ABSTRACT
Yersinia pestis phage is a virus that is parasitic within Yersinia pestis and can specifically lyses Yersinia pestis. The adsorption sites of phage infesting host bacteria are called receptor binding protein (RBP), including extracellular membrane protein, lipopolysaccharide, teichoteic acid, pili, flagella, capsular polysaccharide, etc., of which extracellular membrane protein and lipopolysaccharide are the receptors of Yersinia pestis phage. RBP plays a decisive role in the process of Yersinia pestis phage infecting Yersinia pestis. Therefore, the classification, isolation and application of Yersinia pestis phage are summarized; the research progress in identification and structure of Yersinia pestis phage receptor is analyzed, which is helpful in understanding the cleavage mechanism of Yersinia pestis phage and the interaction mode with Yersinia pestis from the molecular level, and provide more powerful support for in-depth study on Yersinia pestis phage receptor.
ABSTRACT
Objective:To study the basic situation of Yunnan Province Suncus murinus carrying plague phage and to explore its epidemiological significance. Methods:From 2015 to 2018, a survey of plague host animals was carried out in 10 investigation sites in the historical plague foci, new plague foci (after 1982) and stubborn plague foci of domestric mouse in Yunnan Province. The plague phage was isolated and cultured from the intestinal specimens of Suncus murinus. The growth of plaque was observed by double-layer plate method, and the morphology and structure of plague phage were observed under electron microscope. At the same time, intestinal samples were taken to detect the structural gene caf1 of F1 antigen of Yersinia pestis. Results:In this study, a total of 157 Suncus murinus were captured and 16 strains of plague phage were isolated, with a total isolation rate of 10.19%. There was no difference in plague phage isolation rate between historical plague foci (10.00%, 1/10) and stubborn plague foci (16.22%, 12/74), new plague foci (4.11%, 3/73, χ 2 = 0.00, P = 0.965; Fisher test, P = 1.000). However, there was a difference in plague phage isolation rate between stubborn plague foci and new plague foci (χ 2 = 5.88, P = 0.015). There was no significant difference in the isolation rate of plague phage among different sex, growth period and habitat ( P > 0.05). The plaque morphology of the isolated plague phage was diverse, of which four strains were myotavirus phages; and all samples were negative for F1 antigen structural gene caf1. Conclusions:Suncus murinus is widely distributed in the domestic mouse plague foci in Yunnan Province, and the animals carry a certain number of plague phage. Regular surveillance of Suncus murinus and their plague phage has a certain guiding significance for the surveillance and early warning of plague in Yunnan Province.
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Objective:To explore the biochemical characteristics, virulence factors and other phenotypes of the strains of Yersinia pestis isolated in Jianchuan County Yunnan Province in 2017, and to analyze the nature and source of the new plague epidemic. Methods:Three strains of Yersinia pestis (JC109 rat, JC109 fleas and JC113) isolated from Daqing Village, Jinhua Town, Jianchuan County, Dali Prefecture, Yunnan Province in 2017, and 2 associated strains of Yersinia pestis (LJ01 in Yulong County, Lijiang City and LJ04 in Gucheng District of Lijiang City), 5 control strains ( Yersinia pestis JC1332, LJ485, BN2636, EV-76 and Yersinia pseudotuberculosis PST-1), preserved by the Central Laboratory of Yunnan Institute for Endemic Disease Control and Prevention were collected. The biochemical characteristics and ecotypes of Yersinia pestis were analyzed by using arabinose, rhamnose, denbiose, maltose and glycerol fermentation experiments and nitrate reduction experiments. Combining pigmentation factor (pgm), virulence antigen (VW) detection and nutritional requirements test results to determine the virulence of Yersinia pestis. Results:The Yersinia pestis JC109 rat, JC109 fleas and JC113 all fermented arabinose, maltose and glycerol, but didn't ferment rhamnose and denbiose; and the nitrate reduction test was positive. The ecological type belonged to the Himalayan Marmot plague strain of Qinghai-Tibet plateau. The virulence factors pgm and VW tests were positive, the nutritional requirement type was phenylalanine dependent and glutamate independent. It had the same phenotype as the LJ01 strain, but different from the JC1332 strain. Conclusions:The newly isolated strains in Jianchuan County are the same as those in the Lijiang Yulong wild rodent plague foci. This outbreak may have been imported from the Lijiang Yulong wild rodent plague foci to the south.
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Objective:To investigate whether the squirrels in Yunnan Province carried Yersinia pestis phages and their epidemiological significance. 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 Yersinia pestis phages were observed under electron microscope. Meanwhile, spleen, liver and intestinal specimens were taken for detection of Yersinia pestis specific marker gene caf1. Results:A total of 10 squirrels were captured (8 Callosciurus erythraeus and 2 Dremomys pernyi), and four Yersinia pestis phages were isolated (2 in Callosciurus erythraeus and 2 in 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 Yersinia pestis phages were of typical 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 caf1 gene. Conclusions:The proportion of plague phages carried by Yunnan squirrels is relatively high. Although the detection of caf1 is negative. Squirrels may be a carrier of plague transmission due to the existence of Yersinia pestis phages.
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Objective@#To investigate whether plague phages were present in the indicator animals of plague foci in Yunnan Province, and to explore their epidemiological significance.@*Methods@#Anus swabs were collected from indicator animals (dogs or cats) of the 41 plague affected villages in 26 towns of 10 cities (counties, districts) of Yunnan plague foci from November of 2015 to March of 2018. The Yersinia pestis phages were isolated by plague vaccine strain EV76. The isolation of plague phages from different plague foci, the isolation of plague phages from different canine species (cats), the polymorphism of plaque and the host spectrum of phages were analyzed.@*Results@#A total of 1 014 indicator animals (1 003 dogs and 11 cats) were studied, and 102 of plague phages were isolated. In the 10 cities (counties, districts), plague phages were only not isolated from Lancang County, and the plague phages were isolated from the other 9 cities (counties, districts). The separation rates from high to low were as follows: Yiliang County (21.00%, 21/100), Menghai County (19.23%, 25/130), Yuanjiang County (11.63%, 10/86), Midu County (11.50%, 13/113), Wenshan County (10.10%, 10/99), Mile Country (7.07%, 7/99), Lianghe County (6.67%, 7/105), Baoshan Longyang District (4.90%, 5/102) and Gengma County (3.81%, 4/105). Of the 102 plague phages, 75 were isolated from the native dogs (Chinese pastoral dogs, 9.32%, 75/805), 20 from the pug dogs (13.70%, 20/146), 5 from the wolf dogs (17.24%, 5/29), 1 from Samoye (1/4) and 1 from Alaska dog (1/2). The plaque of the phage was divided by five appearance of complete lysis (the plate was clear), large (2.5-4.0 mm), big (1.5-< 2.5 mm), middle (0.5-< 1.5 mm) and small (< 0.5 mm). The representative phages were all of the Myoviridae family. Most of the phages could lysis the strains of Yersinia pestis, and some phages could lysis Shigella and type 5 Yersinia pseudotuberculosis (PST5).@*Conclusion@#The plague phages are present in the plague foci of Yunnan, and the phages are polymorphic.
ABSTRACT
Objective To investigate whether plague phages were present in the indicator animals of plague foci in Yunnan Province,and to explore their epidemiological significance.Methods Anus swabs were collected from indicator animals (dogs or cats) of the 41 plague affected villages in 26 towns of 10 cities (counties,districts) of Yunnan plague foci from November of 2015 to March of 2018.The Yersinia pestis phages were isolated by plague vaccine strain EV76.The isolation of plague phages from different plague foci,the isolation of plague phages from different canine species (cats),the polymorphism of plaque and the host spectrum of phages were analyzed.Results A total of 1 014 indicator animals (1 003 dogs and 11 cats) were studied,and 102 of plague phages were isolated.In the 10 cities (counties,districts),plague phages were only not isolated from Lancang County,and the plague phages were isolated from the other 9 cities (counties,districts).The separation rates from high to low were as follows:Yiliang County (21.00％,21/100),Menghai County (19.23％,25/130),Yuanjiang County (11.63％,10/86),Midu County (11.50％,13/113),Wenshan County (10.10％,10/99),Mile Country (7.07％,7/99),Lianghe County (6.67％,7/105),Baoshan Longyang District (4.90％,5/102) and Gengma County (3.81％,4/105).Of the 102 plague phages,75 were isolated from the native dogs (Chinese pastoral dogs,9.32％,75/805),20 from the pug dogs (13.70％,20/146),5 from the wolf dogs (17.24％,5/29),1 from Samoye (1/4) and 1 from Alaska dog (1/2).The plaque of the phage was divided by five appearance of complete lysis (the plate was clear),large (2.5-4.0 mm),big (1.5-＜ 2.5 mm),middle (0.5-＜ 1.5 mm) and small (＜ 0.5 mm).The representative phages were all of the Myoviridae family.Most of the phages could lysis the strains of Yersinia pestis,and some phages could lysis Shigella and type 5 Yersinia pseudotuberculosis (PST5).Conclusion The plague phages are present in the plague foci of Yunnan,and the phages are polymorphic.
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Objective To investigate whether the host animals of Yulong plague foci carry Yersiniapestis phage,and to identify isolated plague phage.Methods Rodent specimens were collected in 5 villages of Yulong plague foci in spring and autumn of 2016,respectively.Vaccine strain EV76 was used as breeding bacteria.Phage was isolated from the specimens by double-layer plate method and plaque morphology was identified.Results ① Totally 409 samples collected in spring failed in phage isolation.A total of 40 of Yersinia pestis phages were isolated from 444 samples in autumn,and the total isolation rate was 9.01％ (40/444).② The Yersinia pestis phages were isolated in all of 5 villages,and the isolation rate was of no significant difference (x2 =5.055,P ＞ 0.05).③ Of the 40 strains of phage,37 strains were isolated from Apodemus chevrieri,2 strains from Eothenomys Miletus and 1 strain from Crocidura Dracula.④Based on the appearance,the plaque of the phage was divided into three:large (diameter 1.5-2.5 mm),middle (0.5-＜ 1.5 mm) and small (＜ 0.5 mm).Conclusion There is a higher number of plague phage in the host animals of the plague foci in Yulong County of Yunnan Province,the plaques are diverse in morphology,and their biological characteristics may be polymorphic.
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Objective To identify the causes of nonspecific bands in the detection of a industry standard caf1 gene by polymerase chain reaction(PCR),and to propose a solution to this problem. Methods A total of 112 strains were selected for the experiment, including 40 strains of Yersinia pestis, 72 strains of non-Yersinia pestis;DNA was extracted,and caf1 gene was amplified by PCR;seven non-specific strips were recovered,purified and TA cloning and sequencing; the primer of the caf1 gene was redesigned and validated using all of the strains. Results Using the industry standard caf1 gene primer,DNAs of 40 Yersinia pestis and 72 non-Yersinia pestis were amplified by PCR, 58 non-Yersinia pestis could be amplified with non-specific bands, they were about 400, 500, 600, 700, 800, 900, 1 000 bp. By TA cloning and sequencing, the non-specific bands in the downstream of the industry standard caf1 primer and its reverse complement were amplified. Using the new designed caf1 primer to amplify, 72 non-Yersinia pestis strains showed no non-specific bands. Conclusion Non-specific bands has been amplified in the screening of Yersinia pestis using the primer of the industry standard caf1, and the new caf1 primer can effectively avoid this problem and improve the accuracy of detection.