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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.

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., Author summary Plague is mainly a disease of wild rodents, and their parasitic fleas are considered the transmitting vectors. However, human plague originating from Ovis aries (Tibetan sheep) is found in the Qinghai-Tibet plateau in China, where Marmota. himalayana is the primary plague host. Tibetan sheep-related human plague infection is always associated with slaughtering or skinning diseased or dead Tibetan sheep. The plague in Tibetan sheep clearly lags that in M. himalayana. In this study, we performed a genome-wide single nucleotide polymorphism analysis of Tibetan sheep-related plague events, including pathogens isolated from humans, Tibetan sheep, and marmots. Through genomic analysis, together with the epidemiological connections, we confirmed that human plague came from Tibetan sheep, and the Tibetan sheep plague originated from marmots. Tibetan sheep account for about 1/3 of the total number of sheep in China. Tibetan sheep and goats are important domestic livestock on the Qinghai-Tibet plateau. Therefore, the hazards of Tibetan sheep plague should not be underestimated.

To identify the epidemiology and etiology characteristics of Tibetan sheep plague in Qinghai plateau.The background materials of Qinghai Tibetan sheep plague found during 1975 to 2009 were summarized, the regional, time and interpersonal distribution, infection routes, ecological factors for the spread were used to analyze; followed by choosing 14 Yersinia pestis strains isolated from such sheep for biochemical test, toxicity test, virulence factors identification, plasmid analysis, and DFR genotype.From 1975 to 2009, 14 Yersinia pestis strains were isolated from Tibetan sheep in Qinghai province. Tibetan sheep, as the infection source, had caused 10 cases of human plague, 25 plague patients, and 13 cases of death. All of the initial cases were infected due to eating Tibetan sheep died of plague; followed by cases due to contact of plague patients, while all the initial cases were bubonic plague. Cases of bubonic plague developed into secondary pneumonic plague and septicemia plague were most popular and with high mortality. Most of the Tibetan sheep plague and human plague occurred in Gannan ecological zone in southern Gansu province, which was closely related to its unique ecological and geographical landscape. Tibetan sheep plague coincided with human plague caused by Tibetan sheep, especially noteworthy was that November (a time for marmots to start their dormancy) witnesses the number of Yersinia pestis strains isolated from Tibetan sheep and human plague cases caused by Tibetan sheep. This constituted the underlying cause that the epidemic time of Tibetan sheep plague lags obviously behind that of the Marmot plague. It was confirmed in the study that all the 14 strains were of Qinghai-Tibet Plateau ecotype, with virulence factors evaluation and toxicity test demonstrating strains as velogenic. As found in the (Different Region) DFR genotyping, the strains isolated from Yushu county and Zhiduo county were genomovar 5, the two strain isolated from Nangqian county were genomovar 5 and genomovar 7, while those isolated Delingha region were genomovar 8.Tibetan sheep were vulnerable to plague infection, hence causing human plague as the infectious source. The Yersinia pestis strains isolated from Tibetan sheep plague carried pathogen characteristics of Qinghai-Tibet plateau plague, developing many new characteristics of such plague.

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 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.