Seroprevalence of Toxoplasma gondii infection in domestic cats in central China

To investigate the seroprevalence of Toxoplasma gondii infection in domestic cats in central China, 843 serum samples were collected in Henan province between March 2015 and May 2016 and tested for IgG antibodies against T. gondii using the enzyme-linked immunosorbent assay (ELISA). The overall seroprevalence of T. gondii was 21% (178/843). No significant difference was observed based on the sex of cats (p > 0.05). Significantly higher seroprevalence (p < 0.05) was observed in mixed-breed cats (24%) compared to purebred cats (17%). Seroprevalence in rural cats (29%) was significantly higher (p < 0.01) than in urban cats (16%), and increased significantly (p < 0.01) with age. These results showed that T. gondii was highly prevalent in domestic cats in Henan province, central China, which might have important implications for public health.


Introduction
Toxoplasma gondii is an obligate intracellular protozoan parasite that has a worldwide distribution and infects a wide range of warm-blooded vertebrates, including humans [3]. The ingestion of undercooked meat containing tissue cysts and the ingestion of food or drink contaminated with sporulated T. gondii oocysts are important modes of transmission of T. gondii. As definitive hosts of this parasite, cats play an important role in the life cycle of T. gondii, facilitating the genetic recombination between strains, as well as environmental contamination [12]. Antibodies to T. gondii have been reported in cats worldwide and seroprevalence in cats was recently summarized [2,4,8,9]. The objective of the present survey was to determine the seroprevalence of T. gondii in domestic cats in Henan province, central China, and to evaluate the main associated risk factors related to exposure to T. gondii in this region [6,14].

Ethics statement
The study was reviewed and approved by the Ethics

Study site
The study was conducted in Henan province in the central part of mainland China, covering an area of 167, 000 km 2 and having a population of approximately 106 million. Its geographical position is at east longitude 110°21 0 -116°39 0 and at north latitude 31°23 0 -36°22 0 . The Yellow River passes through central Henan. The area has a continental monsoon climate, with four distinctive seasons. The average annual temperature is 12.1-15.7°C, with a mean annual rainfall of 532.5-1380.6 mm. There are 17 provincial cities distributed in Henan province, with the city of Zhengzhou as its capital. Five cities, , located in the northern, western, central, southern, and eastern parts of Henan province, were selected for sample collections.

Sample collection
A total of 843 blood samples of domestic cats were collected from these five cities in Henan province between March 2015 and May 2016. Cat owners were asked for details of the animal's age, sex, source, and breed using a structured questionnaire. Blood samples were centrifuged and sera were recovered and transferred to 1.5 mL Eppendorf tubes. All the sera were then stored at À80°C until tested for anti-T. gondii antibodies.

Determination of antibodies to T. gondii
Antibodies to T. gondii were determined using the commercial T. gondii IgG ELISA (enzyme-linked immunosorbent assay) Kit (Combined Company, Shenzhen, Guangdong Province, China) according to the manufacturer's instructions [5,13]. The manufacturer's test report showed that the sensitivity and specificity of this ELISA kit were 95% and 100%, respectively. Positive and negative control sera were provided in the kit. Briefly, the T. gondii specific antigen was used to coat a 96-well ELISA plate. After incubation of the diluted serum sample (1:100) in the test well and subsequent washing, a conjugate was added. The plate was washed again and the chromogenic enzyme substrate was added. Finally, the optical density (OD) values of each well were measured using an ELISA plate reader (Multiskan MK3; Thermo Scientific, Waltham, MA, USA) at 450 nm.
A relative rate percent (IRPC) value was obtained using the following formula:

Statistical analysis
Differences in T. gondii prevalence for different variables such as age, breed, and sex were analyzed using a chi square test. Statistical analysis was performed using SPSS 20 software for Windows (SPSS Inc., Chicago, Illinois, USA). The differences were considered statistically significant if p < 0.05.
The prevalence of T. gondii infection in cats increased significantly (p < 0.01) with the increase in age.

Discussion
The present survey showed that T. gondii seroprevalence in cats in rural areas was significantly higher compared to urban cats (p < 0.01), which is consistent with reports by other authors [1,10]. The difference in seroprevalence in urban and rural areas might be explained by differences in local reservoirs of the parasite, both in prey animals and in the environment, which serve as local infection sources for the cats. In Henan, domestic cats are usually kept outdoors in rural areas and often roam more freely with greater access to parasites. In addition, the number of rodents in rural areas is much higher than that in urban areas. Higher T. gondii seroprevalence found in cats in rural areas probably results from the carnivorous behavior of cats living outdoors and eating prey animals such as rodents and birds. The higher seropositivity with increasing age of the cats supports the hypothesis that most cats acquire T. gondii infection after weaning [1,7,13].
In order to protect public health, more measures should be taken. The proper disposal of cat litter, keeping cats indoors to minimize their acquisition of infection from prey or the environment, and reducing the feral cat population are recommended. Reducing close contact with cats and protecting the play areas of children might potentially reduce the oocyst burden [11]. What is more, children, pregnant women, and immunocompromised people should adhere to hygiene principles after contact with soil and cats. Given the high seroprevalence of T. gondii IgG antibodies in cats, performing a screening test and determination of the IgG antibody titer in high-risk populations such as pregnant women in Henan province, central China is recommended.