Seroprevalence of Toxoplasma gondii in slaughtered horses and donkeys in Liaoning province, northeastern China

Background Toxoplasma gondii is an important zoonotic pathogen infecting humans and almost all warm-blooded animals. The most common sources of human infection are ingestion of tissue cysts in raw or undercooked meat. However, limited information is available about T. gondii infection in horses and donkeys in China. In the present study, we report the seroprevalence of T. gondii infection in horses and donkeys in Liaoning province, northeastern China. Methods Serum samples were collected from 711 and 738 slaughtered horses and donkeys, respectively, in 13 regions of Liaoning province. The modified agglutination test (MAT) was used to test the specific antibodies to T. gondii. Results In this study, 178 of 711 (25.0%) horses were seropositive for T. gondii with titers of 1:25 in 81, 1:50 in 62, 1:100 in 33, and 1:200 in 2, and seroprevalence of T. gondii infection from 13 regions ranged from 8.2% to 37.0%. Antibodies to T. gondii were found in 174 of 738 (23.6%) donkeys with titers of 1:25 in 66, 1:50 in 54, 1:100 in 49, and 1:200 in 5, and seroprevalence varied in 13 different regions, ranging from 9.1% to 32.6%. Conclusions The results of the present study indicated that the rate of infection with T. gondii in horses and donkeys is a little high in Liaoning province, northeastern China in comparison to other surveys in China, which suggests that consumption of horse and donkey meat in this area may represent a potential source for human infection with T. gondii.


Background
Toxoplasma gondii parasites are obligate intracellular apicomplexans that can infect virtually all warm-blooded animals; felids are definitive hosts. The most common sources of human infection are ingestion of tissue cysts in raw or undercooked meat or of food or water contaminated with oocysts shed by felids and transplacental transmission [1]. T. gondii infection may cause serious disease in pregnant women and immunocompromised patients, such as AIDS patients and organ transplant recipients [2][3][4][5][6].
Meat from birds and warm-blooded animals traditionally has been considered a major source of T. gondii infection [7]. Viable parasites have been isolated from the meat of these animals, such as sheep, goat, cow, pig, chicken and horse [8]. Horse meat and donkey meat are also important foodstuff worldwide, and humans can acquire infections with T. gondii by ingesting raw or undercooked horse and donkey meat containing tissue cysts.
In equids, T. gondii infections generally display a subclinical course, and serological techniques for the detection of specific antibodies produced in the body against the parasite have a great diagnostic value [8]. Serum antibodies to T. gondii have been found in horses and donkeys in many surveys worldwide [8]. Table 1 summarizes the reports of T. gondii infection in horses and donkeys from China because these papers were published in the Chinese language in local journals and are not easily accessible to foreign scholars. In view of this background, the objective of this study was to survey the seroprevalence of T. gondii infection in horses and donkeys in Liaoning province, northeastern China.

The study area
Liaoning province is located in northeastern China, its geographical position is at east longitude 118°50′ -125°4 7′ and at north latitude 38°43′ -43°29′. It has an area of 145,900 km 2 and a population of approximately 44 million. The area has a temperate monsoon climate with abundant sunshine, a long winter, hot summer, a short spring and autumn. The annual average temperature is 7-11°C, with a highest temperature of 40°C and a lowest temperature of −30°C.

Serological examination
Sera were tested for T. gondii antibodies using 2-fold serial dilutions from 1:25 to 1:3,200 with the modified agglutination test (MAT), as described previously [14]. Briefly, the harvested parasites were kept in 6% formaldehyde solution at 4°C overnight, and suspended in the alkaline buffer at 20,000 parasites/mL. Two-fold dilutions of sera were performed using the serum diluting buffer, and agglutination was performed in U-bottom 96-well microtiter plates using a mixture of 50 μL antigen and 50 μL diluted sera. The plates were incubated at 37°C overnight. The test was considered positive when a layer of agglutinated parasites was formed in wells at dilutions of 1:25 or higher; positive and negative controls were included in each test.

Statistical analysis
Statistical analysis of T. gondii seroprevalence between horse and donkey was performed using a Chi square test with SPSS (SPSS Inc., Chicago, Illinois). A P-value < 0.05 was considered statistically significant.

Ethics statement
All animals were handled in strict accordance with good animal practice according to the Animal Ethics Procedures and Guidelines of the People's Republic of China, and the study was approved by the Animal Ethics Committee of Shenyang Agricultural University (Permit No. SYXK < Liao > 2011-0001).
Some serological tests, including the Sabin-Feldman dye test (DT), latex agglutination test (LAT), indirect hemagglutination (IHA), indirect fluorescent antibody (IFA), enzyme-linked immunoabsorbent assay (ELISA), and modified agglutination test (MAT), have been used to detect T. gondii antibodies in equids; the latter (MAT) is both  sensitive and specific [8]. Worldwide seroprevalence of T. gondii infection in horses has been summarised prior to 2010 by Dubey, ranging from 0.4% to 48.1% [8]; and since then, it has been reported to be 24% in the Czech Republic by LAT [15], 32% in Riyadh Province, Saudi Arabia by DT [16], 34.0% in Costa Rica by MAT [17], 69.6% in the Fernando de Noronha, Brazil by MAT [18], and 10.8% in Southern Spain by MAT [19]. Worldwide seroprevalence of T. gondii infection in donkeys was reported as 65.6% in Egypt by ELISA [8], 11.0% in Gemlik, Turkey by LAT [8], 25.6% in Southern Spain by MAT [19], and 43.2% in Northeast of Brazil by indirect immunofluorescence reaction (IFI) [20]. In China, some surveys have previously reported prevalence of T. gondii infection in horses and donkeys (Table 1), however, it is difficult to compare results of the present study with other surveys in China because of different serological tests used and due to the different regions of China studied. Most surveys used the IHA test, and one survey used ELISA. In this study, we used the MAT for the detection of T. gondii seroprevalence in horses and donkeys because it is the major recommended test for diagnosis of T. gondii infection in several animals and man [8,14]. MAT has high sensitivity and specificity among all serologic methods, and this method is cheaper, easier than other tests and does not need special sophisticated equipment [21,22]. In addition, many surveys in China were conducted many years ago.
In this study, we found that equids with outdoor access may have a greater chance of being exposed to the food and water contaminated with T. gondii oocysts, which had been excreted in feces by cats and might be present in forage and water. This is the reason that seroprevalence of T. gondii infection in equids was relatively high in the present study.
Results of the present study indicated that T. gondii infection is common in horses and donkeys in Liaoning province, and the parasite will remain present in their tissues for life. Horse meat and donkey meat were mostly consumed by local people. Therefore, animals such as horse and donkey are at a high risk of infection and act as a transmission route to humans. Further research on the role of equid meat in human infections and the pathogenesis of T. gondii is needed.
In this study, the seroprevalence results of T. gondii infection in equids were higher than other food animals, including pig, cattle, sheep, chicken, duck, and goose in Liaoning province, northeasern China (Table 4) [22][23][24]. These results also confirm that T. gondii infection in food animals is widespread in northeastern China and should be of public health concern.  MAT [22]