Seroprevalence of Toxoplasma gondii Infection in Pet Dogs in Anhui Province, China

Background: Toxoplasma gondii is an obligate intracellular parasite, which can infect all nucleated cells in a variety of vertebrate animals, including human, causing toxoplasmosis. Although a number of studies have reported on the seroprevalence of T. gondii infection in dogs in China, however, information about T. gondii infection in pet dogs in Anhui, China is not available. Methods: The modified agglutination test (MAT) was used to detect antibodies in sera samples from 468 pet dogs at Anhui Province in China from November 2013 to April 2017. Results: 18.6% animals were T. gondii seropositive, indicating a slightly higher prevalence of T. gondii infection in pet dogs in Anhui, China in comparison with other provinces in China. Conclusion: Our present study provided epidemiological data on T. gondii seroprevalence in pet dogs in Anhui, China for the effective prevention and control of the parasite prevalence in this area.


Introduction
oxoplasma gondii is an obligate intracellular parasite, which infects all nucleated cells in humans and a broad spectrum of vertebrate hosts, leading to zoonosis termed toxoplasmosis (1). About one third of the human population was chronically infected with this parasite, and most of them were asymptomatic (2).
Currently, little national data has been available on the chronic infection rate of T. gondii in China, and a large-scale serological survey of T. gondii infection among persons participated in health screening demonstrated that the total seroprevalence of T. gondii infection was 6.67% in Yunnan Province, Southwestern China (3).
Human can be infected by eating undercooked meat containing tissue cysts or oocyst contaminated food and water. Moreover, Toxoplasma can be transmitted congenitally, and has been associated with severe disease, including toxoplasmic encephalitis, blindness, abortion, fetal abnormalities, and even prenatal death could be caused in Toxoplasmainfected pregnant women were infected with T. gondii (4). Toxoplasma is an opportunistic pathogen, which can be lethal for immunocompromised patients (e.g. HIV/AIDS patients) (5).
With the current economic and social development of China, pet ownership has increased in China, and dogs are the most preferred pets.
However, previous studies have shown that dogs may be involved in the mechanical transmission of a number of zoonotic diseases, including T. gondii to humans (6). Thus, it is important to survey the seroprevalence of T. gondii infection in dogs and assess to what extent dogs constitute a reservoir and source of infection for humans. A number of studies have been reported about the T. gondii infection in dogs in China over the last 10 years (7-11), but only limited surveys of T. gondii infection in dogs in Anhui Province are available (12).
The aim of present study was to investigate the seroprevalence of T. gondii infection in pet dogs in Anhui province of China.

Ethics statement
The present study was approved by the Animal Ethics Committee of Anhui Medical University (Permit code AhMU20130086). Dogs from which sera samples were collected were handled in accordance with the Animal Ethics Procedures and Guidelines of the People's Republic of China.

The site
Anhui province is located at Central China, covering an area of 140,100 km 2 and a population of approximately 70.7 million people ( Fig.  1).

Fig. 1: Geographic location of the present survey
Anhui is situated between east longitudes of 114°54' to 119°37'and north latitudes of 29°41' to 34°38'. It has a typical subtropical monsoon climate, with an annual rainfall of 773-1670 mm, and an average annual temperature of 14-17°C. The average annual rainfall of Anhui is 773-1, 670 mm, and the average annual temperature is 14-17 °C, both of which are suitable conditions for the development of T. gondii oocysts in environments.

Collection and preparation of serum samples
Our survey was conducted between November 2013 and April 2017. A total of 468 blood samples were collected from pet dogs. These pet dogs were admitted into pet hospitals located in eight cities of Anhui Province, namely Hefei, Huainan, Fuyang, Bozhou, Wuhu, Xuancheng, Huangshan and Chizhou. Information regarding the ages and genders of the pet dogs was provided by dog owners. For each dog, at least 3 ml of blood was drawn by jugular venipuncture into a syringe. Blood samples were transferred to tubes and kept at 37 °C for 2 h (h), centrifuged at 2,000 g for 5 min, and the sera samples were stored at -20 °C until further processing.

Serological examinations
The modified agglutination test (MAT), a sensitive and specific technique was used for detecting T. gondii antibodies (13). Briefly, a suspension of Toxoplasma tachyzoites was prepared and fixed with formalin. Sera samples were diluted in phosphate buffered saline (PBS, pH 7.2). Other materials used in this study included positive and negative control sera (kindly provided from Jilong Shen group, Anhui Medical University), antigen diluting buffer containing bovine serum albumin (BSA), 2-mercaptoethanol, and Evans blue dye solution. MAT titers of 1:20 or higher were considered as positive (14), and those sera with dubious results were re-tested. Positive and negative controls were incorporated in each test and investigated at the same dilutions of serum samples.

Statistical analysis
Differences in the seroprevalence of T. gondii infection between male and female dogs, and among different age groups were analyzed using a Chi square test using the SPSS (Release 20.0 standard version, SPSS Inc., Chicago, Illinois). Differences were considered statistically significant at P < 0.05.

Results
Out of 468 pet dogs in Anhui Province, 87 (18.6%, 95% CI 15.1~22.1) were seropositive for T. gondii (Table 1). Antibody titers of 1:20 were found in 42 dogs, 1:40 in 31, 1:80 in 14, while 1:160 or higher was not detected. The variables associated with T. gondii seroprevalence in pet dogs were also analyzed in present study. We found that the seroprevalence of T. gondii infection in males was slightly higher than that in females (
Although there was no significant difference among age groups, the seroprevalence of tox-oplasmosis in pet dogs increased progressively with age ( Table 2, except 2-3 years group), indicating that the exposure of pet dog to T. gondii oocysts in the environments or tissue cysts in meat may contribute to the spread of toxoplasmosis. A large-scale investigation is needed to understand why seroprevalence of T. gondii infection in pet dogs aged 2-3 years was lower.
Consistent with results from previous study (16), the seroprevalence of T. gondii infection in males was slightly higher than that in females. This may be ascribed to male dogs having more opportunities to come into contact with environments, which may be contaminated with oocysts of T. gondii. Anhui province is geographically divided by Yangzi River into two parts, North Anhui and South Anhui (Fig.  1). These two parts are different in climate. The climate is dry in North Anhui area, while South Anhui is moist, which is more suitable for T. gondii oocyst development. This may explain the difference between seroprevalence of T. gondii in pet dogs in North Anhui and that in South Anhui.

Conclusion
The overall seroprevalence of T. gondii in pet dogs in Anhui Province, China was 18.6%. Our study provides baseline data for the effective prevention and control of the parasite prevalence in this area. Further studies will be conducted to isolate T. gondii strain from infected dogs and determine the genotypes of T. gondii infected dogs in Anhui area.