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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.

Background Plague, caused by the bacterium Yersinia pestis, is a highly infectious, zoonotic disease. Hundreds of human plague cases are reported across the world annually. Qinghai Plateau is one of the most severely affected plague regions in China, with more than 240 fatal cases of Y. pestis in the last 60 years. Conventional epidemiologic analysis has effectively guided the prevention and control of local plague transmission; however, molecular genetic analysis is more effective for investigating population diversity and transmission. In this report, we employed different genetic markers to analyze the population structure of Y. pestis in Qinghai Plateau. Methodology/Principal finding We employed a two-step hierarchical strategy to analyze the phylogeny of 102 Qinghai Plateau isolates of Y. pestis, collected between 1954 and 2011. First, we defined the genealogy of Y. pestis by constructed minimum spanning tree based on 25 key SNPs. Seven groups were identified, with group 1.IN2 being identified as the dominant population. Second, two methods, MLVA and CRISPR, were applied to examine the phylogenetic detail of group 1.IN2, which was further divided into three subgroups. Subgroups of 1.IN2 revealed a clear geographic cluster, possibly associated with interaction between bacteriophage and Y. pestis. More recently, Y. pestis populations appear to have shifted from the east toward the center and west of Qinghai Plateau. This shift could be related to destruction of the local niche of the original plague focus through human activities. Additionally, we found that the abundance and relative proportion of 1.IN2 subgroups varied by decade and might be responsible for the fluctuations of plague epidemics in Qinghai Plateau. Conclusion/Significance Molecular genotyping methods provided us with detailed information on population diversity and the spatial-temporal distribution of dominant populations of Y. pestis, which will facilitate future surveillance, prevention, and control of plague in Qinghai Plateau., Author summary Plague is a highly infectious disease caused by the Yersinia pestis bacterium. Since the first strain of Y. pestis was isolated in Qinghai in 1954, confirmed plague cases have occurred nearly every year, and more than 240 people have died from plague over the past 60 years. In this study, we analyzed 102 Y. pestis strains collected from Qinghai Plateau between 1954 and 2011. We determined their genetic diversity and inferred their spatial-temporal distribution, based on genetic markers including SNPs, VNTRs and CRISPRs. Our results indicate that 1.IN2 is the dominant group of Y. pestis in Qinghai Plateau, and its three subpopulations revealed clear geographic clustering that might be driven by interaction with bacteriophages. We observed that the Y. pestis population has moved from the east of Qinghai Plateau to central and western regions over the past 60 years. We also found that the abundance and relative proportion of 1.IN2 subgroups varied over time, leading to fluctuations in plague epidemics. These results extend our knowledge of the genetic diversity of Y. pestis, and its population dynamics in natural plague foci over a number of years. With ongoing risk of outbreaks, we recommend enhanced surveillance in this region.

To analyze the plasmid features and geographical distribution characteristics of Yersinia pestis of different plague foci in China.A total of 2 213 Yersinia pestis strains were colected from 11 Chinese plague foci separated during 1943 to 2012, and plasmid DNA according to alkali cracking method, and measured the relative molecular mass (Mr) of plasmid DNA based on the standard plasmid contrast method, then analyzed the plasmid profiles by agar gel electrophoresis.A total of 2 213 strains had 16 kinds of plasmids with different Mr, including 4×10(6), 6×10(6), 7×10(6), 13×10(6), 16×10(6), 20×10(6), 22×10(6), 23×10(6), 27×10(6), 30×10(6), 36×10(6), 45×10(6), 52×10(6), 65×10(6), 72×10(6) and 90×10(6). Plasmid were classified into 26 kinds of plasmid profiles. A total of 2 213 Yersinia pestis strains contained 4 large plasmids, 52×10(6), 65×10(6), 72×10(6) and 90×10(6), whose ratio was 22.10% (589/2 213), 75.60% (1 672/2 213), 0.17% (4/2 213), 2.12% (47/2 213), respectively. Among which, strains with plasmid 52×10(6), 65×10(6), 90×10(6) distributed in Qinghai-Tibet plateau Himalayan Marmot natural plague foci, strains with 72×10(6) plasmid only distributed in Inner Mongolia Meriones unguiculatus natural plague foci and Junggar Basin R. opimus natural plague foci, and 65×10(6) plasmid distributed in all the other foci.Strains in Chinese 11 plague foci contained 4 kinds of large plasmid, the Mr respectively were 52×10(6), 65×10(6), 72×10(6), 90×10(6), which were classified into 26 kinds of plasmid profiles with other plasmid. These plasmid profiles distributed in relatively independent epidemic focus.

To type Yersinia (Y.) pestis isolates under different regions (DFR) and to observe their geographical distributions in China.23 DFRs primers and PMT1 (plasmid) primer were used to verify the DFR genomovars of Y. pestiss strains from 11 plague foci in China. A total of 3 044 Y. pestis isolates were involved for analysis on DFR profiles with the characteristics of geographical distribution.52 genomovars were verified in 3 044 Y. pestis strains in China in which 19 genomovars as major and 33 genomovars as minor genomovar. 21 new genomovars, namely genomovar 32 to genomovar 52 were described on the basis of 31 genomovars previously confirmed. Three new genomovars belonged to new major genomovars, namely Himalayan marmot natural plague foci of the Qinghai-Tibet plateau newly added genomovar 32 and genomovar 44 as major genomovars. Mongolian gerbil natural plague foci of Inner Mongolia plateau were newly added genomovar 50 as one of the major genomovars.Among 21 new genomovars, 3 were major genomovars, with Chinese Y. pestis DFR as the major genomovars which had obvious distribution characteristics.

To study the biological and genetic characteristics of 119 strains of Yersinia (Y.) pestis isolated from plague patients in Qinghai province, from 1958-2012.Both regular methods and different region(DFR)molecular typing techniques were used to study the epidemiological characteristics on 119 strains of Y. pesticin Qinghai during 1958-2012. Sources of Y. pestis from two outbreaks, in Nangqian county in 2004 and in Xinghai county in 2009,Qinghai province were also analyzed.105 strains of Y. pestis were identified as Qinghai-Tibet Plateau Ecotype while the other 6 strains as Qilian Mountains Ecotype. 84.03% (100/119) of the tested strains carried 4 virulence factors F1(+), Pst I(+), VW(+) and Pgm(+)). 97.30% (72/74) of the tested strains showed high virulence. Strains that carrying 52×10(6), 65×10(6), 92×10(6) plasmids were distributed in Hainan, Haibei, Haixi,Yushu,Guoluo, Huangnan and Huangyuan counties. Genomovar 5 and 8 were the main gene types that circling around Qinghai Lake. Genomovar 10 was found in strains of Y. pesticin Nangqian county while Genomovar 8 was found in the strains isolated from human plague patient during the epidemics in Xinghai county in Qinghai.Data from biological and genetic analyses on the epidemics of human plague in Nangqian county in 2004 and in Xinghai county in 2009 demonstrated that methods as DFR genotyping and virulence factors profiles, as well as plasmids profiles were powerful tools in confirming the human plague epidemics and sources of infection.