Prevalence and genotyping of Toxoplasma gondii in naturally-infected synanthropic rats (Rattus norvegicus) and mice (Mus musculus) in eastern China

Background Synanthropic rats and mice share the same environment with humans and play an important role in epidemiology of toxoplasmosis; however, there is limited information about prevalence and genetic characterization of Toxoplasma gondii in synanthropic rats and mice in China. Findings In the present study, the prevalence and genetic characterization of T. gondii naturally infected synanthropic rodents (Rattus norvegicus and Mus musculus) were investigated in the urban area of Xuzhou city, Eastern China between June 2013 and August 2014. DNA from the brain of each animal was prepared and screened by specific PCR assay targeting 35-fold repeated B1 gene (B1-PCR). PCR positive DNA samples were further genotyped by multi-locus PCR-RFLP. Overall, out of 123 synanthropic rodents, 29 samples were positive by B1 gene-targeted PCR (23.6%). Of these, 7 out of 31 (22.3%) M. musculus were positive, whereas the positive rate of R. norvegicus was 23.9% (22/92). Multi-locus PCR-RFLP analysis reveals that seven PCR-positive samples were completely genotyped and they were identified as type China 1 (ToxoDB# 9). Conclusion To our knowledge, this is the first report of molecular detection and genetic characterization of T. gondii infection in synanthropic rodents in Eastern China. The results of the present study showed a high infection pressure of T. gondii exists in the environment and synanthropic rodents infected by T. gondii may be an important source of infection for cats and other animals.


Background
Toxoplasmosis caused by the obligatory intracellular protozoan Toxoplasma gondii is a widespread zoonosis [1]. It is estimated that up to 30% of the human population of the world is suffering chronic infection with generally benign or mild nonspecific clinical symptoms [2]. Moreover, deaths and great morbidity can be brought about in fetuses and immunocompromised patients [3]. Humans and other animals can get infected mainly through consumption of undercooked meats containing cysts of T. gondii and ingestion of oocysts in environment. In addition, T. gondii can be also transmitted vertically from an infected mother to her baby during her first gestation [4].
Synanthropic rodents are widely distributed in China and it has been reported that the main species of rodents distributed in China may vary due to different climates, food sources and other factors [5]. However, brown rats (Rattus norvegicus) and synanthropic mice (Mus musculus) are widely distributed in the urban area of Noth China [6]. Naturally-infected rodents serving as important reservoir hosts play a key role in dissemination of T. gondii to other animals including cats since they are the main prey for cats and other stray carnivorous animals [7]. Furthermore, free-living animals such as stray cats and rodents could be used as sentinels of environmental spreading with T. gondii in densely built urban areas as they are exposed without any protection to all the infective forms of the parasite and feed on various sources of food on the ground [8][9][10].
Our previous study showed that there was a high prevalence of T. gondii in stray dogs and cats both in urban and rural areas of Xuzhou city, suggesting a high infection pressure for both animals and humans in that area [11]. However, the sources for this relative high prevalence of T. gondii in stray dogs and cats remain unclear. Moreover, knowledge about the prevalence and genetic characterization of T. gondii in synanthropic rodents in China is rather limited. Therefore, in the present study, we determined the prevalence of T. gondii in synanthropic rodents in Eastern China by detecting T. gondii DNA using specific PCR targeting 35-fold repeated B1 gene (B1-PCR), and genotyped T. gondii in synanthropic rodents.

Sample collection and preparation
A total of 123 rodents were randomly collected from Tongshan District, Yunlong District, GuLou District and Peixian County in Xuzhou City, Jiangsu Province, Eastern China during July 2013 to August 2014. The geographical information of Xuzhou City was described in detail elsewhere [11]. Animals were trapped and transported to our laboratory where the animals were anaesthetized and whole brain from each animal was obtained and stored at −20? C until use. The age of animals was estimated by body? s length as description elsewhere [6], and these animals were divided into four groups according to their ages: Juvenile group (with the body length < = 110 mm), Sub-adult group (with the body length 111? 150 mm), adult group (with the body length 151? 175 mm) and old group (with the body length >175 mm).

DNA extraction and specific polymerase chain reaction
DNA extraction was performed using a commercial DNA extraction kit (Shanghai sangon biotech, Shanghai, China) according to manufacturer? s recommendations. Briefly, about 50 mg of each brain tissue was cut into small pieces, homogenized in 200 μl of DNA extraction buffer and proteinase K, and added for ingestion at 55? C for 4 h. Subsequently, 500 μl buffered phenol was added and centrifuged at 12,000 g for 5 min. DNA was extracted twice using phenol-chloroform, and stored at −20? C until use after precipitation by sodium acetate and ethanol.
To estimate the prevalence of T. gondii in synanthropic rodents in Eastern China, specific PCR that targets 35-fold repeated B1 gene (B1-PCR) was employed to detect the possible infection with T. gondii in synanthropic rats and mice [12,13]. Positive control of DNA from T. gondii infected mice experimentally and negative controls were included in each test.
Genetic characterization of T. gondii in positive DNA samples Genetic characterization of T. gondii in randomly selected positive DNA samples in synanthropic rodents were performed using the multilocus PCR-RFLP method [14][15][16][17]. Briefly, a total of 10 genetic markers (i.e., SAG1, SAG2, alter.SAG2, SAG3, BTUB, GRA6, c22-8, L358, PK1, and Apico) were amplified by multiplex PCR using external primers. The PCR reaction (25 μl) consisting of 1 ? PCR buffer, 0.2 mM of each primer, 200 μM dNTPs, 2 mM MgCl 2 , 0.2 U of HotStart Taq DNA polymerase (TAKARA, Japan) were carried out using a thermal cycler (PTC 200, Bio-RAD) under the reaction conditions as follows: 95? C for 5 min to activate the DNA polymerase, then 30 cycles of PCR at 95? C for 30 s, 55? C for 60 s and 72? C for 90 s. Six references including GT1, PTG, CTG, MAS, TgCgCa1 and TgCatBr5 were employed as positive controls in each reaction. The amplified DNA fragments were diluted 1:1 in sterile, double-distilled water and then were amplified by internal primers for each locus, respectively [14][15][16][17]. A similar approach was used for nest PCR, the annealing temperature of which is at 60? C for 60 s for all the markers except Apico (55? C for 60 s). The nest PCR amplified products were then digested with   II/III  II  II  II  II  II  II  II  II  II  II  Reference, Type II, ToxoDB #1   CTG  Cat  United States  II/III  III  III  III  III  III  III  III  III  III I  III  III  III  III  III  I  I  I  u-1* I  Reference, ToxoDB #19   TgWtdSc40 WTD  USA  u-1  II  II  II  II  II  II  II  I  II  I  Type 12, ToxoDB #5   TgCatBr64  Cat  Brazil  I  I  u-1  III  III  III  u-1  I  III  III  I  Reference, ToxoDB #111   TgRsCr1  Toucan  Costa Rica  u-1  I  II  III  I  III  u-2  I  I  III  I  Reference, ToxoDB #52   TgRn05  Rat  Brain  Xuzhou, China u-1  II  II  III  III  II  II  III  II  II  I  ToxoDB #9   Tgmouse07 Mouse  Brain  Xuzhou, China u-1  II  II  III  III  II  II  III  II  II  I  ToxoDB #9   Tgmouse08 Mouse  Brain  Xuzhou, China u-1  II  II  III  III  II  II  III  II  II  I  ToxoDB #9   TgRn13  Rat  Brain  Xuzhou, China u-1  II  II  III  III  II  II  III  II  II  I  ToxoDB #9   TgRn14  Rat  Brain  Xuzhou, China u-1  II  II  III  III  II  II  III  II  II  I  ToxoDB #9   TgRn15  Rat  Brain  Xuzhou, China u-1  II  II  III  III  II  II  III  II  II  I  ToxoDB #9   Tgmouse23 Mouse  Brain  Xuzhou, China u-1  II  II  III  III  II  II  III  II  II  I  ToxoDB #9 *u-1 and u-2 represent unique RFLP genotypes, respectively. WTD: White-tailed Deer. nd: not determined.
restriction enzymes for 1 h at recommended temperature according to the instructions for each enzyme. The digested fragments were resolved in 2.5%-3% agarose gel and visualized using a gel documentation system (UVP GelDoc-ItTM Imaging System, Cambridge, U.K.).

Statistical analysis
Differences in the prevalence of T. gondii-infected rats and mice among different variables including location, age and gender were analyzed using a Chi-square test by SPPS (Release 16.0 standard version, SPPS Inc., Chicago, America). Statistical differences were found when P < 0.05.

Results and discussion
T. gondii DNA in brains was demonstrated in 29 out of 123 (23.6%) rodents collected. Of these, 7 out of 31 (22.6%) mice were T. gondii DNA-positive whereas the prevalence of T. gondii in rats was 23.9% (22 out of 92 animals examined, Table 1). There was no statisticaldifference in T. gondii prevalence in species, genders and regions where samples were collected. However, statistically significant difference was found in the prevalence of T. gondii in synanthropic rodents of different ages (P < 0.01), in which the prevalence of T. gondii in old group (75.0%) and juvenile group (23.1%) were significantly higher than that of sub-adult group (19.0%) and adult group (19.2%), respectively ( Table 1), suggesting that both congenital infection and acquired infection of T. gondii existed in naturally infected synanthropic rodents in this area. DNA samples were selected for genetic characterization using multilocus PCR-RFLP. Multilocus PCR-RFLP results showed that only seven DNA samples showed complete genotyping results and the genotypes were identified as ToxoDB #9 (China 1, Table 2). Unfortunately, other DNA samples showed no results or part genotype results, thus the genotypes could not be determined.
Synanthropic rodents which share the same environment with humans are considered as an important reservoir of T. gondii for cats and other animals because naturally infected rodents constitute important prey for wild and synanthropic felids [18]. However, there is limited information about prevalence of T. gondii in synanthropic rodents in China [19]. In addition, the relative high prevalence of T. gondii in synanthropic rodents in the present study might partly account for the high infection rates of T. gondii in cats and dogs in the same area [11]. However, PCR assay can only provide a suggestive, but not conclusive evidence for T. gondii infection in rodents. Therefore, our results in the present study showed the preliminary but fundamental data for further studies which aim at isolating live parasites in synanthropic rodents.
The information of genetic characterization of T. gondii in synanthropic rodents is rather limited [20]. The genotypes identified in this study were the genotype ToxoDB #9, which is dominantly prevalent in cats and other animals in most parts of China [21,22]. More importantly, this genotype which was also identified in naturally infected cats in this region (Eastern China) showed a moderate or high virulence to mice, indicating that the circulating T. gondii in cats and synanthropic rodents could cause severe toxoplasmosis in humans if it were to spread to humans [22]. Surprisingly, ToxDB #10 and ToxDB #205 which were also prevalent in naturallyinfected cats in this region were absent in our study, suggesting that there might be additional sources for cats infected by T. gondii [23].

Conclusions
This is the first report of molecular detection and genetic characterization of T. gondii in synanthropic rodents in Eastern China. The results of the present study revealed a wide distribution of T. gondii in synanthropic rats and mice in China and an identical genotype circulating in rodents in this region, which provide basic information for further prevention and control of toxoplasmosis in humans.