Bacterial Typing Techniques ; Genotype ; Minisatellite Repeats ; Plague ; Yersinia pestis ; genetics

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.

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 type Yersinia pestis isolated from Gansu Province,and to study the trend of diffreent strains in different administrative regions and different years.Methods Totally 193 strains were enrolled in this study,including 9 strains of Ganning Dauricus type,18 strains of Aerjin type,45 strains of Qilian type and 121 strains of Qingzang type.These strains were genotyped by clustered regularly interspaced short palindromic repeats (CRISPR),and genotypes were named according to international standard.Genotyping by CRISPR in different administrative regions and different years of Gansu Province was explored.Results Two clusters (Ca7 and Cb4),including four genotypes (genotypes 7,22,24 and 26) were classified by CRISPR.From the point of view of origin,genotype 24 was the main genotype in Akesai 36.36％ (16/44),Subei 36.17％ (17/47),Yumen 50.00％ (5/10) and Su'nan 38.67％ (29/75);the main genotype of Xiahe and Huining was genotype 26 (4/7);the main genotype of Shandan was genotype 22 (1/1).From the point of view of time,the main genotype of Yersiniapestis in Gansu Province during the years of 1960-1969,1970-1979 and 1980-1989 was genotype 26 [53.33％ (8/15),60.00％ (6/10) and 48.28％ (14/29)];the main genotype was genotype 22 [40.91％ (18/44)] during the years of 1990-1999;and the main genotype was genotype 24 [43.16％ (41/95)] during the years of 2000-2009.Conclusion Four genotypes of Yersiniapestis in Gansu Province are quite different in different administrative regions and different years.

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 infection status of Yersinia in the main host animals of plague in Xiahe and Luqu counties, the Himalayan marmot plague foci of Gansu Province, and to provide a basis for exploring the epidemic status of plague in these foci. Methods:Samples of the ileocecal region and contents, pharyngeal swabs (or tongue roots), and blood of the main host animals of plague in Xiahe County and Luqu County where the plague were active in the 1950s and 1960s were collected from 2014 to 2018. The Yersinia isolation, virulence determination and F1 antibody detection were performed, respectively. Results:Totally 24 strains of Yersinia were detected in 958 samples of ileocecal region and contents with a bacterial detection rate of 2.51%, which were 13 strains of Yersinia enterocolitia (Y.e), 1 strain of Yersinia kristensenii (Y.k), 2 strains of Yersinia frederiksenii/ intermedia (Y.f/i), 6 strains of Yersinia intermedia (Y.i), 1 strain of Yersinia aldouae (Y.a) and 1 strain of Yersinia massiliensis (Y.m). Totally 19 strains of Yersinia were detected in 958 samples of pharyngeal swabs (or tongue roots), and the detection rate was 1.98%, which were 8 strains of Y.e, 1 strain of Yersinia pseudotuberculosis (Y.p), 4 strains of Y.k, 1 strain of Y.f/i, 4 strains of Y.i, and 1 strain of Yersinia ruckeri (Y.r). The virulence types of 21 strains of Y.e were ail -ystA -ystB +yadA -virF -rfbc -, ail -ystA -ystB -yadA -virF -rfbc -, respectively, accounting for 9.52% (2/21) and 90.48% (19/21), none were pathogenic. The results of F1 antibody in 1 079 serum samples were all negative. Conclusions:Yersinia are widely found in the pharynx and intestines of the main host animals of plague in Xiahe and Luqu counties, and the Y.e detected are all non-pathogenic strains. The results of this investigation can provide clues for further study on the preservation of Yersinia pestis in host animals and their living environment.

Objective To apply the DNA barcoding technology for identification of rodent animals and to establish a rodent animal DNA barcode database in Gansu Province.Methods A total of 54 rodent animals were detected.DNA barcoding technology was used to analyze the DNA mitochondrial cytochrome C oxidase subunit Ⅰ (CO Ⅰ) gene sequence in Gansu Province.Results The intra-specific genetic distance was 0-2％ while the interspecific distance ranged from 18％ to 30％.Eight major clusters were apparently showed on a Neighbor joining tree.Conclusion DNA barcoding technology could overcome the shortcomings of the morphological identification,so it could be used to identify the rodent animals and has important implications for disease control and prevention in the natural focus of Gansu Province.

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.

Plague natural foci in Gansu Province are large in area and widely distributed, and the epidemic status is different in each focus. Some plague natural foci have been resting for many years and are under stable control. In other plague natural foci, the epidemic of plague among animals is relatively active, and dozens of strains of Yersinia pestis can be isolated every year. The plague among animals spreads to the humans from time to time, and the cases of human plague are more severe, short in course of disease, and high in mortality. In recent years, with the sharp increase of migrant workers engaged in grazing and engineering construction in plague natural foci, opportunities to actively contact infected animals such as marmots, hares and sheepdogs have increased, resulting in an increase of human plague cases. In addition, the behaviors of illegal hunting, trafficking and sale of marmots cannot be stopped, resulting in human plague throughout the year. Therefore, the party committees and governments at all levels and the health administrative departments in Gansu Province have formulated the plague prevention and control measures suitable for this region according to the epidemic characteristics of plague among animals and humans. In this paper, the characteristics of plague epidemic situation at present and the prevention and control measures adopted in Gansu Province are discussed, in order to provide scientific basis and guidance for optimizing plague prevention and control measures in the future.