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Toxoplasmosis is a zoonotic disease caused by the obligate intracellular protozoan parasite T. gondii which is widely prevalent in humans and animals worldwide. The diagnosis of toxoplasmosis and distinguishing acute or chronic T. gondii infections have utmost importance for humans and animals. The TgSAG1, TgGRA7, and TgBAG1 proteins were used in the present study to develop the serological rSAG1-ELISA, rGRA7-ELISA and rBAG1-ELISA methods for the testing of T. gondii specific IgG and IgM antibodies and differentiating acute or chronic toxoplasmosis in 3733 animals, including Tibetan sheep, yaks, pigs, cows, cattle, horses, chickens, camels and donkeys from the Qinghai-Tibetan Plateau. The ELISA tests showed that the overall positivity of IgG antibody was 21.1% (786/3733), 15.3% (570/3733) and 18.2% (680/3733) for rSAG1-, rGRA7- and rBAG1-ELISA, respectively, and the positivity of IgM antibody was 11.8% (439/3733), 13.0% (486/3733) and 11.8% (442/3733) for rSAG1-, rGRA7- and rBAG1-ELISA, respectively. A total of 241 animals (6.5%) positive for all rSAG1-, rGRA7- and rBAG1-IgG were found in this study, and the 141 animals (3.8%) tested were anti-T. gondii IgM positive in all three ELISAs. Moreover, the 338, 284 and 377 animals were IgG positive in rSAG1 + rGRA7-, rBAG1 + rGRA7- and rSAG1 + rBAG1- ELISAs respectively, and the 346, 178 and 166 animals in rSAG1 + rGRA7-, rBAG1 + rGRA7- and rSAG1 + rBAG1-ELISAs were IgM positive respectively. The results confirmed that the application of SAG1, GRA7, and BAG1 recombinant antigens could successfully be used in the detection of specific IgG and IgM antibodies for distinguishing between acute or chronic T. gondii infections. It is inferred that the forms in which current animal species in the plateau area were infected with T. gondii, and the period of infection or the clinical manifestations of the current infections may be different. The present study provides substantial clinical evidence for the differential diagnosis of toxoplasmosis, and the classification of acute and chronic T. gondii infections.

Neosporosis is a worldwide infectious disease caused by intracellular parasite Neospora caninum that is a major pathogen of abortion in cattle and neurological disorders in other hosts. However, limited data are available on animals exposed to N. caninum in the Qinghai-Tibetan Plateau Area (QTPA), and little is known about whether animals in the plateau area play an important role in the epidemiology of N. caninum. Therefore, indirect ELISAs based on a combination of NcSAG1 and NcGRA7 antigens were developed to examine both N. caninum-specific IgG and IgM antibodies in Tibetan sheep, yak, cow, pig, cattle, horse, chicken, camel, and donkey from the QTPA in this study. The results showed that all current species present- IgG and IgM-positive animals, and that the overall seroprevalence of N. caninum were 18.6 (703/3,782) and 48.1% (1,820/3,782) for the IgG and IgM antibodies, respectively. Further analysis found significant differences from different altitudes in IgG in Tibetan sheep and IgM in the yak. Hence, the present serological results indicate that the tested animal populations in the QTPA are suffering from N. caninum infections or have become carriers of N. caninum antibodies. To the best of our knowledge, this is the first report on current N. caninum-infected animals in the QTPA, the first epidemiology of neosporosis in cow and camel in China, and the first record of N. caninum IgM antibodies in all the surveyed animals in China. This study provides the latest valuable data on the epidemiology of neosporosis in China and in plateau areas of the world.

Anaplasma genus infects the blood cells of humans and animals by biting, causing zoonotic anaplasmosis. However, limited data are available on carrier animals for Anaplasma spp. antibodies in the Qinghai–Tibetan Plateau Area. Therefore, a serological indirect ELISA diagnostic method based on the major surface protein 5 (MSP5), derived from Anaplasma phagocytophilum, was developed in this study to analyze both IgG and IgM antibodies of Anaplasma spp. in a total of 3952 animals from the Qinghai–Tibetan Plateau, including yaks (Bos grunniens), cows (Bos taurus), cattle (Bos taurus domesticus), Tibetan sheep (Ovis aries), horses (Equus ferus caballus), pigs (Sus domesticus), chickens (Gallus gallus domesticus), donkeys (Equus asinus), stray dogs (Canis sp.), and stray cats (Felis sp.). The results showed that recombinant MSP5 protein was expressed and was successfully used to establish the indirect ELISA methods. The overall positivity for Anaplasma IgG and IgM antibodies was 14.6% (578/3952) and 7.9% (312/3952), respectively, and a total of 123 animals (3.1%) were both IgG- and IgM-positive. Moreover, the most prevalent Anaplasma IgG positivity was exhibited by donkeys (82.5%), followed by stray dogs, Tibetan sheep, pigs, chickens, horses, yaks, cows, cattle, and stray cats. The analysis for IgM antibody positivity revealed that IgM positivity was the most prevalent in the stray dogs (30.1%), followed by horses, yaks, Tibetan sheep, cows, stray cats, and cattle. Moreover, the results revealed significant differences (p < 0.05) at different altitudes in Anaplasma-specific IgG in the yaks, Tibetan sheep, and horses, and in IgM in the yaks and Tibetan sheep. In conclusion, this study is the first to demonstrate that yaks, cows, cattle, Tibetan sheep, horses, donkeys, stray dogs, stray cats, pigs, and chickens living in the Qinghai–Tibet Plateau are carrier animals for Anaplasma spp. IgG or IgM antibodies. The current findings provide valuable current data on the seroepidemiology of anaplasmosis in China and for plateau areas of the world.

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.