First report of Toxoplasma gondii infection in market-sold adult chickens, ducks and pigeons in northwest China

Background Toxoplasma gondii infection is a global concern, affecting a wide range of warm-blooded animals and humans worldwide, including poultry. Domestic and companion birds are considered to play an important role in the transmission of T. gondii to humans and other animals. However, little information on T. gondii infection in domestic birds in Lanzhou, northwest China was available. Therefore, this study was performed to determine the seroprevalence of T. gondii infection in domestic birds in Lanzhou, northwest China. Methods In the present study, the seroprevalence of T. gondii antibodies in 413 (305 caged and 108 free-range) adult chickens, 334 (111 caged and 223 free-range) adult ducks and 312 adult pigeons in Lanzhou, northwest China, were examined using the modified agglutination test (MAT). Results 30 (7.26%) chickens, 38 (11.38%) ducks and 37 (11.86%) pigeons were found to be positive for T. gondii antibodies at the cut-off of 1:5. The prevalences in caged and free-range chickens were 6.23% and 10.19% respectively, however, statistical analysis showed that the difference was not significant (P > 0.05). The seroprevalences in caged and free-range ducks were 6.31% and 13.90% respectively, but the difference was not statistically significant (P > 0.05). Conclusions The results of the present survey indicated the presence of T. gondii infection in adult chickens, ducks and pigeons sold for meat in poultry markets in Lanzhou, northwest China, which poses a potential risk for T. gondii infection in humans and other animals in this region. This is the first seroprevalence study of T. gondii infection in domestic birds in this region.


Conclusions:
The results of the present survey indicated the presence of T. gondii infection in adult chickens, ducks and pigeons sold for meat in poultry markets in Lanzhou, northwest China, which poses a potential risk for T. gondii infection in humans and other animals in this region. This is the first seroprevalence study of T. gondii infection in domestic birds in this region.

Background
Toxoplasma gondii is an important intracellular protozoan parasite, widely prevalent in humans and animals, including poultry throughout the world [1][2][3]. T. gondii infection is generally transmitted either congenitally, or via ingestion of undercooked or raw meat from infected animals, or ingestion of food or water contaminated with oocysts excreted by infected felids [1,2,4]. Free-range (FR) chickens, an important intermediate host, are considered one of the best indicators for soil contamination with T. gondii oocysts because of their feeding style [2]. Low numbers of exposed poultry develop clinical symptoms, such as encephalitis, chorioretinitis and neuritis, however, poultry meat is an important part of cuisine, consumed widely all over the world, and consumption of uncooked poultry meat or not properly cooked poultry meat is a risk factor for T. gondii infection in humans or other animals [1,2].
In recent years, seroprevalence studies of T. gondii in chickens, ducks and pigeons have been conducted extensively in various parts of the world [1,2,[5][6][7], there have been some surveys in mainland China [8][9][10]. However, little is known about the prevalence of T. gondii in chickens, ducks and pigeons in northwest China. Here, we report T. gondii seroprevalence in domestic birds in Lanzhou, northwest China for the first time.

Methods
The study area The survey was conducted in Lanzhou City (35°5′~38°N , 102°30′~104°30′ E), the Capital of Gansu Province, northwest China. Lanzhou is situated in the geometric center of China, covering an area of approximately 13,000 square kilometers, with an average altitude of 1,500 meters. The climate of this city is typically temperate and monsoonal continental, with an average annual temperature of 9.3°C, and an annual precipitation of 360 mm.

Blood samples
A total of 413 blood samples from adult chickens, 334 blood samples from adult ducks and 312 blood samples from adult pigeons were collected from animals slaughtered and sold for meat in four poultry markets (poultry market A, B, C, D) in Lanzhou, northwest China between April and November 2011. Blood samples were transported to the laboratory in Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou, Gansu Province, China, kept at room temperature for 2 hr, centrifuged at 3,000 g for 10 min, then clear serum was separated. The serum samples obtained were stored at −20°C until further analyzed. Owners of poultry were asked for information of animal husbandry practices.

Serological examination
Antibodies to T. gondii were determined in chicken, duck and pigeon sera by the modified agglutination test (MAT) as described previously [2,8,11,12]. In brief, sera were added to the "U" bottom of 96 well microtiter plates, and diluted two-fold starting from 1:5 to 1:160. Bird sera with MAT titers of 1:5 or higher were considered positive for T. gondii antibodies based on previous studies [2,8,[13][14][15], those sera with doubtful reactions were re-tested, and positive and negative controls were included in each test.

Statistical analyses
Differences in the seroprevalence of T. gondii-infected chickens and ducks and between free-range and caged groups were analyzed using a Chi square test in SPSS for Windows (Release 18.0 standard version, SPSS Inc., Chicago, Illinois). The differences were considered statistically significant when P < 0.05.

Results
One hundred and five (9.92%) out of 1059 serum samples were assayed positive for T. gondii by MAT (Table 1). Of these, a total of 30 (7.26%) out of 413 chickens were seropositive and antibody titers were 1:5 in 20, 1:10 in seven, 1:20 in one, 1:40 in one and 1:80 in one chicken ( Table 1). As shown in Table 2, the seroprevalence varied in different poultry markets, ranging from 1.73% to 10.19%, and the seroprevalence in caged and free-range chickens was 6.23% and 10.19%, respectively.
Antibodies to T. gondii were found in 38 (11.38%) out of 334 ducks, and antibody titers were 1:5 in 33 and 1:10 in five ducks (Table 1). T. gondii seroprevalences in ducks came from four different poultry markets ranging from 0% to 18.07% ( Table 2). The investigation revealed that the prevalence in caged and free-range ducks was 17.61% and 12.72%, respectively.

Discussion
In this investigation, seroprevalence of T. gondii infection in chickens was 7.26%, which was lower than that observed in other countries [2], and also lower than that reported in Jingzhou city (25.17%) [10], but similar to that in Zhangjiakou city (7.41%) [9], and Guangzhou city (8.43%) [8]. The seroprevalence in caged chickens and free-range chickens was 6.23% and 10.19%, respectively, although the difference was not statistically significant (P > 0.05). The overall T. gondii seroprevalence in ducks in Lanzhou was 11.38%, which was lower than that reported in other countries [16][17][18], and also lower than that reported in Guangzhou city (16%) [8]. The differences in seroprevalence may due to differences in ecological and geographical factors. Of these, the T. gondii seroprevalence in caged ducks and free-range ducks were 6.31% and 13.90%, respectively, but the difference was not statistically significant (P > 0.05), probably related to different life styles of the examined ducks.
To our knowledge, there was only one report regarding toxoplasmosis in pigeons in mainland China [14]. In the present study, antibodies to T. gondii were found in 37 (11.86%) pigeons at the cut-off of 1:5, but most had low titres, with MAT titres of 1:5 in 35 pigeons, only 2 pigeons had titres of 1:10 or higher. This seroprevalence was higher than that in Guangdong Province (8.7%) [14], possibly indicating geographical differences. Pigeon meat can serve as a source of T. gondii infection for hunters and other animals, so it would be a risk factor for T. gondii infection in humans or other animals.
In this study, we chose MAT because it is sensitive and specific for detecting T. gondii antibodies in bird species [2,8,[13][14][15], compared to other serologic methods. Moreover, geographical conditions, feeding and living styles, and number of cats and rodents may contribute to the differences in T. gondii seropositivity in birds.

Conclusions
The present survey revealed the seroprevalence of T. gondii in chickens, ducks and pigeons in Lanzhou, northwest China for the first time, which indicated the potential risk of domestic birds as a source of T. gondii infection in humans and other animals in this region.