Seroprevalence of Toxoplasma gondii in horses and donkeys in Yunnan Province, Southwestern China

Background Toxoplasma gondii is an intracellular protozoan parasite that infects almost all warm-blooded animals, including humans, with a worldwide distribution. There have been limited reports about the seroprevalence of T. gondii infection in equids around the world and little is known about the seroprevalence of T. gondii in equids in southwestern China, in particular in Yunnan Province. The objective of the present investigation was to estimate the seroprevalence of T. gondii infection in equids in this area. Methods A total of 399 serum samples (266 from horses and 133 from donkeys) were collected in 2012, and assayed for T. gondii antibodies by Indirect Haemagglutination (IHA) test using a commercially available kit. Results A total of 108 (27.1%) equids, including 81 (30.5%) horses and 27 (20.3%) donkeys were positive for T. gondii antibodies, and the seroprevalence ranged from 18.8% to 37.5% among different sampling areas. The seroprevalence was 27.4% and 26.8% for male and female equids, respectively, and the difference was not statistically significant (P > 0.05). The seroprevalence ranged from 21% to 32.9% among different age groups, and the difference was not statistically significant (P > 0.05). Conclusions The results of the present survey indicated the existence of high T. gondii seroprevalence in Yunnan Province, southwestern China, which has significant public health concern. Therefore, it is imperative that improved integrated measures be carried out to prevent and control T. gondii infection in equids in the studied region.


Background
Toxoplasma gondii is an important zoonotic parasite, which can infect humans and almost all warm-blood animals, with a worldwide distribution [1][2][3][4][5]. Toxoplasmosis is not only of great importance for livestock and causes huge economic loss to the livestock industry, it is also a public health problem owing to its transmission to humans by ingestion of uncooked meat containing tissue cysts, or consuming food or drink contaminated with oocysts, or accidental ingestion of sporulated oocysts from the environment [1][2][3][4][5]. Although T. gondii infection rarely displays obvious clinical symptoms in adults, it may lead to severe consequences in an immunodeficient person such as an AIDS patient, and infection in pregnant women may lead to abortion, still birth, or other serious congenital consequences in newborns [1][2][3].
Horses and donkeys are important and useful animals to humans in many ways, such as sport competitions, police work, carriage, and so on. Horse and donkey meat are also the popular and delicate food for people worldwide. Infection of T. gondii is subclinical in horses, atypical clinical signs includes fever, ataxia, retinal degeneration and encephalomyelitis, and abortion or stillbirth in pregnant equids. Human toxoplasmosis cases associated with consumption of horse meat have been reported in some countries [6,7]. Yunnan Horse and Yunnan Donkey are fine equine breeds in China, they play very important roles in agriculture and tourism, and meat of horse and donkey are considered as special delicacy dishes in local restaurants in Yunnan Province. However, data on horse and donkey infection with T. gondii is limited in southwestern China. The objective of the present survey was to examine the seroprevalence of T. gondii infection in horses and donkeys in Yunnan Province, southwestern China, and the results would provide fundamental data for prevention and control of T. gondii infection in equine animals, and also will provide base-line information on potential risk factors associated with public health.

Ethics statement
The collection of serum samples from equids in the present study was consented by owners of equids, and all horses and donkeys were handled in strict accordance with good animal practice according to the Animal Ethics Procedures and Guidelines of the People's Republic of China.

Blood samples
A total of 399 blood samples (266 from horses and 133 from donkeys) were collected via jugular vein of animals in 2012 in Yunnan Province. Whenever possible, data regarding species/breed, geographic origin, age and gender of each animal were collected. The ages of animals were classified into four categories according to their growth cycle: foal (0 < yr ≤ 1, 73 samples), adolescent (1 < yr ≤ 5, 108 samples), middle age (5 < yr ≤ 10, 138 samples) and elderly age (yr > 10, 80 samples), 190 were males and 209 were females. All samples were sent to the laboratory in Kunming and centrifuged (3,000 rpm) for 5 min,  and the serum samples were kept at −20°C until assayed for antibodies to T. gondii.

Serological assay
Antibodies to T. gondii were detected in serum samples by an indirect hemagglutination antibody (IHA) test using a commercially available kit (Veterinary Research Institute, Jiangsu Academy of Agricultural Sciences, Nanjing, China) according to the recommended protocol of the manufacturer for the detection of antibodies to T. gondii in animals. The serum sample was considered as positive when a layer of agglutinated erythrocytes was observed in wells with dilutions of 1:64 or higher.

Statistical analysis
Statistical analysis of T. gondii seroprevalence in different regions, ages and genders of animals were performed using Generalized Lineal Model (GLM) test in the SPSS software (Release 18.0 standard version, SPSS Inc., Chicago, Illinois), and P < 0.05 was considered statistically significant.
The different seroprevalence results may due to differences in hygiene conditions, climates, and the prevalence of T. gondii in cats, as well as the sensitivity of the serological methods. Our preliminary survey showed that the prevalence of T. gondii oocysts in the faeces of stray cats in Yunnan was 25.6% (unpublished data), indicating a high risk as a source of T. gondii infection for equids, other animals and humans. In the present study, we used IHA to detect antibodies to T. gondii in horse and donkey serum samples because IHA is considered one of the most sensitive and specific serological methods for detecting T. gondii antibodies in equids, other animals and humans [31][32][33][34][35], and it have been extensively used in China ( Table 2, [32][33][34][35]). The cutoff value of 1:64 was used according to the national standard (GB/T 18448.  of China for detection of T. gondii antibodies in humans and animals. Due to the population size of donkeys in Yunnan Province, the sampled number (No = 133) of donkeys in the present study was small, and the results of the present survey may not reflect the accurate T. gondii seroprevalence in donkeys. Nevertheless, the present investigation revealed that horses and donkeys in Yunnan Province had a high T. gondii seroprevalence, indicating a potential threat to public health in this province, which is one of the most famous tourist destinations in China.

Conclusions
The results of the present study indicate that T. gondii seroprevalence in horses and donkeys in Yunnan Province is quite high, consumption of horse or donkey meat is likely to be a risk factor for human infection with T. gondii. Therefore, it is imperative to take prevention and control measures to reduce T. gondii prevalence in equine animals in this province.