We studied Yersinia infection and carriage in shepherd dog on the natural foci of Marmota himalayana plague and investigated the substitution or antagonism relationship among three pathogenic Yersinia in nature,providing a novel approach for prevention and control of plague.Blood sample and rectal swab specimens from shepherd dogs were collected.Rectal swab specimens were detected and isolated for Y.enterocolitican and Y.pseudotuberculosis.IHA was used to measure the positive rate of F1 antibody.Results showed that among 88,94,70 and 64 serum specimens respectively from Akesai,Subei,Sunan,and Tianzhu,IHA positive rates were 31.82％,32.98％,2.86％ and 1.56％ separately.A total of 236 anal swab specimens of shepherd dog were collected,among which one Y.kristensenii strain and 2 non-pathogenic Y.enterocolitica strain were recovered from Akesai and Subei respectively,where plague was violently prevalent in animals.All the results indicate that shepherd dogs in natural foci of M.himalayana plague where plague in animals are prevalent with non-pathogenic Y.enterocolitica,is an evidence of antagonism relationship in three pathogenic Yersinia.

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Abstract: Objective To conduct a molecular epidemiological tracing and analysis of Yersinia pestis strains isolated from two human plague outbreaks with unknown sources in Gansu Province, China. The results of this analysis would provide a basis for isolating and controlling the sources of Yersinia pestis. Methods The strains of Yersinia pestis isolated from two human plague outbreaks occurring on December 12, 2017, and September 27, 2019 were genotyped by the different region (DFR) and the clustered regularly interspaced short palindromic repeats (CRISPR). The repeat numbers of the variable number tandem repeat (VNTR) loci in the tested strains of Yersinia pestis were calculated by the multiple variable number tandem repeats analysis (MLVA), and the location of the phylogenetic tree of the tested strains was determined with the method of minimum spanning tree (MST) by the software BioNumerics 6.6. Results The strain of 20171212 lacked DFR01, DFR02, DFR03, DFR04, DFR13, DFR23, and the DFR type was identified as type 8. The space sequence of YPa was a1'-a2-a3-a4-a5-a6-a7-a35, the space sequence of YPb was b1-b2-b3-b4, the space sequence of YPc was c1-c2-c3, the gene cluster of CRISPR was Ca35', the genotype of CRISPR was 26'. MLVA clustering analysis showed that the strain clustered within in the cluster of Yuerhong pasture in Subei County and formed an independent branch. On the other hand, the strain of 20190927 lacked DFR01, DFR13 and DFR23, with the DFR type identified as type 1b. The space sequence of YPa was a1-a2-a3-a4-a5-a6-a7, the space sequence of YPb was b1-b2-b3-b4, the space sequence of YPc was c1-c2-c3, the gene cluster of CRSIPR was Ca7, the genotype of CRSIPR was 22 MLVA clustering analysis showed that the strain was located close to the cluster of Dangjinshan in Akesai County, and relatively distant from the cluster of Yuerhong pasture in Subei County. Conclusions The genotypes of strain 20171212 by DFR and CRISPR were consistent with the main genotypes of Y. pestis from Himalayana Marmota foci in Subei County, which confirmed that the human plague cases were naturally occurring locally. However, the strain gathered the cluster of Yuerhong pasture in Subei County, which indicated that the source of infection was not in Yanchiwan Town, but in the surrounding area of the Yuerhong pasture. The genotypes of strain 20190927 by DFR and CRISPR were in accordance with the main genotype of Y. pestis from Himalayana Marmota foci in Akesai County and were closer to the cluster of Dangjinshan in Aksai County than to

Objective:To understand the plague epidemic characteristics in the natural foci of the Qilian Mountains-A-erh-chin Mountains Himalayan marmot plague in Gansu Province, and to provide scientific basis for innovative prevention and control of the plague in combination with local conditions. Methods:A retrospective study was used to collect the monitoring data of the natural foci of plague in Gansu Province from 2011 to 2018 (from the epidemic surveillance files of the Center for Disease Control and Prevention of Gansu Province and direct network reporting information). Descriptive epidemiological method was used to analyze the plague epidemic characteristics of natural foci of the Qilian Mountains-A-erh-chin Mountains Himalayan marmot plague in Gansu Province from 2011 to 2018, including the distribution of host animals, pathogenic and serological testing of the plague bacteria, and the epidemic characteristics of human plague. Results:From 2011 to 2018, the total average marmot density in the natural foci of the Qilian Mountains-A-erh-chin Mountains Himalayan marmot plague in Gansu Province was 0.21/hm 2, of which Tianzhu County had the highest average marmot density of 0.58/hm 2, and Jiayuguan City had the lowest average marmot density of 0.01/hm 2. A total of 381 strains of Yersinia pestis were isolated in the foci, of which 4 were isolated from human corpses, 298 were host animals, and 79 were infectious vectors. Among them, the top 3 counties (cities) of isolated strains were Aksai County (38.85%, 148 strains), Subei County (31.50%, 120 strains) and Yumen City (16.27%, 62 strains). A total of 6 860 marmot serum, 1 769 dog serum were tested, the F1 antibody positive rates were 2.70% (185/6 860), 8.42% (149/1 769); and the F1 antigen positive rate of 814 animal materials was 4.30% (35/814), respectively. There were 4 times of human plague, 4 cases occurred and 4 cases died; 3 times occurred in Subei County and 1 time in Yumen City. The onset months were July, September, November and December. Active contact with infected animals such as shepherd dogs was the main route of infection, and migrant herders were the key occupation population. Conclusions:The animal epidemic situation in the natural foci of the Qilian Mountains-A-erh-chin Mountains Himalayan marmot plague in Gansu Province is active, and the plague presents different epidemic states in different regions. The prevention and control measures should be taken according to local conditions and guided by classification to strictly prevent the occurrence and transmission of the plague.

Objective: To investigate the effects and related mechanism of bivalirudin on the survival of random skin flap on the back of rat. Methods: Thirty SD rats were divided into bivalirudin group and normal saline group according to the random number table, with 15 rats in each group. The random flap model with size of 9 cm×3 cm was reproduced on the back of rats in two groups. Immediately post injury, rats in bivalirudin group were intraperitoneally injected with 5 mg/mL bivalirudin (0.8 mL/kg), while rats in normal saline group were intraperitoneally injected with normal saline (0.8 mL/kg) once a day. The continuous injection lasted for 7 days. The flap was divided into distal area, middle area and proximal area averagely based on the flap blood supply. On post injury day (PID) 1, 3, and 7, the overall survival of each area of flap was observed with naked eyes. On PID 7, the survival rate of flap was calculated, and then the morphology of skin tissue at the center of the three areas of flap was observed by HE staining, the microvessel density (MVD) of the middle area of flap was calculated, and the expression of vascular endothelial growth factor (VEGF) of the middle area of flap was detected with immunohistochemical staining. Data were processed with t test. Results: (1) On PID 1, flaps of rats in two groups had different degrees of swelling, mainly concentrated in distal area, but there was no obvious necrosis. The middle area and proximal area of flaps in two groups were survived. On PID 3, the necrosis of flaps of rats in two groups was concentrated in the middle area, while the proximal area of flap was still in survival state, and most distal area of flap was necrosis with a little scab. On PID 7, the necrosis of middle area of flaps of rats in two groups was gradually fused, and the survival area of flap of rats in bivalirudin group was larger than that in normal saline group. The distal area of flap was almost necrotic, and the proximal area of flap was almost survived. (2) On PID 7, the survival rate of flap of rats in bivalirudin group was (64±4)%, significantly higher than that in normal saline group [(45±3)%, t=13.49, P<0.01]. (3) On PID 7, the histological morphology of distal area of flap of rats in two groups was similar, the inflammatory cells were infiltrated abundantly, and tissue edema was obvious. A large number of new blood vessels appeared in the middle area of flap of rats in bivalirudin group, with the formation of collateral vessels, and basic dilation of new blood vessels was seen. There were fewer new blood vessels appeared in the middle area of flap of rats in normal saline group, and dilation of new blood vessels was not obvious. There was little inflammatory cells infiltration in the proximal area of flap of rats in two groups. Compared with that in normal saline group, tissue edema extent of proximal area of flap of rats in bivalirudin group was less, and expansion was observed in more blood vessels. (4) The MVD of middle area of flap of rats in bivalirudin group was (26±5)/mm^2, significantly higher than that in normal saline group [(18±3)/mm^2, t=5.43, P<0.05]. (5) The expression of VEGF of middle area of flap of rats in bivalirudin group was 6 534±384, significantly higher than that in normal saline group (4 659±448, t=12.31, P<0.05). Conclusions Bivalirudin can promote the survival of random skin flap in rats, and the mechanisms may include reducing the formation of thrombosis, improving the blood supply of flap, and increasing the expression of VEGF, promoting the formation of new blood vessels.

Objective To summarize and analyze the epidemic situation and trend of animal plague in plague foci of Gansu section of Golmud-Dunhuang railway,and provide basic reference for prevention and control of the plague.Methods The quantity of Marmot,rate of carrying fleas and flea index,plague serological and etiology detection results,the cross analyzing results of the railway section and the animal plague foci in Gansu section of Dolmud-Dunhuang railway (Akesai and Subei counties) from 2010 to 2015 were analyzed via the retrospective analysis method.Results The total average density of Marmot of Golmud-Dunhuang railway crossing area Akesai and Subei counties was between 0.23-0.41 per hectare between 2010 and 2015.The rate of parasitic dye fleas in plague foci of Akesai was between 11.30％ and 32.65％,body flea index was 0.74-1.84.The rate of parasitic fleas dye fleas in plague foci of Subei was between 5.34％ and 14.07％,the flea index was 0.25-2.04.The etiology detection Marmot plague of Akesai and Subei was 2 049,142 of them were positive,the positive rate was 6.93％;592 groups of vector were detected,44 of them were positive;the positive rate was 7.43％.There was no statistically significant difference between inspection bacteria rate and parasitic vector inspection rate of Marmot (x2 =0.014,P ＞ 0.05).Totally 1 719 serums of Marmot were tested,132 of them were positive,the positive rate was 7.68％.Totally 421 serums of Canis lupus familiaris were detected,61 of them were positive,the positive rate was 14.49％.There was statistically significant difference between the serum positive rate of Marmot and Canis lupus familiaris (x2 =19.116,P ＜ 0.05).Totally 17 animal foci were found along the railway in 2013,the nearest distance of the foci of bid from construction was 0.5 km,popular area was 61.02 km2,and 1 360 people were directly threatened.Conclusions Animal plague in Akesai and Subei regions along Golmud-Dunhuang railway is highly active.So we must take effective measures to guarantee the smooth progress of railway construction.

Objective To analyze the relationship between meteorological variables and the prevalence of marmot plague in the Himalayan marmot foci of Subei County,Gansu Province.Methods The surveillance data (from Gansu Provincial Center for Disease Control and Prevention) on human plague,the meteorological factors (average rainfall,temperature,relative humidity) data (from China Meteorological Data Sharing Service Network) of the current and preceding (relative to current year as a reference) first,second and third years and meteorological factors data of the current and preceding first,second and third months in Subei of Himalayan marmot foci from 1973 to 2014 were collected.The association of positive rate of plague bacteriological detection and meteorological factors was evaluated by using SAS 9.3 software.The influence of meteorological factors on the epidemic of marmot plague was analyzed by the logistic regression analysis.Results From 1973 to 2014,6 human plague cases with 5 dead cases occurred in Subei County.The positive rate of plague bacteriological detection was positively correlated with the average relative humidity of current year and the average temperature of preceding first year (r =0.366,0.385,P ＜ 0.05),and it was negatively correlated with the average relative humidity of preceding third year (r =-0.304,P ＜ 0.05);the positive rate of plague bacteriological detection was positively correlated with the average rainfall of preceding first month (r =0.212,P ＜ 0.05),and it was negatively correlated with the average relative humidity of preceding second and third months (r =-0.196,-0.201,P ＜ 0.05).The risk factor of animal plague epidemic in marmot foci in Subei County was average temperature of current month [odds ratio (OR) =1.304,95％ confidence intervals (CI):1.195-1.422],and the protective factor was average relative humidity of preceding third month (OR =0.960,95％CI:0.932-0.988).Conclusions The prevalence of marmot plague is closely associated with meteorological factors in the natural plague foci of Subei County of Gansu Province.The temperature not only plays a role in promoting the prevalence of plague,but also has a lagging effect.Therefore,the meteorological factors can be considered as a reference of the plague prevalence in Himalayan marmot plague foci.

Objective: To investigate the correlation of meteorological factors with the numbers of marmots and parasitic fleas in the plague foci of Himalayan marmot in Subei County and Sunan County of Gansu Province. Methods: The surveillance data(from Gansu Provincial Center for Disease Control and Prevention) of natural plague foci of Subei, Sunan of Himalayan marmot from 1982 to 2016 and the meteorological factors data (from China Meteorological Data Sharing Service Network) such as annual average temperature, rainfall and relative humidity during the same period were collected. The correlation between marmot density, body flea index and meteorological factors were analyzed by SAS 9.3 software. Based on the meteorological data as an independent variable in current year and previous 1, 2, and 3 years (relative to previous 1, 2, and 3 years as the reference), the marmot density as the dependent variable, the time series semiparametric generalized additive model (GAM) was established to quantitatively study the nonlinear effect of meteorological factors on the marmot density by R2.3.2 software. Results: From 1982 to 2016, the marmot density in Subei County of Gansu Province was 0.29-6.63 per hectare, and the body flea index range was 0.04-4.56; the marmot density in Sunan County of Gansu Province was 1.22-7.92 per hectare, and the body flea index range was 0.06-0.42. In Subei County, the marmot density was correlated with the previous 1 year rainfall and relative humidity, respectively (r= 0.43, 0.36, P < 0.05), and negatively correlated with the average temperature of the year and the previous 1, 3 years (r=-0.58,-0.55,-0.41, P < 0.05). In Sunan County, the marmot density was correlated with the relative humidity of the year and the previous 1 year rainfall and relative humidity, respectively (r= 0.36, 0.57, 0.43, P < 0.05). In Subei County, the body flea index was negatively correlated with the average temperature of the year and the previous 1, 2, 3 years, respectively(r=-0.57,-0.43,-0.31,-0.37, P < 0.05). In Sunan County, there was a negative correlation between the body flea index and the average temperature of the year and the previous 1, 2, 3 years (r=-0.71,-0.46,-0.38,-0.40, P < 0.05). After controlling other influencing factors, the results of the GAM showed that for every 1 mm increase in rainfall of the year in Subei County, the marmot density increased 0.93%; for every 1% increase in relative humidity of the previous 3 year in Subei County, the marmot density increased 25.34%; for every 1 mm increase in rainfall of the previous 3 year in Sunan County, the marmot density increased 1.69%. Conclusions The numbers of marmots in the plague foci of Himalayan marmot in Subei County and Sunan County of Gansu Province are closely related to rainfall and relative humidity, and the influence has different hysteresis effects. Surface temperature is a important factor affecting the population of parasitic fleas.

Objective To explore the effects of meteorological factors on population densities of the host and the prevalence of animal plague in the natural plague foci of Spermophilus alaschanicus.Methods The surveillance data of seroprevalence rate in natural plague foci of Spermophilus alaschanicus of Gansu Province in current year,one previous year (relative to current year as a reference),as well as two and three previous years’ meteorological data such as average annual rainfall,temperature,relative humidity were collected from 1962 to 2014.SAS 9.3 software was used for correlation and regression analysis of the influencing meteorological factors on the density of Spermophilus alaschanicus and prevalence of animal plague;generalized additive model (GAM) was used for quantitative study of meteorological factors on density of Spermophilus alaschanicus;logistic regression analysis was used to study the effect of meteorological factors on prevalence of Spermophilus alaschanicus plague.Results Serum positive rate of Spermophilus alaschanicus was positively correlated with average annual rainfall of current and previous one year,and with relative humidity of current year (r =0.279,0.341,0.654,P ＜ 0.05);serum positive rate was negatively correlated with average annual temperature of current and preceding first,second and third years (r =-0.360,-0.339,-0.273,-0.323,P ＜ 0.05).The average annual rainfall of preceding second and third years was significantly associated with the densities of Spermophilus alaschanicus by GAM parameter estimation (t =2.88,2.42,P ＜ 0.05).The average annual rainfall of preceding second and third years was significantly associated with the densities of Spermophilus alaschanicus by GAM nonparameter estimation (x2=31.580,13.428,P ＜ 0.05),but the effect was nonlinear.Multivariate logistic regression analysis showed that average annual rainfall of preceding first year [odds ratio (OR) =2.475,95％ confidence interval (C/):1.175-5.213] was risk factor for the prevalence of the animal plague in the natural plague foci of Spermophilus alaschanicus.Conclusions Rainfall is risk factor for the prevalence of the animal plague in the natural plague foci of Spermophilus alaschanicus.There is a lag effect of rainfall on the numbers of hosts and the prevalence of Spermophilus alaschanicue plague.When the rainfall in the region (not continuously) reaches a certain amount,it will provide favorable conditions for the spread of Yersinia pestis.

Objective To investigate the relationship between fleas density of Spermophilus alaschanicue and host density as well as meteorological factors.Methods The densities of fleas of Spermophilus alaschanicue and meteorological temperature,precipitation,relative humidity data in the same period from 1977 to 2014 in plague natural foci of Spermophilus alaschanicue of Gansu Province were collected.In order to further establish the multiple linear regression and the stepwise regression analysis model,SAS 9.3 software was used to explore the linear correlation analysis between fleas index number,density of Spermophilus alaschanicue and meteorological factors.Results The average body flea index positively correlated with the density of Spermophilus alaschanicue (r =0.34,P ＜ 0.05),in addition,hole fleas index was in negative correlation with the average temperature of the current and preceding first,second and third years (r =-0.44,-0.39,-0.45,-0.54,P ＜ 0.01).However,hole fleas index positively correlated with the average rainfall of preceding first year (r =0.38,P ＜ 0.05).More specifically,the monthly body flea index was negatively correlated with the average rainfall and the average temperature of current,preceding first,second,and third months,and the average relative humidity of current month (r =-0.23,-0.34,-0.43,-0.29,-0.25,-0.33,-0.36,-0.38,-0.26,P ＜ 0.01).After multiple linear regression analysis,the following equations were obtained:body fleas index =3.983 1 + 0.695 1 × Sperrnophilus density-0.004 9 × average annual rainfall;hole fleas index =10.780 4 + 0.009 1 × average annual rainfall-0.216 2 × annual average relative humidity.Conclusions The relationship between fleas density of Spermophilus alaschanicue and the environment is complex,they have mutual connection as well as mutual restriction.Except Spermophilus density affects the number of fleas,the two most important meteorological factors which affect the flea body index are temperature and rainfall.In addition,the relationship between meteorological factors and flea body index shows somewhat lagging behind for two to three months.