Genetic Characterization of Toxoplasma gondii from Wild Rodents in Sichuan Province, Southwestern China.

Background
Wild rodents are the intermediate hosts of Toxoplasma gondii. The distribution of genetic diversity of T. gondii in wild rodents is of importance to understand the transmission of this parasite. This study aimed to genetically characterize T. gondii isolates from wild rodents in Sichuan province, southwestern China in 2013.


Methods
Genomic DNA was extracted from 10 g wild rodents' brain samples. Semi-nested PCR and multilocous PCR-RFLP technology were performed to examine genetic diversity of T. gondii isolates as described previously.


Results
Overall, 181 brain tissues of different wild rodents, including Eothenomys miletus (n=88), Crocidura attenuate (n=9), Rattus rattus sladeni (n=46), Mus musculus Linnaeus (n=6) and R. niviventer (n=32) were tested for T. gondii DNA, respectively. Six of them were positive for the T. gondii B1 gene by semi-nested PCR amplification, 4 showed complete genotyping results for all 11 polymorphic loci (SAG1, SAG2, alt. SAG2, SAG3, BTUB, GRA6, L358, PK1, C22-8, C29-2 and Apico) by PCR-RFLP, determined to represent a potential new genotype (http://toxodb.org/toxo/).


Conclusion
These results documented genetic characterization of T. gondii in wild rodents from Sichuan province, and enriched the genetic diversity of T. gondii in China.


Introduction
oxoplasma gondii is an obligatory intracellular protozoan that causes a widespread zoonosis-toxoplasmosis (1). This disease is mostly subclinical and unnoticed in healthy individuals, however, other immunocompromised patients such as AIDS patients or cancer patients with undergoing immuno-suppressive therapy can get severe diseases, even death. T. gondii can infect the fetus via the placenta by transplacental transmission and damage the baby.
Wild rodents are the intermediate hosts of T. gondii and play important role in the transmission. There were different T. gondii genotypes in distinct areas of wild rodents in China (9)(10)(11). Sichuan with a unique ecosystem is one of provinces located in southwestern China. However, there is little genetic information on T. gondii diversity in this province. This research focused on the genetic characterization of T. gondii isolates from wild rodents in Sichuan Province. These findings would provide baseline data for improving prevention and control of T. gondii infection in wild rodents.  These four locations were located in the southwest of Sichuan Province, sharing borders with Yunnan Provinces in the south, with an average annual rainfall of 1000~1100 mm.
All rodents were handled in accordance with regulations laws required by the Animal Ethics Procedures and Guidelines of the People's Republic of China. This study received ethical approval by the Animal Ethics Committee of Dali University.
Ten grams of each animal's brain tissue was digested with proteinase K for 2 h, then TIANamp Genomic DNA kit (Tiangen ™, Beijing, China) was used to extract the genomic DNA. Semi-nested PCR targeting the T. gondii B1 gene was performed to detect T. gondii infection as described previously (10). DNA samples showing positive T. gondii B1 gene amplification were then used for further genetic characterization.

Statistical analysis
The prevalence of T. gondii-infected wild rodents among different variables including species, region and age were analyzed using multinomial logistic regression by SPSS for Windows (Release 16.0 standard version, Chicago, IL, USA). Statistical differences were considered to show statistically significant when P< 0.05.

Results
The prevalence of T. gondii B1 gene in wild rodents as determined by PCR amplification is shown in Table 1 There was no statistical difference in T. gondii prevalence among species, regions and age.
Among the 6 T. gondii-positive DNA samples, only 4 positive DNA samples were genotyped completely at 11 genetic loci. The 4 samples represent a new genotype (Table 2). Due to low DNA concentration, other 2 samples were identified at less than 6 loci, and the results were not shown.
The study area in the present study is a region of Sichuan Province with plain, tableland, high hills, lower and medium height mountains that has typical complex and diverse landscape and rich in biodiversity. However, little information about the prevalence and genetic characterization of T. gondii is known in this ecological diversity environment. Rat is one of the food sources of cats, and naturally infected rat plays an important role in T. gondii transmission. The seroprevalence of T. gondii in rats in southern China was 3.2% (7/217) by modified agglutination test (MAT) (13); in eastern China, T. gondii prevalence in rats was 23.6% (29/123) by B1 gene-targeted PCR amplification, and 7 PCR-positive samples were completely genotyped and they were identified as genotype China 1 (ToxoDB# 9)(10). In another study, 11 out of 183 were found to be positive for the T. gondii B1 gene in wild rodents from northwest China, 4 samples belonged to ToxoDB Genotype #10 and two samples were identified as two new geno-types (11). Our findings indicated that 6 out of 181 wild rodents were positive for the T. gondii B1 gene and all the 4 successfully genotyped belong to the same type, representing a potentially new genotype. This is the first report of genetic typing of T. gondii isolates in rats in Sichuan Province.