.So far, the phage lytic experiment still is the key diagnostic test method to Y. pestis. So, once the test be failure, the whole plague diagnostic system would receive serious challenge. Now, some Y. pestis isolates have be found existing resistance against Y. pestis phage in nature during the animal plague surveillance practices. Therefore an alternative of Y. pestis phage would be significant for the plague diagnosis and for understanding the interaction of the pathogen and bacteriophage.The study includes three section:(1) Establishing and assessing the isolating method of Y. pestis phage from soil samples, which is the basis of further study on the role of the Y. pestis phage in microecological environment. (2) The isolation of wild Y. peslis phages from Yunnan natural plague focus. (3) Identifying the biological characteristics of Y. pestis phage YL060 isolated from mice nest in natural plague focus.1. Using the spot method and the two-layer plating method, the isolating method of Y. pestis phage from the simulated soil that had mixed the known Y. pestis phage (used in the lysis test in diagnosis of Y.pestis) in advance was established in lab. Two methods (spot method, two-layer plates) all could obtain obvious plaques from filtering medium after series phage dilution (5μL. 50μL,500μL, 1000μL, respectively). And the minimum detectable quantity of phage was 30PFU in 2g soil samples. The titer of plague phage reduced more than two orders of magnitude in soil samples when the two-layer plating method was used (comparing the spot method). Still another, pre-cultivating could enhance the yield of phage when phage were rarely in the environmental samples. As so as the two-layer plating method concerned, the method not only could detect the phage, but also could accurately measure its titer.2. Using the attenuated vaccine Yersinia pestis strain EV76 as host bacterium, seven Y. pestis phages were isolated from 261 biology material and non-biology material (including mice nest, soil of mice hole, faeces of mice, cecum of mice) collected from Yulong natural plague focus, Yunnan. Four Y. pestis phages isolated from mice nest and three from cecum of mice. It is the first time that the Yersinia pestis phages were reported isolated from mice nest in plague natural focus, and such results were considered related to recent animal plague epidemics in Yulong plague focus.3 The morphology of the phage YL060 under electron microscope showed the particles presented tadpoles-like features with an icosahedra head of 60 nm diameter, and a long flexible tail about 140 nm. According to the classification standards of the international commission on virus classification, it belongs to Myoviridae in Caudovirales. The optimal multiplicity of infection was 0.1; Latent period and burst period was 50 min and 80 min, respectively; In addition, the average burst index of phage YL060 is 7.1. YL060 has narrow host lysis spectrum, i.e. it is only sensitive to Yersinia pestis. The phage can endure the temperature below 50 ℃, and it contains stronger lysis activities in a range of pH 5 to 10. The phage is sensitive to ultraviolet ray while insensitive to chloroform. The aga-gel electrophoresis showed that the genome size of YL060 is more than 23 kb. |
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