Seroprevalence and Epidemiology of Toxoplasma gondii in Animals in the Qinghai-Tibetan Plateau Area, China

Toxoplasma gondii belongs to the Apicomplexan protozoa—an obligate intracellular parasite—causing toxoplasmosis that has a worldwide distribution and is very harmful to both human health and the livestock industry. However, the information on toxoplasmosis in the Qinghai-Tibetan Plateau Area (QTPA) and the seroprevalence of T. gondii in the food-borne animals in that area has been limited. Therefore, this study focused to T. gondii and toxoplasmosis to perform an indirect ELISA test based on recombinant TgSAG2 protein to establish a comprehensive record of the seroprevalence of T. gondii infections in a wide range of animals, including Tibetan sheep (Ovis aries), yaks (Bos grunniens), cows, chicken, pigs, and horses, in the QTPA. Overall, the seropositive rates of the specific-T. gondii IgG and IgM antibodies in all investigated animals were 44.1% (1179/2673) and 18.0% (469/2612), respectively. The 14.9% (389/2612) sera were determined to be both IgG and IgM positive samples, 30.2% (789/2673) were single-IgG seropositive, and a total of 80 in 2612 animals (3.0%) were single-IgM seropositive. Moreover, for the animal species, the pig was the most prevalent animal (90.2%, 304/337) for IgG positivity, followed by Tibetan sheep (50.7%, 460/907), chickens (45.8%, 229/500), yaks (21.1%, 140/663), cows (18.5%, 38/205) and horses (13.1%, 8/61), respectively. For the IgM antibody positivity, the pig was also the most prevalent animal (41.8%, 141/337), followed by Tibetan sheep (21.2%, 191/907), cows (15.1%, 31/205), chickens (12.4%, 62/500) and yaks (6.6%, 44/663), respectively. The significant differences in the prevalent distribution of T. gondii were found in the different altitudes. In conclusion, this study found the high seroprevalence for T. gondii infections among these animal species in the QTPA, and provides new data to facilitate further research for development of control measures against T. gondii infections in the surveyed locations.


Introduction
The Qinghai-Tibetan Plateau Area (QTPA), the largest plateau with the highest average altitude on the planet, is located in northwestern China [1]. The Qinghai province, located on the northeastern side of QTPA, has a unique and vigorous natural ecosystem due to the high altitude (an average elevation of more than 2000 m above sea level) and cold climate (an average annual temperature below 10 • C) [2,3]. Therefore, a variety of unique livestock has been domesticated in the province, including Tibetan sheep (Ovis aries), yaks (Bos grunniens), cows, chicken, pigs, and horses [4], which are the important economic livestock animals and food animals in this special area [5]. These animals share water and food in the plateau area [6,7].
Toxoplasma gondii is a very important food-borne zoonotic pathogen that can infect almost all warm-blooded animals, including humans, livestock and birds [8][9][10][11][12][13][14] and also causes economic losses in the livestock industry [15][16][17]. Tibetan sheep and yaks are indigenous species raised under extensive animal systems, while cows, chickens, pigs, and horses are important economic livestock animals in the QTPA. The animals could be the intermediate hosts of T. gondii in this plateau area. Humans or these animals might be infected by ingesting water and foods which contain infecting oocysts from cats to develop toxoplasmosis [8,14,16,17]. Therefore, this study focused on T. gondii and toxoplasmosis to investigate the seroprevalence of T. gondii IgG and IgM antibodies in a wide range of animals in the QTPA.
The serological tests play a crucial role in the diagnosis of toxoplasmosis and were widely used [18]. In China, some studies have been reported that humans and animals were infected by T. gondii with different prevalence, of which the prevalence in food animals (such as cattle, sheep, goat, chickens, and swine) was higher than that in humans [19,20]. Although there are several reports on the seroprevalence and epidemiology of T. gondii in animals from the different regions in the QTPA, most attempts of testing toxoplasmosis have been done in yaks which are the important economic animals and food-borne animals in the QTPA [21][22][23][24][25][26]. To date, information on toxoplasmosis in the QTPA and the seroprevalence of T. gondii in other food animals in that area has been limited.
Among several serodetection tests used for diagnosis of toxoplasmosis, the latex agglutination test (LAT) and indirect haemagglutination (IHA) were shown to be insensitive in their present form, the indirect fluorescent antibody test (IFAT) and modified agglutination test (MAT) are considered time-consuming and expensive though specific; hence, the indirect ELISA test has been accepted as the most practical test especially for a large number of samples [9,11]. The surface antigen 2 (SAG2) of T. gondii has an added diagnostic relevance, as it is exposed to the immune system of the hosts [18], and has been identified and tested as an important candidate for the serological diagnosis for toxoplasmosis in many studies [27]. Furthermore, the ELISA based on recombinant SAG2 fusion protein has been used as an effective field serodiagnostic test for the detection of T. gondii infection in different animal species, such as cat, cattle, sheep, goat and pig [18]. Thus, the aim of this study was to perform an indirect ELISA test based on recombinant TgSAG2 protein (rTgSAG2) to establish a detailed record of the seroprevalence of T. gondii-specific IgG and IgM antibodies in Tibetan sheep, yaks, cows, chicken, pigs, and horses in the QTPA.

Results
In this study, the seroprevalence of T. gondii in the animals from the QTPA ( Figure 1, Table 1), including Tibetan sheep, yaks, cows, chickens, pigs, and horses, was investigated by the indirect ELISA.
To confirm the rTgSAG2-based indirect ELISA in this study, the positive and negative serum samples of mice for T. gondii or the positive serum samples of mice for N. caninum were used to develop the ELISA assay. The results showed that rTgSAG2 protein could determine T. gondii positive sera but no any reaction with T. gondii negative or N. caninum sera (Figure 2A), suggesting current recombinant protein could be studied with animal sera for detecting the T. gondii specific-antibody. Moreover, the cut-off value for judging positive samples was determined by using negative controls. The cut off values were determined as 0.120, 0.114, 0.246, 0.482, 0.423 and 0.110 for IgG antibodies based on rTgSAG2-ELISA in Tibetan sheep, yaks, cows, chicken, pigs, and horses; and 0.411, 0.234, 0.306, 0.468 and 0.065 for IgM antibodies based on rTgSAG2-ELISA in Tibetan sheep, yaks, cows, chickens and pigs, respectively ( Figure 2B,C).     In this study, a total of 2673 animal serum samples were tested in a detailed survey of specific anti-T. gondii IgG antibodies in different sampling sites among Tibetan sheep, yaks, cows, chickens, pigs, and horses, and the 2612 animal serum samples were investigated the specific anti-T. gondii IgM antibodies in different areas among Tibetan sheep, yaks, cows, chickens and pigs from Qinghai. The overall seropositive rates of IgG and IgM in all investigated animals were 44.1% (1179/2673) and 18.0% (469/2612), respectively (Table 2). Moreover, as shown in Table 3, the 47.1% (1258/2673) animals showed at least one T. gondii specific-IgG or IgM was positive. Analysis of the positive animals, showed that the 14.9% (389/2612) sera were determined to be both IgG and IgM positive samples, 30.2% (789/2673) were single-IgG seropositive, and a total of 80 in 2612 serum samples (3.0%) were single-IgM seropositive which contains 3.7% (34/907), 2.0% (14/663), 8.8% (18/205), 0.6% (3/500) and 3.3% (11/337) single-IgM positive animals for Tibetan sheep, yaks, cows, chickens and pigs, respectively (Table 3).     (Figure 3A,B). For the IgM antibody positivity, the pig was also the most prevalent animal (41.8%, 141/337), followed by Tibetan sheep (21.2%, 191/907), cows (15.1%, 31/205), chickens (12.4%, 62/500) and yaks (6.6%, 44/663) ( Figure 3C,D), respectively. To analyze the effect of altitude in the seroprevalence, all serum samples were divided into three groups, 2000-3000, 3000-4000, and 4000-5000 m ( Table 4). As shown in Table 4, the analyzed results showed that the T. gondii IgG and IgM seroprevalence among the altitude groups was significantly different (p < 0.05). Moreover, the seroprevalence of IgG and IgM significantly differed among animal species (p < 0.05).

Discussion
Tibetan sheep, yaks, cows, chickens, pigs, and horses play an important role in the agricultural economy in the QTPA, and are the main food-borne animals. Tibetan sheep, famous for their high-quality pelage and their nutritious delicious meat, are widely distributed in Qinghai. The majority of yaks in the world are living on the QTPA, while approximately 35.0% (~4.9/14 million of the yaks) of the yaks in a total of the QTPA are distributed in Qinghai province [22]. Toxoplasmosis has a wide distribution in this plateau area and is very harmful to both human health and the livestock industry. The current study provides a valuable note of the serological prevalence of T. gondii IgG antibodies and the first record of epidemiology of T. gondii IgM antibodies in all these surveyed animals on the whole Qinghai province, which contains two cities and six prefectures.
Since the indirect ELISA test has been accepted as the most practical test in the diagnosis of toxoplasmosis especially for a large number of samples, it has been used for seroprevalence and epidemiology of T. gondii in humans and animal species such as cat, cattle, sheep, goat and pig [18,27]. Therefore, in this study, we choose the indirect ELISA method to sero-detect the antibodies of T. gondii in sera samples from different animal species. The SAG2-based ELISA, presents its immunodominant nature, could determine T. gondii-specific antibodies with the high sensitivity from both acute and chronic T. gondii infection when used either alone or in combination with the other proteins. Importantly, the recombinant SAG2 protein has adequate diagnostic sensitivity and specificity for both T. gondii-specific IgG and IgM antibodies without cross reactivity [28]. Hence, all these special characteristics could make rTgSAG2 a promising candidate for the sero-diagnosis of T. gondii IgG and IgM antibodies in the different animal species in this study. Our study confirmed that current indirect ELISA based on rTgSAG2 not only detected the specific-T. gondii IgG and IgM antibodies but also distinguished them well.
In the current study the seroprevalence of T. gondii infection in Tibetan sheep was 50.7% (460/907). Previous studies showed a lower seroprevalence of T. gondii infection in Qinghai Tibetan sheep (29.8%), evaluated by indirect hemagglutination (IHA) test [23]. In the other two equally important provinces in QTPA, Gansu, and Tibet, adjacent to Qinghai, the seroprevalence results were also lower. In Gansu province was found a seroprevalence of 20.3%, evaluated by modified agglutination test (MAT) [29], and in Tibet province, a seroprevalence of 5.7% was found (26/455), using an IHA [26]. Although limited in current information, testing technologies, and the number of samples, our data indicate the epidemiological prevalence of T. gondii infection in Tibetan sheep with a high rate in Qinghai in the QTPA. T. gondii seroprevalence in sheep and goats varies with altitude and climate, and sheep and goats from the eastern coastal locations have a higher prevalence rate than that of sheep from the west plateau part in China [21]. However, the opposite is that for this plateau organism, we found the highest infection rate in the highest altitude in this study (Table 4). Therefore, the prevalence of pathogens will change with the changes in the living environment of animals, especially in the plateau environment. Moreover, the high prevalence of T. gondii infection in Tibetan sheep may be due to not only oocyst original infection but also transplacental transmission.
T. gondii could indirectly infect humans through ingesting the undercooked or raw meat or milk from yaks or cows. Although cattle are not considered to be an outstanding host for T. gondii [21], the positive rates of 21.1% or 18.5% for T. gondii IgG antibody and 6.6% or 15.1% for T. gondii IgM antibody in yaks or cows were found in this study, respectively. Yak is the main meat consumed by humans in Qinghai, while the cow is the source of milk for humans in the tested area [30]. The current seroprevalence of T. gondii in yaks or cows and the living habits (such as some ethnic groups that consume raw or undercooked meat or with milk) in the QTPA, not only cause economic losses, but also seriously threatens the local human health. Furthermore, the seroprevalence of T. gondii infection in the current study in yaks from Qinghai province was consistent with the average prevalence of T. gondii infection, which was 20.8% ranging from 8.3 to 26.4% in this area in 2008-2014 [30][31][32][33]. This suggests steady epidemiology of T. gondii in yaks in this plateau area. The higher seroprevalence of T. gondii in yaks may be due to geographical factors, such as rivers, foods, and wild animals that these animals share in the large ecological environment.
The current study was the first report for the seroprevalence and epidemiology of T. gondii in chickens, pigs and horses in the Qinghai, though these animals infected with T. gondii have been investigated in other province in China [25,34,35]. Of the investigated animal species in this study, the most prevalent animal for T. gondii infections was the pig, for which seroprevalence was significantly higher than other animals. Moreover, the current seroprevalence in pig showed significantly higher than that of previously reported in Shanxi, Jilin, Chongqing, and Tibet provinces in China [34,[36][37][38]. These differences might be due to different climates and rearing systems. More important is that the contribution of cats and rodents could not be ignored in T. gondii infections [34]. Although the high levels of antibodies to T. gondii were not accompanied by severe clinical symptoms and high mortality in pigs in the current study, it could not be ignored that a serious toxoplasmosis outbreak in the neighboring province (Gansu province) led to pig deaths [39]. Therefore, the high seropositivity of IgG and IgM in pigs found in this study should attract more attention to prevent the outbreak of toxoplasmosis in the investigated area. In addition, the current seroprevalence of T. gondii in chickens was tested to be significantly higher than the average prevalence of T. gondii infection from 2000 to 2017 in China [21]. Generally, the chicken might be used as an indicator of environmental and soil contamination with the Toxoplasma oocysts because chickens could ingest the oocysts from soil or environmental food causing T. gondii infections [40]. Therefore, the local peoples should pay attention to the soil and environment polluted with Toxoplasma oocysts. Furthermore, the current ELISA test based on rTgSAG2 found a slightly lower seroprevalence of T. gondii in horses (13.1%, 8/61) than the average seroprevalence of T. gondii infection (18.0%, 615/3413) in China [35]. Although this study was limited to the number of the horse samples, the high prevalence suggests the horses should not ingest food and water that is probably contaminated by the oocysts of T. gondii that might reduce the seroprevalence.
In this study, our data demonstrated all tested animal species showed a positive for T. gondii infection with the high prevalence of IgG and IgM antibodies in two cities and six prefectures of Qinghai, suggesting that the distribution of toxoplasmosis is ubiquitous across Qinghai. Moreover, analysis of the seroprevalence of T. gondii IgG and IgM in the tested animals from the 44 sampling sites were conveniently chosen from 2174 m to 4897 m altitudes, suggesting the significant differences present in the infection rates at different altitudes. This may be due to the high-altitude areas in grazing areas where exist the possibility of sharing common water and food among animals, while in the low-altitude areas are a large number of activities for humans, and definitive-host cats leading to these food-borne animals are frequently exposed to the infecting source, causing T. gondii infections.
Here, we investigated the seroprevalence of the specific-T. gondii IgG and IgM antibodies. The diagnosis of acute T. gondii infection depends on the detection of Toxoplasma-specific IgG and IgM antibodies. The IgG avidity test and IgM analysis are considered to be suitable methods for determining acute infections [28]. The current results showed that all tested animal species present the single-IgG positive animals, both IgG and IgM positive animals, and single-IgM positive animals ( Table 3). Considering that IgM is a T. gondii-specific antibody that appears in the early stage of acute Toxoplasma infection, and as the infection progresses, IgG and IgM antibodies will be observed together, while the IgM will reduce and IgG will keep the high level in the later stage of the infection, and the infection will turn into a chronic T. gondii infection. Therefore, our study attests that these animals from the different altitude sampling areas tested are suffering from acute or chronic Toxoplasma infection or have become carriers of T. gondii antibodies after the infections. To the best of our knowledge, this study is the first record of the epidemiology of T. gondii IgM antibodies and distinguishes T. gondii-specific IgG and IgM antibodies in these food-borne animals in the Qinghai province and in China.

Sample Collection of Tibetan Sheep (Ovis aries), Yaks (Bos grunniens), Cows, Chickens, Pigs and Horses
In this study, a total of 2673 serum samples were collected from the different animals, and the 44 sampling sites were conveniently chosen from 2174 m above sea level to 4897 m in the two cities and six prefectures of the QTPA from June to November 2020 (Figure 1), including apparently healthy Tibetan sheep (n = 907), yaks (n = 663), cows (n = 205), chickens (n = 500), pigs (n = 337), and horses (n = 61) ( Table 1). All procedures were carried out according to the ethical guidelines of Qinghai University.

Serum Harvest
These animal blood samples were kept in an icebox, then sent to the State Key Laboratory of Plateau Ecology and Agriculture, Qinghai University. The samples were centrifuged at 5000 rpm for 10 min, 4 • C to separate and harvest sera and stored at −20 • C until used.

Recombinant Protein Expression
The recombinant TgSAG2 was expressed with previously described methods [27], with slight modifications. The concentration of rTgSAG2 protein was measured with a bicinchoninic acid protein assay kit (Thermo Fisher Scientific, Inc., Rockford, IL, USA).

Indirect ELISA
The 96-well ELISA plates were coated with 1 µg/ml rTgSAG2 protein diluted in coating buffer (0.05 M Carbonate-Bicarbonate, pH 9.6) and incubated at 4 • C overnight. The ELISA plates were washed by PBS-T (0.05% Tween-20) three times, and then blocked with 3% skimmed milk, then washed once. Collected sera were diluted by 1:100 and incubated for 1 h at 37 • C. The plates were washed with PBS-T six times. Then, the HRP conjugated anti-IgG or IgM secondary antibodies of the corresponding species (Bethyl Laboratories, Montgomery, TX, USA), were diluted 1:4000 for the Tibetan sheep, yak, cow, chicken, pig and horse sera, and added and incubated for another 1 h at 37 • C. After washing six times, TMB, (3, 3 , 5, 5 -Tetramethylbenzidine) substrate containing 2.5% H 2 O 2 was used to develop the reaction for 20 min, and 50 µL stop solution (2 M sulfuric acid) was added to each well to stop the action of horseradish peroxidase in the substrate. The results were measured at OD 450 nm. The cut-off point was calculated as the mean values of OD 450 nm for standard Toxoplasma-negative sera kept in our laboratory (ten samples of each animal) plus three times the standard deviations of OD450 values of these negative controls. The positive and negative serum samples of mice for T. gondii (kept at in our Lab) or the positive serum samples of mice for N. caninum (gift from Prof. Lijun Jia from Yanbian University, Jilin, China) were set as control to confirm the rTgSAG2-based indirect ELISA.

Statistical Analysis
To graph and analyze the data, GraphPad Prism 8 software (GraphPad Software Inc., USA) was used. The prevalence and 95% confidence intervals per pathogen species were calculated using the OpenEpi program (https://www.openepi.com/Proportion/ Proportion.htm, accessed on 15 November 2020). The chi-squared test and the logistic regression analysis were used to compare proportions of detected sample positivity in different regions and among different animals. The differences were considered to be statistically significant when the resulting p-values were lower than 0.05.

Conclusions
This study demonstrates the high seroprevalence for the specific-T. gondii IgG and IgM antibodies among Tibetan sheep, yaks, cows, chickens, pigs, and horses in the QTPA. These give the new valuable data on the epidemiology of T. gondii in the plateau area, and suggest that we should pay more attention to improve the animal's survival environment, provide more water sources, avoid exposure to the cat, improve animal welfare, and strengthen future prevention and control of T. gondii infection in these food-borne animals in this region. However, the current study is limited to the range of sampling points, the number of samples, and the lack of records for the definitive host cat's activity around the sampling point; thus, the serological prevalence of T. gondii infections should likewise be extensively investigated in animals of food-borne and economic importance, including cats and other meat-producing animals. Future studies should assess the epidemiology of T. gondii in these local animal species.