The plague, which is caused by the Gram-negative coccobacillus bacterium Yersinia pestis, has been classified as a reemerging infectious disease by the World Health Organization. The Qinghai-Tibet Plateau natural plague focus is the largest plague focus in China, and Marmota himalayana is the primary host of the plague. Tibetan sheep (Ovis aries) were first identified as naturally infected hosts of Y. pestis based on etiological evidence in 1975, and activities such as slaughtering or skinning Tibetan sheep that have been infected by Y. pestis or died from Y. pestis infection had caused severe human plague in Qinghai. Tibetan sheep are important domestic livestock in the Qinghai-Tibet Plateau. Knowledge regarding the infection rate of Y. pestis in Tibetan sheep is important for understanding the range of infection and improving measures to control plague epidemics in this area. In this study, a serological survey involving 12,710 Tibetan sheep in all 44 counties in Qinghai Province was conducted. The total positive rate of indirect hemagglutination assay for Y. pestis in Tibetan sheep in Qinghai was 0.68% (86/12,710). Serological positivity to the Y. pestis F1 antibody was found in Tibetan sheep in all prefectures, except the Haidong and Haibei prefectures in Qinghai, with the seropositive rate in different counties ranging from 0.33% to 5.2% and the titers in the positive sera ranging from 1:20 to 1:5120. In addition, the seropositive rates in animal plague focus counties were higher than the rates in non-animal plague counties. Such results indicated a widespread infection of Tibetan sheep with Y. pestis in Qinghai, even though only sporadic epidemics of Tibetan sheep plague have been reported in Qinghai.

Objective To compare the coding sequences (CDS) of Yersinia pestis D106004 strain from Yulong County in Yunnan Province and Z176003 strain from Qing-Tibet Plateau in order to find the differences between their genomes and the genetic characteristics. Methods The CDS of Yersinia pestis D106004 strain and Z176003 strain were searched and compared by BLAST. Twenty-two differential CDS were selected to design 22 pairs of primers. PCR amplification was carried out in 119 representative plague strains from different isolation sources (natural foci of Himalayan marmot plague in the Qinghai-Tibet Plateau, natural foci of Apodemus chevrieri and Eothenomys miletus plague in Yunnan), time span of about 50 years, and distribution in six ecological types including Tibet, Qinghai, Sichuan, Gansu and Yunnan, and PCR products were sequenced and verified. The strains were all from the State Key Laboratory for Infectious Disease Prevention and Control, National Institute for Infectious Disease Control and Prevention, Chinese Center for Disease Control and Prevention. Results In 119 representative plague strains of 6 ecological types, the cumulative sequence length of 22 differential CDS PCR amplification products was 2.13 × 106 bp. Among the 119 representative plague strains in the foci of Yulong D106004 strain and Qinghai-Tibet Plateau Z176003 strain, 22 differential CDS had high homology, there was no difference in 78.2％ (2047/2618) sequences of differential CDS, and 21.8％ (571/2618) sequences had three types of gene mutations ( deletion , missense and frameshift mutations). The characteristics of the differences were stable in the 6 ecological plague strains of the foci, and they were divided into 6 geographical distributions. Conclusion Yulong D106004 strain and Qinghai-Tibet Plateau Z176003 strain have high homology, close genetic relationship, and little difference in genome, but the genetic characteristics of different ecotype strains are stable.

OBJECTIVE:This study aims to develop a strategy for distinguishing Yersinia pestis of the Xilingele Plateau ecotype from other biovar of Yersinia pestis using insertion sequences.METHODS:Genome computational analysis was used to discover the positions of insertion sequences unique to Yersinia pestis 91001. PCR was used to confirm that these positions were exclusively present in the Xilingele Plateau ecotype of Yersinia pestis, while it was absent in other Yersinia species.RESULTS:It was validated that the amplicons of IS100-45-1 IS100-51-1, IS285-40 and IS285-41 were exclusive to Yersinia pestis of the Xilingele Plateau ecotype.CONCLUSIONS:Insertion sequences could be reliable candidate biomarkers for Yersinia pestis strains of Xilingele Plateau ecotype. We can differentiate Yersinia pestis strains of Xilingele Plateau ecotype from other Yersinia pestis using simple PCR methods.

Plague, caused by Yersinia pestis, was classified as a reemerging infectious disease by the World Health Organization. The five human pneumonic plague cases in Yulong County in 2005 gave rise to the discovery of a Yulong plague focus in Yunnan province, China. Thereafter, continuous wild rodent plague (sylvatic plague) was identified as the main plague reservoir of this focus. In this study, the epizootics in Yulong focus were described, and three molecular typing methods, including the different region (DFR) analysis, clustered regularly interspaced short palindromic repeats (CRISPRs), and the multiple-locus variable number of tandem repeats (VNTR) analysis (MLVA) (14+12), were used for the molecular typing and source tracing of Y. pestis isolates in the Yulong plague focus. Simultaneously, several isolates from the vicinity of Yunnan were used as controls. The results showed that during the 10-year period from 2006 to 2016, an animal plague epidemic occurred in 6 of those years, and 5 villages underwent an animal plague epidemic within a 30-km2 area of the Yulong plague focus. Searching for dead mice was the most effective monitoring method in this plague focus. No positive sample has been found in 6937 captured live rodents thus far, suggesting that the virulence of strains in the Yulong plague focus is stronger and the survival time of mice is shorter after infection. Strains from Lijiang, Sichuan and Tibet were of the same complex based on a typing analysis of DFR and CRISPR. The genetic relationship of Y. pestis illustrated by MLVA “14+12” demonstrates that Tibet and Sichuan strains evolved from the strains 1.IN2 (Qinghai, 1970 and Tibet, 1976), and Lijiang strains are closer to Batang strains (Batang County in Sichuan province, 2011, Himalaya marmot plague foci) in terms of genetic or phylogenic relationships. In conclusion, we have a deeper understanding of this new plague focus throughout this study, which provides a basis for effective prevention and control.

目的 研究青藏高原喜马拉雅鼠疫疫源地那曲和比如地区鼠疫耶尔森菌(鼠疫菌)菌株的分子分型特征,确定具有该分型特征的鼠疫菌在青藏高原鼠疫疫源地的分布情况.方法 应用板块重排、差异区段分析、间区规律短回文重复、多位点可变数目串联重复序列分析方法对分离自青藏高原地区、四川省、云南省的98株鼠疫菌进行分析.结果 那曲和比如地区的鼠疫菌菌株中第57～60板块的排列方式与测序菌株Z176003一致,在其他实验菌株中未发现该重排特征.结论 分离自那曲和比如的鼠疫菌菌株具有独特的分子分型特征,该特征在本地区的菌株中普遍存在,且菌株多态性持续分化.

The Qinghai-Tibet plateau is a natural plague focus and is the largest such focus in China. In this area, while Marmota himalayana is the primary host, a total of 18 human plague outbreaks associated with Tibetan sheep (78 cases with 47 deaths) have been reported on the Qinghai-Tibet plateau since 1956. All of the index infectious cases had an exposure history of slaughtering or skinning diseased or dead Tibetan sheep. In this study, we sequenced and compared 38 strains of Yersinia pestis isolated from different hosts, including humans, Tibetan sheep, and M. himalayana. Phylogenetic relationships were reconstructed based on genome-wide single-nucleotide polymorphisms identified from our isolates and reference strains. The phylogenetic relationships illustrated in our study, together with the finding that the Tibetan sheep plague clearly lagged behind the M. himalayana plague, and a previous study that identified the Tibetan sheep as a plague reservoir with high susceptibility and moderate sensitivity, indicated that the human plague was transmitted from Tibetan sheep, while the Tibetan sheep plague originated from marmots. Tibetan sheep may encounter this infection by contact with dead rodents or through being bitten by fleas originating from M. himalayana during local epizootics.

The third plague pandemic originated from Yunnan Province, China in the middle of the 19th century. The last human plague epidemic in Yunnan occurred from 1986-2005. On June 6, 2016, a case of human plague was reported in the Xishuangbanna Prefecture, Yunnan. The patient suffered from primary septicemic plague after exposure to a dead house rat (Rattus flavipectus), which has been identified as the main plague reservoir in the local epizootic area. Moreover, a retrospective investigation identified another bubonic plague case in this area. Based on these data, human plague reemerged after a silent period of ten years. In this study, three molecular typing methods, including a clustered regularly interspaced short palindromic repeats (CRISPR) analysis, different region analysis (DFR), and multiple-locus variable number of tandem repeats analysis (MLVA), were used to illustrate the molecular characteristics of Yersinia pestis (Y. pestis) strains isolated in Yunnan. The DFR profiles of the strains isolated in Yunnan in 2016 were the same as the strains that had previously been isolated in this Rattus flavipectus plague focus. The c3 spacer present in the previously isolated strains was absent in the spacer arrays of the Ypc CRISPR loci of the strains isolated in 2016. The MLVA analysis using MLVA (14+12) showed that the strains isolated from the human plague case and host animal plague infection in 2016 in Yunnan displayed different molecular patterns than the strains that had previously been isolated from Yunnan and adjacent provinces.

Background Anthrax is an acute zoonotic infectious disease caused by the bacterium known as Bacillus anthracis. From 26 July to 8 August 2015, an outbreak with 20 suspected cutaneous anthrax cases was reported in Ganquan County, Shaanxi province in China. The genetic source tracking analysis of the anthrax outbreak was performed by molecular epidemiological methods in this study. Methods Three molecular typing methods, namely canonical single nucleotide polymorphisms (canSNP), multiple-locus variable-number tandem repeat analysis (MLVA), and single nucleotide repeat (SNR) analysis, were used to investigate the possible source of transmission and identify the genetic relationship among the strains isolated from human cases and diseased animals during the outbreak. Results Five strains isolated from diseased mules were clustered together with patients’ isolates using canSNP typing and MLVA. The causative B. anthracis lineages in this outbreak belonged to the A.Br.001/002 canSNP subgroup and the MLVA15-31 genotype (the 31 genotype in MLVA15 scheme). Because nine isolates from another four provinces in China were clustered together with outbreak-related strains by the canSNP (A.Br.001/002 subgroup) and MLVA15 method (MLVA15-31 genotype), still another SNR analysis (CL10, CL12, CL33, and CL35) was used to source track the outbreak, and the results suggesting that these patients in the anthrax outbreak were probably infected by the same pathogen clone. Conclusions It was deduced that the anthrax outbreak occurred in Shaanxi province, China in 2015 was a local occurrence.

Objectives To identify coding sequences in the genome of 2 strains of Yersinia pestis from the Qinghai-Tibet Plateau,which is a natural focus of the Himalayan marmot,in order to ascertain the genetic diversity of Y.pestis.Methods The software BLAST,the Perl programming language,and the software Excel were used to compare coding sequences and statistically analyze the Z176003 strain of Y.pestis from Tibet,the D106004 strain from Yulong,Yunnan,and a "default coding sequence database." Results Preliminary comparison of the Z176003 strain of Y.pestis from Tibet and the D106004 strain from Yulong,Yunnan yielded 418 different coding sequences.The "default coding sequence database" had 135 different coding sequences.Eighty six coding sequences were ultimately determined to differ between the 2 strains and the database of the Y.pestis genome.Conclusion Coding sequences from the 2 strains of Y.pestis from the Qinghai-Tibet Plateau differed.Coding sequences that truly differed were groups of different coding sequences of Y.pestis.This finding should provide reliable information for further study of the genetic characteristics of Y.pestis from the Qinghai-Tibet plateau.

The SGTs of SNR (CL10, CL12, CL33, CL35) in MLVA15-31 strains. (XLS 28 kb)

目的 了解新疆维吾尔自治区(新疆)鼠疫耶尔森菌(鼠疫菌)规律聚集的间隔短回文重复(CRISPR)位点的遗传多态性,对菌株进行基因分型.方法 利用PCR技术对分离自新疆的58株鼠疫菌的基因组DNA进行扩增,测定扩增产物的核酸序列,分析各菌株CRISPR位点的间区序列种类和排列情况,应用Bionumerics软件制作聚类图,确定菌株基因型.结果 共鉴定42个间区序列和23种间区序列阵列,58株鼠疫菌分为16个基因型,即基因3型、8型、11型和16型,主要分布在新疆乌恰县、和田地区、尼勒克和玛纳斯县;基因7型的鼠疫菌全部分离自新疆准噶尔盆地大沙鼠及其体表寄生蚤;其他型别的鼠疫菌则主要分布在新疆北天山和准噶尔盆地.结论 新疆地区的鼠疫菌CRISPR位点遗传多态性较高,不同基因型的鼠疫菌地理分布有明显的区域性特征.

In 2005, five human cases of primary pneumonic plague were reported in Yulong County in Yunnan province of China and resulted in two deaths. Several Yersinia pestis strains (designated Yulong strains) have been isolated from rats and fleas in this area since 2006, and a new plague focus in Yulong County has been confirmed. However, the source of these Yulong strains and their relationship with strains from nearby natural foci remain unclear. In this study we compared Yulong strains with previously isolated strains from Yunnan province by biochemical analysis, clustered regularly interspaced short palindromic repeats (CRISPR) and pulsed-field gel electrophoresis (PFGE) methods which revealed significant differences between the Yulong strains and early Y. pestis strains. However, the Yulong strains were found to be similar to Himalayan marmot strains. Therefore, Yulong strains are a new type of Y. pestis in Yunnan province and likely represent the first step in the transmission of Yersinia pestis from the Qinghai-Tibet Plateau to Yunnan province. The Yulong focus merits greater attention because of its special status of Yunnan plague epidemiology.

Anthrax is a continuous threat in China, especially in rural regions. In July 2015, an anthrax outbreak occurred in Xifeng County, Liaoning Province. A total of 10 cutaneous anthrax cases were reported, with 210 people under medical observation. In this study, the general characteristics of human anthrax outbreak occurred in Liaoning Province were described, and all cases were caused by butchering and contacting sick animal. Meanwhile, the phylogenetic relationship between outbreak-related isolates/samples of the year 2015 and previous Bacillus anthracis strains was analyzed by means of canonical single nucleotide polymorphisms (canSNP), multiple-locus variable-number tandem repeat analysis (MLVA) with 15 markers and single-nucleotide repeats (SNR) analysis. There are two canSNP subgroups found in Liaoning, A. Br. 001/002 and A. Br. Ames, and a total of six MLVA 15 genotypes and five SNR genotypes were observed. The strain collected from anthrax outbreak in Xifeng County in 2015 was classified as A. Br. 001/002 subgroup and identified as MLVA15-29 genotype, with same SNR profile (CL10: 17, CL12: 15, CL33: 29, and CL35: 13). So we conclude that the same clone of B. anthracis caused the anthrax outbreak in Xifeng County in 2015, and this clone is different to previous isolates. Strengthening public health education in China is one of the most important measures to prevent and control anthrax.

OBJECTIVE:To establish a gene identification method of Yersinia pestis and Yersinia pseudotuberculosis for plague surveillance.METHODS:According to the specific genomic sequences of Y. pestis and Y. pseudotuberculosis, i.e. "pestis Island (PeI)" and "pseudotuberculosis Island (PsI)" and the published genomic sequences of 12 strains of Y. pestis and 4 strains of Y. pseudotuberculosis, the specific identification primers of these sequences were designed.RESULTS:A total of 52 strains of Y. pestis and 57 strains of Y. pseudotuberculosis and other intestinal bacteria strains were tested with PCR. Of the 5 pairs of Y. pestis identification primers, PeI2 and PeI11 were specific for Y. pestis. Besides Y. pestis, the primers PeI1, PeI3 and PeI12 could detect part of 57 Y. pseudotuberculosis strains. Of the 5 pairs of Y. pseudotuberculosis identification primers, PsI1 could detect all the 52 strains of Y. pestis and 57 strains of Y. pseudotuberculosis. PsI7, PsI16, PsI18 and PsI19 were specific for Y. pseudotuberculosis.CONCLUSION:The primers PsI1, PeI 2 and PeI11, PsI7, PsI16, PsI18 and PsI19 can be used in the rapid identification of Y. pestis and Y. pseudotuberculosis, which can be also used to explore the circulation of atypical Y. pestis in quiescent plague foci.

Objective To study the differences of chromosomal structure among Yersinia pestis strains isolated from China,and to investigate the reasons of chromosomal rearrangement events occurred in Yersinia pestis as well as the possibility of strain identification and phylogenetic analysis based on the chromosomal rearrangement features.Methods According to the genome sequence data downloaded from web of National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/genome),alignment of all the coding sequences (CDSs) among five strains(American strain CO92 as reference and other four completely sequenced strains from Inner Mongolia,Jianchuan of Yunnan,Yulong of Yunnan,Naqu of Tibet in China named 91001,D182038,D106004 and Z176003 as comparison strains) was performed,and then the chromosome of Yersinia pestis was divided into several large DNA segments (named chromosomal plate in the text) according to the similarity of CDSs.Plate arrangement patterns in each strain' s chromosome and gene content of breakpoint regions were determined.Finally,genetic relationships among Yersinia pestis strains were analyzed on the basis of rearrangement diversity from paired-comparison.Results Yersinia pestis chromosomes of strains CO92,D182038,D106004,91001 were composed of 44 relatively independent plates,except strain Z176003.Gene order was very stable within each plate,while it was movable between the plates.Comparing with the reference strain CO92,13 rearrangement events occurred in the chromosomes of both strain D182038 and strain D106004,and 14 rearrangement events involved in Z176003,while 37 rearrangement events occurred in 91001.Paired-comparison data showed that only 8 plates order differences were existed between D106004 and Z176003.Forty-three breakpoint regions were identified on the chromosome of strain CO92,and 39 of them contained insertion sequences,and 25 of them were IS100.Conclusions Yersinia pestis genome represents a high degree of genetic flux,and chromosomal structures of strains are significantly different from each other.Chromosomal rearrangement events is closely related to the large number of insertion sequences in the Yersinia pestis chromosome.Rearrangement diversity among Yersinia pestis strains could reflect their genetic relationships.

目的 建立一种快速区分鉴别田鼠型与非田鼠型鼠疫耶尔森菌(鼠疫菌)的分子分型方法.方法 设计特异引物对93株不同来源、不同生物型鼠疫菌的天冬氨酸酶基因(aspA)进行PCR扩增,用限制性内切酶Hpy CH4Ⅳ对扩增产物进行限制性片段长度多态性(RFLP)分析;直接测序法检测aspA基因多态性.结果 aspA基因扩增产物经Hpy CH4Ⅳ酶切后呈现2种基因型:田鼠型鼠疫菌(38株)均为101、126 bp 2个条带;非田鼠型鼠疫菌(55株)均为227 bp单一条带.测序结果证实酶切识别位点发生突变导致带型差异.结论 初步建立了鼠疫菌PCR-RFLP(聚合酶链反应-限制性片段长度多态性)法aspA基因分型方案,通过Hpy CH4Ⅳ酶切带型差异区分高毒力的非田鼠型菌株与低毒力的田鼠型菌株,适用于疫源地流行病学调查.

We analyzed 10 isolates of Francisella tularensis subspecies holarctica from China and assigned them to known clades by using canonical single-nucleotide polymorphisms. We found 4 diverse subtypes, including 3 from the most basal lineage, biovar japonica. This result indicates unprecedented levels of diversity from a single region and suggests new models for emergence.

The Yersinia pestis chromosome contains a large variety and number of insert sequences that have resulted in frequent chromosome rearrangement events. To identify the chromosomal rearrangement features of Y. pestis strains from five typical plague foci in China and study spontaneous DNA rearrangements potentially stabilized in certain lineages of Y. pestis genomes, we examined the linking mode of locally collinear blocks (LCBs) in 30 Y. pestis strains by a polymerase chain reaction-based method. Our results suggest most strains have relatively stable chromosomal arrangement patterns, and these rearrangement characteristics also have a very close relationship with the geographical origin. In addition, some LCB linking modes are only present in specific strains. We conclude Y. pestis chromosome rearrangement patterns may reflect the genetic features of specific geographical areas and can be applied to distinguish Y. pestis isolates; furthermore, most of the rearrangement events are stable in certain lineages of Y. pestis genomes.

Yersinia pestis is the causative agent of bubonic and pneumonic plagues.Strains of Y.pestis are classified into four biovars:antiqua,mediaevalis,orientalis,and microtus[1].There are two microtus-related plague foci in China:the Microtus brandti plague focus in the Xilin Gol Grassland(focus L)and the Microtus fuscus plague focus in the Qinghai-Tibet Plateau(focus M).Microtus strains are avirulent to humans,and experiments have shown that strains isolated from Microtus brandti in Inner Mongolia are virulent to