Skip to main content

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

Abstract

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]-[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.

Findings

Materials and methods

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]-[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 MgCl2, 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]-[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 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.

Table 1 The prevalence of Toxoplasma gondii infection in synanthropic rats and mice in Xuzhou City, Eastern China

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.

Table 2 Summary of genotyping of Toxoplasma gondii in synanthropic rats and mice in China

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 naturally-infected 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.

References

  1. Dubey JP: Toxoplasmosis of Animals and Humans. 2010, CRC Press, Boca Raton, Florida, 2

    Google Scholar 

  2. Tenter AM, Heckeroth AR, Weiss LM:Toxoplasma gondii: from animals to humans. Int J Parasitol. 2000, 30: 1217-1258. 10.1016/S0020-7519(00)00124-7.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  3. Sibley LD, Boothroyd JC: Virulent strains of Toxoplasma gondii comprise a single clonal lineage. Nature. 1992, 359: 82-85. 10.1038/359082a0.

    Article  CAS  PubMed  Google Scholar 

  4. Montoya JG, Liesenfeld O: Toxoplasmosis. Lancet. 2004, 363: 1965-1976. 10.1016/S0140-6736(04)16412-X.

    Article  CAS  PubMed  Google Scholar 

  5. Zhou P, Chen Z, Li HL, Zheng H, He S, Lin RQ, Zhu XQ:Toxoplasma gondii infection in humans in China. Parasit Vectors. 2011, 4: 165-10.1186/1756-3305-4-165.

    Article  PubMed Central  PubMed  Google Scholar 

  6. Zheng ZM, Jiang ZK, Chen AG: Rodent Zoology. 2008, Shanghai Jiaotong University Press, Shanghai Municipality, 2

    Google Scholar 

  7. Dubey JP, Frenkel JK: Toxoplasmosis of rats: a review, with considerations of their value as an animal model and their possible role in epidemiology. Vet Parasitol. 1998, 77: 1-32. 10.1016/S0304-4017(97)00227-6.

    Article  CAS  PubMed  Google Scholar 

  8. Hughes JM, Williams RH, Morley EK, Cook DA, Terry RS, Murphy RG, Smith JE, Hide G: The prevalence of Neospora caninum and co-infection with Toxoplasma gondii by PCR analysis in naturally occurring mammal populations. Parasitology. 2006, 132: 29-36. 10.1017/S0031182005008784.

    Article  CAS  PubMed  Google Scholar 

  9. Meerburg BG, De Craeye S, Dierick K, Kijlstra A:Neospora caninum and Toxoplasma gondii in brain tissue of feral rodents and insectivores caught on farms in the Netherlands. Vet Parasitol. 2012, 184: 317-320. 10.1016/j.vetpar.2011.09.001.

    Article  CAS  PubMed  Google Scholar 

  10. Meireles LR, Galisteo AJ, Pompeu E, Andrade HF:Toxoplasma gondii spreading in an urban area evaluated by seroprevalence in free-living cats and dogs. Trop Med Int Health. 2004, 9: 876-881. 10.1111/j.1365-3156.2004.01280.x.

    Article  CAS  PubMed  Google Scholar 

  11. Yan C, Fu LL, Yue CL, Tang RX, Liu YS, Lv L, Shi N, Zeng P, Zhang P, Wang DH, Zhou DH, Zhu XQ, Zheng KY: Stray dogs as indicators of Toxoplasma gondii distributed in the environment the first report across an urban–rural gradient in China. Parasit Vectors. 2012, 5: 5-10.1186/1756-3305-5-5.

    Article  PubMed Central  PubMed  Google Scholar 

  12. Hill DE, Chirukandoth S, Dubey JP, Lunney JK, Gamble HR: Comparison of detection methods for Toxoplasma gondii in naturally and experimentally infected swine. Vet Parasitol. 2006, 141: 9-17. 10.1016/j.vetpar.2006.05.008.

    Article  CAS  PubMed  Google Scholar 

  13. Homan WL, Vercammen M, De Braekeleer J, Verschueren H: Identification of a 200- to 300-fold repetitive 529 bp DNA fragment in Toxoplasma gondii, and its use for diagnostic and quantitative PCR. Int J Parasitol. 2000, 30: 69-75. 10.1016/S0020-7519(99)00170-8.

    Article  CAS  PubMed  Google Scholar 

  14. Tian YM, Huang SY, Miao Q, Jiang HH, Yang JF, Su C, Zhu XQ, Zou FC: Genetic characterization of Toxoplasma gondii from cats in Yunnan Province, Southwestern China. Parasit Vectors. 2014, 7: 178-10.1186/1756-3305-7-178.

    Article  PubMed Central  PubMed  Google Scholar 

  15. Jiang HH, Huang SY, Zhou DH, Zhang XX, Su C, Deng SZ, Zhu XQ: Genetic characterization of Toxoplasma gondii from pigs from different localities in China by PCR-RFLP. Parasit Vectors. 2013, 6: 227-10.1186/1756-3305-6-227.

    Article  PubMed Central  PubMed  Google Scholar 

  16. Zhang XX, Lou ZZ, Huang SY, Zhou DH, Jia WZ, Su C, Zhu XQ: Genetic characterization of Toxoplasma gondii from Qinghai vole, Plateau pika and Tibetan ground-tit on the Qinghai-Tibet Plateau, China. Parasit Vectors. 2013, 6: 291-10.1186/1756-3305-6-291.

    Article  PubMed Central  PubMed  Google Scholar 

  17. Chen J, Li ZY, Zhou DH, Liu GH, Zhu XQ: Genetic diversity among Toxoplasma gondii strains from different hosts and geographical regions revealed by sequence analysis of GRA5 gene. Parasit Vectors. 2012, 5: 279-10.1186/1756-3305-5-279.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  18. Murphy RG, Williams RH, Hughes JM, Hide G, Ford NJ, Oldbury DJ: The urban house mouse (Mus domesticus) as a reservoir of infection for the human parasite Toxoplasma gondii: an unrecognised public health issue?. Int J Environ Health Res. 2008, 18: 177-185. 10.1080/09603120701540856.

    Article  PubMed  Google Scholar 

  19. Yin CC, He Y, Zhou DH, Yan C, He XH, Wu SM, Zhou Y, Yuan ZG, Lin RQ, Zhu XQ: Seroprevalence of Toxoplasma gondii in Rats in Southern China. J Parasitol. 2010, 96: 1233-1234. 10.1645/GE-2610.1.

    Article  PubMed  Google Scholar 

  20. Wang L, Cheng HW, Huang KQ, Xu YH, Li YN, Du J, Yu L, Luo QL, Wei W, Jiang L, Shen JL:Toxoplasma gondii prevalence in food animals and rodents in different regions of China: isolation, genotyping and mouse pathogenicity. Parasit Vectors. 2013, 6: 273-10.1186/1756-3305-6-273.

    Article  PubMed Central  PubMed  Google Scholar 

  21. Dubey JP, Zhu XQ, Sundar N, Zhang H, Kwok OC, Su C: Genetic and biologic characterization of Toxoplasma gondii isolates of cats from China. Vet Parasitol. 2007, 145: 352-356. 10.1016/j.vetpar.2006.12.016.

    Article  CAS  PubMed  Google Scholar 

  22. Zhou P, Zhang H, Lin RQ, Zhang DL, Song HQ, Su C, Zhu XQ: Genetic characterization of Toxoplasma gondii isolates from China. Parasitol Int. 2009, 58: 193-195. 10.1016/j.parint.2009.01.006.

    Article  CAS  PubMed  Google Scholar 

  23. Wang L, Chen H, Liu D, Huo X, Gao J, Song X, Xu X, Huang K, Liu W, Wang Y, Lu F, Lun ZR, Luo Q, Wang X, Shen J: Genotypes and mouse virulence of Toxoplasma gondii isolates from animals and humans in China. PLoS One. 2013, 8: e53483-10.1371/journal.pone.0053483.

    Article  PubMed Central  CAS  PubMed  Google Scholar 

Download references

Acknowledgments

Project support was provided, in part, by National Training Programs of Innovation and Entrepreneurship for Undergraduates (20130313021), Training Programs of Innovation and Entrepreneurship for College Students in Jiangsu Province (20130313021z) and the Open Funds of the State Key Laboratory of Veterinary Etiological Biology, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences (Grant No. SKLVEB2013KFKT005).

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Kui-Yang Zheng.

Additional information

Competing interests

The authors declare that they have no competing interests.

Authors’ contributions

KYZ, LLF and CY conceived and designed the study, CY, LJL and ZLL performed the experiments, analyzed the data, and drafted the manuscript. BBZ, ZLL, HFZ, XS, YQW and RXT helped in study design, study implementation and manuscript revision. All authors read and approved the final manuscript.

Rights and permissions

Open Access  This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made.

The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

To view a copy of this licence, visit https://creativecommons.org/licenses/by/4.0/.

The Creative Commons Public Domain Dedication waiver (https://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Yan, C., Liang, LJ., Zhang, BB. et al. Prevalence and genotyping of Toxoplasma gondii in naturally-infected synanthropic rats (Rattus norvegicus) and mice (Mus musculus) in eastern China. Parasites Vectors 7, 591 (2014). https://doi.org/10.1186/s13071-014-0591-6

Download citation

  • Received:

  • Accepted:

  • Published:

  • DOI: https://doi.org/10.1186/s13071-014-0591-6

Keywords