Serological Epidemiological Investigation of Tibetan Sheep ( Ovis aries ) Plague in Qinghai,China

Introduction

The plague was responsible for the deaths of millions of people in Europe in the 14th century and tens of thousands of people in China in the 19th century (Morelli et al. 2010). Yersinia pestis (Y. pestis), which is the etiological pathogen of the plague, was transmitted from natural reservoirs primarily by fleas (such as Xenopsylla cheopis or Collopsylla dolabris, and others). Currently, the plague remains endemic in parts of Asia, Africa, and the Americas. The plague is categorized as a Class A infectious disease (the most serious category) under China's Law on the Prevention and Treatment of Infectious Diseases. In total, 15 plague foci covering more than 1.4 million square kilometers are present in China, and the largest focus is the Qinghai-Tibet Plateau Marmota himalayana Plague Focus, which includes the provinces of Qinghai, Gansu, Xinjiang, and Sichuan and the Tibet Autonomous Region. The Qinghai-Tibet Plateau M. himalayana Plague Focus was first identified in Qinghai in 1954 and is listed as the highest risk area for the plague in China because the Y. pestis (biovar Antiqua) in this focus causes pneumonic and septicemic plague with high mortality (Wang et al. 2016).

In 1975, human plague that originated from Tibetan sheep was identified in the Yushu prefecture of Qinghai Province. Three strains of Y. pestis were isolated from one patient and two Tibetan sheep (He et al. 2016). This report provided the first evidence that Tibetan sheep could be naturally infected with Y. pestis and infected Tibetan sheep could cause human plague (Wang et al. 2016). Subsequently, 10 Tibetan sheep-related human plague outbreaks occurred in Qinghai Province between 1975 and 2009, resulting in 25 cases and 10 deaths (He et al. 2016). All index infectious cases had a clear exposure history, such as slaughtering or skinning diseased or dead Tibetan sheep (Wang et al. 2016).

Tibetan sheep are important domestic livestock in the Qinghai-Tibet plateau and have been identified as the second host animal that can cause human plague in Qinghai (the first host animal was marmots) (Wang et al. 2016). Human infections associated with Tibetan sheep result in a high mortality rate (He et al. 2016). However, the infection rate of Tibetan sheep across all areas in Qinghai remains unknown. This study investigated the distribution of the F1 antibody in serum samples from 12,710 Tibetan sheep from all 45 counties in Qinghai between 2013 and 2015. Meanwhile, the positive rate of the F1 antibody in Tibetan sheep was compared with that in the main local host (M. himalayana).

Materials and Methods

Results

Qinghai Province includes the provincial capital (Xining City) and 7 prefectures, encompassing 44 counties. We investigated the F1 antibody in 12,710 Tibetan sheep in Qinghai during 2013–2015. In total, 0.68% (86 of 12,710) of the sera from Tibetan sheep yielded positive results using IHA. In addition, the consistency between the IHA results and the colloidal gold test results was 100%.

At prefecture level, no serological positivity was found in Haidong and Haibei prefectures; however, serological positivity to the Y. pestis F1 antibody was found in Tibetan sheep in five prefectures, as well as Xining City in Qinghai. Three prefectures, that is, Hainan, Haixi, and Huangnan, were first identified as prevalent areas containing Tibetan sheep plague, and the positive rates of serum with the F1 antibody were 1.61%, 0.65%, and 1.44%, respectively (Supplementary Table S1). In the Yushu and Guoluo prefectures, the seropositive rates were 0.26% and 1.01%, respectively. The positive serum rates in the above two prefectures were lower than those found in three other prefectures (Haixi, Hainan, and Huangnan) (χ ² = 8.82, p = 0.003).

The seropositive rate ranged from 0.33% to 5.2% (Fig. 1 and Supplementary Table S1) at the county level in Qinghai. The highest rate was observed in Xinghai County of the Hainan prefecture (5.20%), and the titers in the sera ranged from 1:20 to 1:80. The highest titer was observed in Maqin county of the Guoluo prefecture (1:5120). Such investigation results indicated that the Tibetan sheep infected by Y. pestis were widely distributed in Qinghai and the prevalence of Y. pestis infection varied. Notably, three positive serum samples were collected from Tibetan sheep slaughtered at the local abattoir in the metropolitan area of Xining City, and the positivity rate was 0.5% (3/600). In fact, the three Tibetan sheep were transported from one of the plague epidemic areas (Maqin county of the Guoluo prefecture).

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FIG. 1.

Serological positive rate in Tibetan sheep in Qinghai. Lower left: location of the Qinghai province in the Qinghai-Tibet plateau; the serological positive rate in Tibetan sheep, based on the scale bar (lower right), is listed at the county level in Qinghai. Color images available online at www.liebertpub.com/vbz

In this survey, no positive serum samples were found in Tibetan sheep from the 11 non-animal plague counties (the three positive serum samples from Tibetan sheep in Xining City primarily originated from Maqin county; therefore, Xining City was not included in the animal plague focus counties), whereas in 33 counties that had been identified as M. himalayana plague foci, 8 counties had positive serum samples. A statistically significant difference was observed between the positive rates in animal plague focus counties and the rates in non-animal plague counties (adjusted χ ² = 14.71, p = 0.0001).

According to the routine animal surveillance data in Qinghai, there were nine surveillance counties in Qinghai (Wulan, Gonghe, Tongde, Hannan, Guinan, Qilian, Guide, Huzhu, and Delingha) were considered existing rodents plague epizootics during 2013–2015 for samples collected from M. himalayana or carnivorous were positive (i.e., IHA or RIHA or bacteria isolation positive) (Supplementary Table S2). While in this survey, there were eight counties (Supplementary Table S1) existed serological positivity samples in Tibetan sheep, and the overlapping counties involved in Guide, Delingha, Wulan. In Wulan county, Y. pestis strains were isolated in 2014 and 2015 in routine animal plague surveillance, and RIHA positive was found in 2013 (Supplementary Table S2).

Discussion

The Qinghai M. himalayana plague natural foci were first identified in 1954 following the isolation of Y. pestis from a dead marmot. In total, 468 human plague cases with 240 deaths were reported in Qinghai Province between 1958 and 2014 (Wang et al. 2016). According to the history of the plague in Qinghai, 162 cases originated primarily from marmots (34.62%), 39 cases originated from Tibetan sheep and goats (Procapra picticaudata) together (rate: 8.33%), and 16 cases originated from carnivorous animals (3.42%); 216 cases of pneumonic plague were caused by person-to-person transmission (46.15%) (Wang et al. 2016). M. himalayana is the primary host of the plague in this area. Notably, hunting marmots, particularly processing and skinning marmots, is a primary risk factor for the human plague in the Qinghai-Tibet Plateau natural plague focus, and is a frequent cause of severe pneumonic plague and death. Similarly, Tibetan sheep-related human plague infection has been associated with slaughtering or skinning diseased or dead Tibetan sheep. Eating meat from infected sheep that is not fully cooked is another cause of human plague infection (Wang 1999). Tibetan sheep have been identified as the second host animal that could cause human plague in Qinghai (Wang et al. 2016). Therefore, the infection of Tibetan sheep with Y. pestis could be a potential public health concern in Qinghai and in China.

The detection of the F1 antibody by IHA is a standard animal plague surveillance method and a human plague serological diagnostic method. The F1 antibody in animal serum could be used to evaluate the animal plague prevalence level. In this serological investigation, 0.33–5.20% of sheep (at the county level) were positive in six prefectures in Qinghai. Therefore, there is a widespread infection of Tibetan sheep with Y. pestis in Qinghai, despite a sporadic epidemic of the Tibetan sheep plague in Qinghai. However, the positive serum rates in animals were not completely parallel to human plague infection associated with Tibetan sheep. For instance, the positive serum rates in Yushu and Guoluo prefectures were lower than those found in three other prefectures (Haixi, Hainan, and Huangnan), despite the fact that the Yushu and Guoluo prefectures had human plague cases associated with Tibetan sheep since 1975 (He et al. 2016), whereas non-human plague associated with Tibetan sheep has been reported in the Haixi, Hainan, and Huangnan prefectures (Wang et al. 2016).

Several serological surveys of the F1 antibody in Tibetan sheep have previously been performed. In 2005, an outbreak of Tibetan sheep plague was identified in Yushu, and 13 Tibetan sheep and 1 Tibetan goat from a flock died from Y. pestis infection. Three Y. pestis isolates isolated from Tibetan sheep (n = 2) and Capra aegagrus hircus (Tibetan goat, n = 1) were identified. Following the animal plague, 1051 serum samples were collected from Tibetan sheep on the same farm in 2005, and the infection rate of Y. pestis in the Tibetan sheep was 6.08% (64/1051), with serum titers ranging from 1:20 to 1:1280 (Yu et al. 2006). However, similar investigations in serum were performed in the next year (2006) on the same farm, and the positivity rate was only 0.70% (7/1005) (Wang et al. 1994c). In addition, when Tibetan sheep plagues were not observed in the investigated flocks (i.e., no Tibetan sheep had died or became sick from Y. pestis infection), the serological positive rates of F1 antibody were comparatively low (0.26–0.52%) compared with the occurrence of Tibetan sheep plague in Yushu in 2005 (Wang et al. 2016). In our study, certain areas had comparatively low infection rates, including lower infection rates in some historically endemic counties such as Yushu. This may be due to the short sheep breeding period (2–3 years) and the fact that most sheep in the flocks in the endemic counties were newly bred. Also, there was a reduction in Tibetan sheep during this time.

Even-toed ungulates (Artiodactyla) (Edmunds et al. 2008), including camels (Arbaji et al. 2005, Leslie et al. 2011) and goats (Christie et al. 1980), can be naturally infected by Y. pestis and are considered important plague hosts in certain areas. Donkeys, pigs, and cows could also be naturally infected with Y. pestis (Lien-Teh et al. 1936). Several researchers have isolated Y. pestis in P. picticaudata (Tibetan Gazelle), Pseudois nayaur (blue sheep), Cervus elaphus (red deer), and C. aegagrus hircus (Tibetan goat) in China (Wang et al. 2016). Previous symptoms observed in Tibetan sheep challenged with Y. pestis wild strain (strain name 141) indicated that the Tibetan sheep were susceptible animals with a high susceptibility and moderate sensitivity (Wang et al. 1994a). In a previous study, a total of 21 Tibetan sheep were infected with 3.5 × 10²–3.9 × 10⁸ CFU of the Y. pestis by the intravenous injection (i.v.) and subcutaneous injection (s.c.) route; the incidence rate was 90.47% (i.v. route) and 100% (s.c. route), respectively, and the mortality rate was 47.36% (i.v. route) and 38.09% (s.c. route) (Wang et al. 1994a). Corresponding clinical observation on these experimental Tibetan sheep showed that the incubation period of infected sheep was 1–5 days through i.v. and s.c., and 78% (62/79) Tibetan sheep exhibited medium fever with 1–2°C above normal sheep body temperature (38–40°C); 22% (17/79) Tibetan sheep exhibited high fever (above 2–3°C). The fever duration was 3–8 days, with individual tested sheep up to 14 days (Wang et al. 1994b). Acute and subacute clinical features were observed in 31.65% of challenged sheep; corresponding clinical features included high body temperature, shiver, increasing breaths, and symptoms of lymphadenopathy corresponding to the site of infection (Wang et al. 1994b). The clinical features of Tibetan sheep plague were similar to pasteurellosis (Wang et al. 1994b), whereas 68.35% of the tested Tibetan sheep developed delayed clinical features. Some sheep could carry the bacteria for more than 1 month, and 58.96% of challenged Tibetan sheep healed naturally (Wang et al. 2001). The distribution of Tibetan sheep plague broadly overlapped with the natural focus of marmots in the Qinghai-Tibet Plateau M. himalayana Plague Focus (Wang 1999). Also, a previous study indicated that the infection with Y. pestis in Tibetan sheep might be due to transmission from marmots (Wang et al. 2016). More detailed investigations, including ongoing ecological investigations and genome-based investigations, could provide more detailed information regarding the origin of Tibetan sheep and human plague cases.

Tibetan sheep account for ∼1/3 sheep in China (Wang 1999), and Tibetan sheep and goats are important domestic livestock in the Qinghai-Tibet Plateau. Tibetan sheep with Y. pestis infection make the plague epidemics more complex in Qinghai, including a more complex infection route and transmission over long distance. In addition, the people involved in slaughtering or skinning domestic livestock are considered at risk in epizootic areas.