Seroprevalence of Toxoplasma gondii infection in sheep in Inner Mongolia Province, China

Toxoplasma gondii is an important zoonotic parasite that can infect almost all warm-blooded animals, including humans, and infection may result in many adverse effects on animal husbandry production. Animal husbandry in Inner Mongolia is well developed, but data on T. gondii infection in sheep are lacking. In this study, we determined the seroprevalence and risk factors associated with the seroprevalence of T. gondii using an indirect enzyme-linked immunosorbent assay (ELISA) test. A total of 1853 serum samples were collected from 29 counties of Xilin Gol League (n = 624), Hohhot City (n = 225), Ordos City (n = 158), Wulanchabu City (n = 144), Bayan Nur City (n = 114) and Hulunbeir City (n = 588). The overall seroprevalence of T. gondii was 15.43%. Risk factor analysis showed that seroprevalence was higher in sheep ≥12 months of age (21.85%) than that in sheep <12 months of age (10.20%) (p < 0.01). Seroprevalence was higher in male sheep (18.76%) than females (12.80%) (p < 0.01). Barn-feeding sheep (23.13%) had higher prevalence than grazing sheep (10.94%) (p < 0.01). The seroprevalence was significantly different in different districts (p < 0.01). This study shows that sheep are exposed to T. gondii in Inner Mongolia, and provides a data reference for public health and disease control.


Introduction
Toxoplasma gondii is a food-borne intracellular parasite that can infect nearly all warm-blooded animals worldwide, even humans [9]. Approximately one-third of the human population has been exposed to T. gondii. Infection by the parasite may cause cerebral and ocular damage and even death, especially in immunodeficient patients [8,18]. Humans are mainly infected with T. gondii by ingesting uncooked meat and water contaminated by oocysts from the environment, or by vertical transmission [2,3,7]. In addition, T. gondii can also have a negative influence on animal growth, development and reproduction, and cause great economic loss to livestock husbandry [6]. Livestock become infected mainly by ingesting food and water contaminated with sporulated oocysts [12]. *Corresponding author: yanxinlei1987620@foxmail.com a These authors contributed equally to this work.
Toxoplasma gondii infection in sheep can cause a wide variety of non-specific symptoms (fever and dyspnoea), and specific symptoms (depression, lethargy, vomiting, diarrhea, chorioretinitis, and lymphadenopathy), and can even cause abortions and stillbirths [10]. Recently, increasing consumption of mutton has raised the risk of T. gondii infection. Sero-epidemiological surveys have reported a global distribution of T. gondii in sheep ranging from less than 4.4% to over 80.0% [5,14,19].
As a major livestock husbandry Province in China, the number of sheep stocks in Inner Mongolia has reached over 100 million in recent years, and the animal production economy is one of the most important pillar industries in this district. In the past, many studies have examined the seroprevalence of T. gondii infection in livestock, including horses and cattle in Inner Mongolia [16,21]. However, data on the seroprevalence of T. gondii in sheep in Inner Mongolia are not comprehensive nor detailed [4]. The natural grassland of Xilin Gol League and Hulunbeir City is famous throughout the world for its high quality, and sheep production here is prosperous. The population in Hohhot City, Ordos City, Wulanchabu City, and Bayan Nur City accounts for half of the total population in Inner Mongolia. Therefore, samples were collected from these areas, which makes the results more representative. The aim of this study was to determine the current status of the prevalence of T. gondii in sheep in Inner Mongolia, and to provide a reference for the prevention and control of T. gondii. The results will serve as baseline comparison data for future industrial development and safety assessments, and provide information to public health departments, wildlife managers and researchers.

Serum samples
Blood samples were collected from 29 counties of Xilin Gol League, Hohhot City, Ordos City, Wulanchabu City, Bayan Nur City, and Hulunbeir City (Fig. 1) to investigate the presence of serum antibodies against T. gondii. A total of 1853 blood samples were selected randomly from September 2018 to November 2019, and the background information of each sample was obtained mainly from the hosts or breeders.
No special criteria were applied to different farms. During visits to the localities, blood samples were collected from the jugular vein into a centrifuge tube. These centrifuge tubes filled with blood samples were quickly frozen in a local freezer once collected, and brought back to the laboratory in an incubator. Each of the blood samples was centrifuged at 4000 rpm for 8 min, and serum was separated and stored at À20°C until further analysis.

Determination of antibodies against T. gondii
Antibodies against T. gondii from serum samples were detected by an indirect enzyme-linked immunosorbent assay (ELISA) test, using a commercially available kit (CK-DN74810, 96T), which was obtained from Quanzhou Ruixin Biotechnology Co., Ltd. The detection procedure was carried out in accordance with the protocol described by the manufacturer. When the reaction was complete, the optical density (OD) value was measured at 450 nm using a Microplate Reader within 15 min. Positive and negative controls provided within the kit were included in each test. The serum samples were considered positive if the sample OD value was greater than the cut-off (the cut-off was the sum of the average value of the negative control OD value and 0.15).

Statistical analysis
Statistical analysis was carried out by chi-square (v 2 ) testing with SPSS (Statistical Analysis System, Version 20.0). When p < 0.01, the difference was considered extremely significant; when 0.01 < p < 0.05, the difference was considered significant; when p > 0.05, the difference was not significant. The odds ratio (OR) at the 95% confidence level was used for the determinants influencing the epidemiology of parasites.

Results
Antibodies against T. gondii were found in 286 of the 1853 sheep by the ELISA kit (Table 1); the overall seroprevalence was 15.43%. On the basis of values for T. gondii antibody detection, the seroprevalence of four districts was higher than the overall seroprevalence, and Bayan Nur City had the highest seroprevalence (23.68%). Across all districts, Xilin Gol League had the lowest seroprevalence (12.02%). Chi-square test analysis showed that there were significant differences in the prevalence of T. gondii infection in different districts (v 2 = 112.010, p value = 0.000) ( Table 1).
In this study, 154 of the 821 male sheep serum samples tested were positive, with a positive rate of 18.76%, and 132 of the 1032 female sheep serum samples tested were positive, with a positive rate of 12.80% (Table 2). There was a significant difference in the seroprevalence of T. gondii infection between the sexes (p < 0.01). Sheep !12 months of age were at higher risk (21.85%, 182/833) than sheep <12 months (10.20%, 104/1020). Barn-feeding sheep were at higher risk (23.13%, 158/683) than grazing sheep (10.94%, 128/1070). There were significant differences in the seroprevalence of T. gondii infection in different ages and rearing models (p < 0.01). Risk factor analyses showed that sex (OR = 0.682), age (OR = 0.467), and rearing model (OR = 0.473) were risk factors for T. gondii infection in sheep (Table 2).
Over a period of one year, we collected blood samples from different districts every month. Compared with other months, October had the highest prevalence (21.69%, 41/189), and February had the lowest prevalence (10.74%, 13/121). Chi-square test analysis showed that there was no significant difference in the seroprevalence of T. gondii infection in different months (v 2 = 23.157, p value = 0.393) ( Table 3).

Discussion
Antibodies against T. gondii were found in 286 out of 1853 sheep (15.43%) in this study, which was higher than that reported in Shandong in 2019 (9.84%) and Yunnan in 2015 (9.70%) [1,22]. Moreover, Gao et al. reported a prevalence of 17.10% (13/76) for T. gondii infection in Chifeng, Inner Mongolia, which was in the same range as the prevalence of infection in this study [4]. In this study, the seroprevalence of T. gondii in sheep varied from 5.00% to 29.63% among different counties. There was a great difference in the prevalence of T. gondii infection in different districts. We speculated that many factors contributed to this difference, such as climate, elevation, sheep strain, feeding model, and level of disease prevention and control, bearing in mind the vast size of Inner Mongolia Province (1.18 million km 2 ). Therefore, in order to make the data more accurate, samples will be collected from more districts in the future. Moreover, there were significant differences in the seroprevalence of T. gondii by sex. Males (18.76%) had a higher risk than females (12.80%) (p < 0.01). However, studies in Henan, China, revealed a higher prevalence of T. gondii in females than in males [20]. Studies in Yunan, China found no association between sex and the prevalence of T. gondii [22]. According to Romanelli et al. the presence of oestrogen in females normally increases immunity, and androgen in males decreases immunity [13]. Therefore, we suspect that sex is likely to work in conjunction with other unknown factors. Moreover, we found that the seroprevalence of T. gondii infection had a significant difference concerning age (sheep !12 months: 21.85%, and sheep <12 months: 10.20%). A total of 10.20% (104/1020) of sheep <12 months were seropositive. The higher prevalence in sheep !12 months was likely due to the prolonged time of exposure and repeated exposure to the oocyst-contaminated environment, resulting in a greater possibility of infection. In this study, there were significant differences in the seroprevalence between barn feeding and grazing rearing systems. Other studies have also shown that barn feeding involves a higher risk than grazing [11]. Since grazing sheep are pastured in comparatively large grazing areas, these sheep are exposed to T. gondii oocysts at a low level. However, barn feeding sheep were raised in a concentrated manner, which may increase the chances of T. gondii infection among sheep once food, water, or the environment was contaminated by oocysts. This may be the reason why the T. gondii infection prevalence in Bayan Nur City was the highest in our study (barn feeding sheep: 114, and no grazing sheep). At the same time, there was no significant difference in the seroprevalence in different months. However, some studies have shown that the seroprevalence is probably related to seasons [17], and the reason for this difference may be due to different environments, temperatures, and various sample qualities. Moreover, studies have shown that high temperatures have little impact on the reduction in viability of T. gondii [15].
In this study, seropositive samples were found in 29 counties of all six districts, which suggested that T. gondii infection was common in sheep in Inner Mongolia. As an important foodborne zoonotic parasite, T. gondii is seriously harmful to people and animals with various routes of infection. Therefore, great attention should be paid to the prevention and control of T. gondii in sheep. Certain measures can be taken to reduce the prevalence of T. gondii infection in sheep, such as strengthening the management of sheep farms, keeping the barn clean, and preventing feline excreta from polluting sheepfolds, food, or drinking water.

Conflict of interest
All individual authors declare that they have no conflict of interest (financial, personal, or other).