Introduction

Streptococci are Gram-positive suppurative cocci, which are widely distributed in nature. They are isolated in the digestive tract and nasopharynx of humans and other animals, composing of pathogenic and non-pathogenic types (YAO, H C, et al., 1999). Pathogenic Streptococci can cause suppurative diseases in humans and livestock, such as mastitis, septicemia, equine plague, and human scarlet fever (COLMAN, G, 1990). Streptococcosis is a potentially epidemic disease of sheep. It is an acute febrile infectious disease caused by hemolytic Streptococci and usually occurs during winter and spring (WANG, M Q, et al., 2021). The condition is mainly caused via the digestive tract, respiratory tract, and through injured skin. The clinical manifestations in sheep and goats are swelling of the mandibular lymph nodes and associated throat region and fibrinous pneumonia (LI, X Y, et al., 2014).

The local sheep found in Tibet are a unique and rare breed that have lived in the Qinghai-Tibet Plateau region for many decades (SU, Y, et al., 2022). The breed is a dual-purpose breed providing meat and milk as well as contributing to the local economic development of the region. Therefore, safe and effective improvement of Tibetan sheep breeding and ensuring the quality of its by-products (meat, milk, etc.) is of great significance to meet the market needs of the people and high-quality development of animal husbandry (ZHANG, X, et al., 2022). Moreover, sheep streptococcosis perennially perplexes local animal husbandry conditions and green growth in the plateau (FOSTER, G, et al., 2020). There are more than 100 identified species of Streptococcus, first reported by Weimann in Germany in 1910 (YANG, S D, et al., 1953). It was reported in China in 1952 and then spread all over the country. Furthermore, with the development of the breeding industry, disease occurrence showed an obvious upward trend, causing great harm to the overall GDP of the country. In recent years, sheep streptococcosis has been prevalent every year in pastoral areas of Tibet. In 2017, the incidence of Streptococcus ovis (S. ovis) in Shannan prefectural city of Tibet was 12.3%, with a fatality rate of 18.6% for the first time (BIANBA, Z M, et al., 2017).

The different species of Streptococcus, e.g., Streptococcus mastidis (S. mastidis) and Streptococcus equi (S. equi), are the primary pathogens, causing a decrease in milk production and the quality of dairy products with severe economic loss to breeding farms of dairy sheep and goats (ROSA, N M, et al., 2021; VEZINA, B, et al., 2022). According to some researchers, S. equi may also cause fibrin polyarthritis in goats (STEWARD, K F, et al., 2017). In previous studies, humans were considered the only biological host of suppurative Streptococci (BESSEN, D E, 2009). However, it has occasionally been identified in samples of milk from sheep with mastitis (VELA, A I, et al., 2017). With this in mind, the Tibetan government has taken joint measures (vaccination and destruction of infected animals) in infected areas, but the epidemic still occurs frequently due to the development of animal husbandry in the region. Hence, more information is always required to control the outbreak of the disease. This study was designed to determine the prevalence of potential risk factors for sheep streptococcosis for the very first time in the Tibetan region.

Materials and methods

Study areas and sampling

A study was conducted in five counties of Shigatse prefectural city (Sangzhuzi (E: 89.05°, N: 29.35°), Bailang (E: 89.17, N: 28.95°), Lazi (E: 89.31°, N: 29.26°), Saga (E: 84.47°, N: 29.54°), Zhongba (E: 84.02°, N: 29.76°) from October 2021 to April 2022. A total of 1458 sera samples from non-vaccinated (Streptococcus) sheep and goats were collected from 50 flocks. Samples were collected from two types of animal-rearing systems: intensive farms and scattered herds. The intensive farms are managed by the relevant departments of the local government. There are full-time managers and qualified veterinarians working on the farms with experience in disease prevention and control and management. Herders graze free-range their sheep/goats. The areas of Saga and Zhongba are pastoral regions, while Sangzhuzi, Bailang, and Lazi areas are semi-agricultural and semi-pastoral areas. The average altitude of the sampling sites was 4000 m above sea level, where animals are grazed openly all year round, with extra supplementary feeding being provided in winter (Fig. 1).

Fig. 1
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Map showing the location of the selected counties in Tibet, China

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Sampling quantity and distribution of sheep serum

The sample size was calculated using a design prevalence of 20% with a 95% confidence interval (CI) and 5% precision (SAVILLE, W J, et al., 2004). A total of 720 sheep serum samples were collected in Sangzhuzi (n = 197), Lazi (n = 350), and Bailang (n = 175) areas during 2021, and a further 738 samples were collected in Sangzhuzi (n = 122), Bailang (n = 108), Lazi (n = 147), Saga (n = 201), and Zhongba County (n = 158) areas during 2022. Overall, 1458 samples were collected from 13 intensive farms and 37 scattered herds in the Shigatse area. Among them, 938 samples were assocaited with sheep and goat farms, while 520 were associated with scattered herds (n = 192).

Serological tests

Approximately 5 mL of whole blood was collected from the jugular vein. The samples were kept at room temperature for several hours and then centrifuged at 3000 rpm for 10 min. The serum was then collected in 2 mL centrifuge tubes and stored at −20 °C. The frozen samples were thawed and tested with a commercial ELISA kit for detecting antigens of Streptococcus ovis (Batch number: 202107; Shanghai enzyme-linked Biotechnology Co., Ltd.).

Statistical analyses

Data obtained from both the serological test and questionnaire survey was stored in a Microsoft Excel spreadsheet (Microsoft Corp.), and statistical analysis was performed using the statistical software R (R Development Core Team 2022). Chi-square test was used to analyze the association between the presence of serum antibodies against Streptococcus and breeds, ages, sex, altitude, sampling years, and management type. The statistical significance was set as p < 0.05. In univariate analysis, variables with p ≤ 0.20 were offered to a multivariate logistic regression analysis. A multivariable logistic regression model was generated by using a backward stepwise process to control for confounding and test for effect modification, and the variables with p < 0.05 were retained in the final model. The likelihood ratio test was conducted to evaluate the model and was regarded as the most appropriate model with the smallest AIC (Akaike information criterion) value (BOUKARY, A R, et al., 2013). The model was then assessed by the Hosmer-Lemeshow goodness-of-fit test.

Results

Among all the screened sheep, 6.93% (101/1458, 95% CI: 5.7–8.4) were found as positive for Streptococcus, and the flock prevalence of Streptococcus was 84% (42/50, 95% CI: 70.9–92.8).

At the individual level, there was no significant difference in the prevalence of Streptococcus in different breeds, ages, and sex. The individual prevalence of sheep and goats at the altitude of 4000 m was 5.61%, and 8.79% respectively (Table 1). Statistically, the Streptococcus from different altitudes above sea level was significant (OR = 0.62, 95% CI 0.41–0.93; p < 0.05). There was a significantly higher animal level prevalence in 2022 (8.29%) than in 2021 (5.54%) (OR = 1.52, 95% CI: 1.02–2.33; p < 0.05). Moreover, the prevalence of Streptococcus in pastoral households was 10% (95% CI: 7.6–12.9), and at intensive farms was 5.22% (95% CI: 3.9–6.8). A significant difference was noticed in individual positive rates between the two management types (OR = 0.50, 95% CI: 0.33–0.74; p < 0.01) (Table 1). In the final model, only the sampling year and the management type had significant differences, among which the odds of Streptococcus in 2021 were significantly different from that in 2022 (OR = 2.16, 95% CI: 1.39–3.39). The odds of Streptococcus at intensive farms significantly differed from that of streptococci in herders (OR = 2.64, 95% CI: 1.71–4.10). The likelihood ratio test, χ2 = 23.14 (p < 0.001), AIC value of the final model, 716.97, and a Hosmer-Lemeshow goodness-of-fit value (p = 0.34), indicated that the model was a good fit of the data (Table 2)

Table 1 The univariate analysis of risk factors associated with Streptococcus seropositivity among sheep and goats in Shigatse area by enzyme-linked immunosorbent assay
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Table 2 Multivariable logistic regression analysis of risk factors related to Streptococcus seropositivity in sheep and goats
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Discussion

China has a long history of raising sheep, regardless of quantity or breed diversity. Tibet region is one of the five major pastoral areas in China, where livestock rearing is an important industry for farmers and herders (ZAMORA, L, et al., 2017). Sheep are considered as important as yaks to farmers and herders in the plateau regions. Different pathogens might affect the growth of the sheep industry, e.g., Streptococcosis. There are few studies investigating the prevalence and risk factors for infection with Streptococci in sheep. This study provides preliminary information on infection in the Tibet region. The smaller number of studies on the disease has urged researchers to focus more on other potential factors to control the disease’s impact. The current study was conducted in five different areas of Shigatse prefectural city. Results showed that the animal level seroprevalence in sheep and goats was relatively high at 6.3%, with the herd level (84%). At the animal level, the seroprevalence detected in the current study was slightly higher than that in Qinghai Province (3.13%) (LI, X Y, MA, R L, WANG, Y P, WANG, S X, FU, Y J and ACKERMANN, K, 2014), although both regions are located in the Qinghai-Tibet Plateau. The difference may be due to the grazing environment, breeds, nutritional status, and sample size. There are few reports on the epidemiology and risk factors for infection with S. ovis compared to S. suis and S. equi in pigs and horses. For example, the prevalence of S. equi in Croatia (STRITOF, Z, et al., 2021) and Lesotho (LING, A S, et al., 2011) were 16.5% and 10.1%, respectively. The prevalence of S. suis in Tibet was reported to be 60.13% (LAN, L, 2017). This may be related to the different types of livestock breeds in different regions affected by various Streptococci species.

From the results of this study, it can be seen that S. ovis infection was endemic, with the close connection between Tibet and other provinces in recent years and with the passing of National Highway 318 and National Highway 219 in the sampling area. At the same time, the average elevation of Shigatse is more than 4000 m, the climate is changeable all year round, the temperature difference between day and night, summer, and winter is large, and the grass withering period is long. These provide convenience for the spread and occurrence of Streptococcus sheep. Because the sampling site is in an environment of high cold and hypoxia, and the results of this study show that the disease is still widespread in the Shigatse area, Streptococcus has adapted to this environment, and its growth and transmission are basically not restricted by this kind of cold and hypoxia. Through the investigation, the seroprevalence in sheep sampled in 2022 (8.29%) was significantly higher than that in 2021 (5.54%, p < 0.05). The sampling time was April 2022 and October 2021, which indicated that sheep streptococcosis was more prevalent in early spring than in winter in the Shigatse area. At the same time, Tibetan forage gradually turned green in April, leading to significant diarrhea in sheep. According to some studies, it may decrease sheep resistance, providing favorable conditions for breeding Streptococcus in the animals’ bodies.

Another finding observed in our study is that the seroprevalence in animals from pastoral herds (10%) was significantly higher than in animals from intensive farms (5.22%). It may be understood that the education level of local herdsmen is generally low, and they know little about the prevention and control of livestock epidemic disease, which may also be one of the reasons for the widespread occurrence of the disease in the local area. Moreover, herders do not have proper supplementary feeding but instead rely on grazing to maintain their nutritional status.

Conclusion

Streptococcus ovis is prevalent in the Shigatse area of Tibet, China. Using a logistic regression model, it was found that only sampling time and management type of Tibetan sheep were risk factors for seropositivity to streptococcus. Therefore, preventive measures such as immunization plans, proper animal housing, monitoring and surveillance, proper carcass disposal, training, and education of farmers regarding the spread of disease, especially during early spring, should be strengthened, and also, reasonable supplementary feeding needs to be provided.