Abstract
Background: Toxoplasma gondii
is a foodborne zoonotic parasite that can infect humans and a variety of warm-blooded animals. Wild rodents play an important role as intermediate hosts of T. gondii, and the survey data of T. gondii in wild rodents in China are limited.
Methods:
Therefore, from August 2023 to May 2024, a total of 510 brain tissue samples were collected from nine species of wild rodents, and T. gondii was detected using a nested PCR targeting the B1 gene. All positive samples were genotyped at 11 PCR-RFLP markers (i.e., SAG1, 5′-and 3′-SAG2, alternative SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico) to determine their genotype.
Results:
In the present study, out of a total of 510 samples analyzed by nested PCR, three (0.59%) tested positive for T. gondii. It is noteworthy that all three positive samples were derived from female Rattus flavipectus rodents captured in Yunnan Province, where the detection rate was 3.41% (3/88; 95% confidence interval: 0–6.96). Complete genotyping was successfully performed on these three samples, revealing two to be of the ToxoDB #9, which is a common and widely distributed strain in China. The third sample presented a novel genotype.
Conclusion:
This finding suggests that wild rodents could be harboring a variety of T. gondii genotypes, including new genotypes, which may have significant implications for understanding the epidemiology of the parasite and its potential to cause disease in humans and animals.
Introduction
Toxoplasma gondii is a significant foodborne zoonotic parasite that infects a wide range of hosts, including humans and rodents (Dubey et al., 2021). This protozoan parasite is known for its extraordinary ability to infect almost any warm-blooded animal, making it one of the most widespread zoonotic pathogens worldwide (Dubey, 2010). In humans, infection with the parasite T. gondii can cause toxoplasmosis, which may lead to serious health complications, particularly in immunocompromised individuals and pregnant women (Ajzenberg et al., 2002; Hill and Dubey, 2002).
Wild rodents play a crucial role in its environmental transmission, as they are not only widely distributed and characterized by high population densities but also frequently interact with final hosts (cats) (DeFeo et al., 2002). Felids, as the definitive hosts of T. gondii, shed oocysts in their feces, infecting various warm-blooded intermediate hosts, such as rodents and humans, leading to tissue cyst formation (Tenter et al., 2000; Bodaghi et al., 2012). Transmission to intermediate hosts can also occur through two other primary routes: congenital transmission or consumption of undercooked meat containing tissue cysts (Jittapalapong et al., 2011; Khademvatan et al., 2017).
Moreover, wild rodents exhibit a remarkable ability to thrive and reproduce in environments associated with human activity, such as farms and peri-urban regions. This characteristic increases the likelihood of disease transmission to both humans and animals (Hosseini et al., 2021). Their adaptability, combined with their frequent interactions with domestic animals and humans, makes them ideal sentinel animals for assessing the risk of T. gondii oocyst contamination in the environment (Zhu et al., 2023). Understanding the prevalence and genetic diversity of T. gondii in wild rodents is essential for developing effective strategies to mitigate the risk of zoonotic transmission and protect public health.
In recent years, the prevalence of T. gondii in wild rodents has been documented globally, highlighting their role as potential reservoirs for the parasite (Ivovic et al., 2019; Ode et al., 2022). In China, however, studies on T. gondii infection in wild rodents are limited, with reports primarily from Guangdong, Jilin, Jiangsu, Hubei, Hunan, Yunnan, and Qinghai provinces (Yin et al., 2010; Zhang et al., 2013; Yan et al., 2014; Lv et al., 2021; Li et al., 2024). Notably, a study by Dong et al. reported the overall seroprevalence of T. gondii in Chinese is 8.2%, but data on the genotypic characteristics of T. gondii in wild rodent populations remain scarce (Dong et al., 2018). In this study, we investigated the prevalence and genotyping of T. gondii B1-positive samples from brain tissues of nine wild rodent species collected across three provinces in China. Our findings provide novel insights into the distribution and genetic diversity of T. gondii in these regions, emphasizing the potential role of wild rodents as reservoirs for diverse parasite strains, including T. gondii genotypes previously described in human strains. These results highlight the need for continued surveillance and research to address the public health implications of toxoplasmosis.
Materials and Methods
Sample collection and DNA extraction
A total of 510 brain tissues were collected from nine wild rodent species (n = 287, Microtus fortis; n = 23, Niviventer lotipes; n = 41, Rattus norvegicus; n = 22, Apodemus agrarius; n = 39, Rattus flavipectus; n = 39, Bandicota indica; n = 5, Rattus rattus sladeni; n = 41, Rattus losea; n = 13, Mus musculus) three provinces (n = 88 from Yunnan, n = 319 from Hunan, n = 103 from Guangxi) in China between August 2023 and May 2024 (Table 1). Data of the wild rodents (regions, species, sex, sampling time, and environments) were recorded. The investigated wild rodents were handled in accordance with good animal practices required by the Animal Ethics Committee of Qingdao Agricultural University (Approval No. QAU-AEW-20210701001). The wild rodents from which the brain tissues were collected, were conducted according to the good animal practices required by the Animal Ethics Procedures and Guidelines of the People’s Republic of China. Genomic DNA was extracted from each brain sample using the TIANamp Genomic DNA kit. All DNA extractions were performed with extraction controls to ensure the reliability of the process.
The Prevalence of T. gondii Infection in Wild Rodents in China
CI, confidence interval; T. gondii, Toxoplasma gondi.
PCR amplification and genotyping of T. gondii
Then the B1 gene of T. gondii was amplified to detect the status of the T. gondii infection in investigated wild rodents as previously described (Hill et al., 2006). The DNA sample of the B1 gene positive was further analyzed for genetic characterization of T. gondii.
Multilocus polymerase chain reaction-restriction fragment length polymorphism with 11 genetic markers (i.e., SAG1, 5′-and 3′-SAG2, alternative SAG2, SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico) was used to genotype the B1 gene positive samples as described previously (http://toxodb.org/toxo/) (Su et al., 2006; Su et al., 2010). Briefly, the genomic DNA of B1 gene-positive samples was amplified by multiplex PCR with external primers targeting 11 markers. The PCR reaction was conducted in a 25 μL volume containing 1 × PCR buffer, 0.2 mM of each primer, 200 μM deoxyribonucleotide triphosphates (dNTPs), 2 mM MgCl2, 0.2 U of HotStart Taq DNA polymerase (TAKARA, Japan), and 2 μL DNA template. All the DNA was incubated at 94°C for 10 min, followed by 30 PCR cycles at 95°C for 30 s, 55°C for 60 s, 72°C for 1.5 min. The PCR product of the first PCR reaction was used for nested PCR amplification with internal primers for each marker, separately. Every amplification included eight reference strains as positive controls (Table 2). The second PCR products were digested at a suitable temperature with restriction enzymes for 2 h. The enzymatic products were subjected to electrophoresis on 2.5–3.0% agarose gel and were visualized under UV light (Zheng et al., 2016).
Genetic Characterization of T. gondii Infection in Wild Rodents in China
u-1 and u-2 represent unique RFLP genotypes, respectively.
Statistical analysis
Prevalence (percentage indicating number of infected specimens relative to total studied specimens) and its 95% confidence interval (CI) were calculated using the meta package in R software (v.4.2.1). To evaluate the influence of multiple factors on T. gondii infection rates, logistic stepwise regression analysis and Fisher’s exact test were performed using SAS (v. 9.1, SAS Institute Inc., USA), with region (χ1), species (χ2), gender (χ3), sampling time (χ4), and environment (χ5) as independent variables. The best-fitting model, determined by stepwise selection, retained region as the primary predictor of infection prevalence.
Results
Prevalence and risk factor of T. gondii
Results show that 3 out of 510 samples (0.59%) tested positive for T. gondii (95% CI: 0.16–1.02). All positive samples were obtained from R. flavipectus captured in Yunnan Province (Table 1). Logistic regression analysis showed that region was the main risk factor for T. gondii infection, and the equation was described as y = −5.0813 χ3 + 3.0144.
Genotyping of T. gondii
Two of the positive samples were identified as ToxoDB #9, while one sample represented a novel genotype (Table 2).
Discussion
In this study, the overall prevalence of T. gondii in wild rodents was 0.59% (3/510), which is lower than that reported in recent surveys both regionally and internationally. For example, a study in China reported an overall prevalence of 5.24% (20/382) in five species of wild rodents across five provinces in 2021 (Lv et al., 2021). Similarly, a study in Romania found a prevalence of 7.3% in 20 small mammal species from 63 locations (Kalmar et al., 2023). Another study reported the prevalence of T. gondii infection among adult Rattus rattus in Brazil was 1.36% (1/73) (Nunes et al., 2025). It is well known that the detection rates of T. gondii in wild rodents may vary significantly by region, possibly due to differences in environmental conditions, rodent species composition, or the presence of definitive hosts.
Logistic regression analysis revealed the region as the primary risk factor for T. gondii infection in wild rodents. Specifically, the model indicates that certain regions exhibit a significantly lower likelihood of T. gondii infection, likely influenced by regional factors such as climate or the distribution of feline definitive hosts, rather than rodent population abundance. These findings suggest that environmental and ecological conditions play a critical role in shaping T. gondii prevalence, highlighting the need for region-specific control measures. Notably, all positive samples in this study were from female R. flavipectus captured in Yunnan Province (3.41%; 3/88; 95% CI: 0–6.96), located in southwestern China and renowned for its high biodiversity. A previous investigation conducted in Zhaotong City, Yunnan Province, within the breeding environment of semifine wool sheep, reported a T. gondii B1 gene positivity rate of 3.23% (1/31) in the brain tissue of wild rodents. (Li et al., 2024). This stark contrast within Yunnan likely stems from localized differences: Zhaotong’s breeding environment, with high cat density, dense rodent populations, and poor sanitation, fosters intense oocyst transmission, whereas the current study’s sampling areas, possibly in less contaminated habitats with lower cat activity or different rodent behaviors, exhibit reduced prevalence.
In China, multiple T. gondii genotypes have been reported, including ToxoDB #1, ToxoDB #2, ToxoDB #3, ToxoDB #9, ToxoDB #10, ToxoDB #20, ToxoDB #204, ToxoDB #205, and ToxoDB #225 (Dubey et al., 2007; Zhou et al., 2009; Shwab et al., 2014). Among these, ToxoDB #9 is the predominant strain in China, comprising approximately 60% of all T. gondii isolates (Wang et al., 2013). In this study, ToxoDB #9 was identified in two positive samples from wild rodents. In addition, ToxoDB #9 has been previously documented in various hosts, such as chicken, pork, cat, sika deer, minks, and humans (Wang et al., 2013; Cong et al., 2016; Zheng et al., 2016; Li et al., 2022). These findings emphasize the extensive distribution and dominance of ToxoDB #9 among T. gondii isolates in China and underscore the potential role of wild rodents as reservoirs for this genotype.
Notably, a novel genotype detected in one of the samples highlights the genetic diversity of T. gondii in wild rodents. Several studies have reported the discovery of new T. gondii genotypes in wild rodents, which is consistent with our study (Zhang et al., 2013; Wang et al., 2019). The identification of this new genotype underscores the dynamic nature of T. gondii populations and suggests that wild rodents may serve as reservoirs for previously undescribed strains. The genetic diversity of T. gondii in wild rodents is likely influenced by multiple factors, including the presence of definitive hosts and the complex ecological interactions within the rodent community (Sohn-Hausner et al., 2024). The discovery of new T. gondii genotypes in wild rodents highlights the parasite’s genetic diversity and underscores the importance of ongoing genetic surveillance. This monitoring is crucial for understanding the population structure of T. gondii and informs effective disease prevention and control strategies.
This study provides novel insights into the prevalence and genetic diversity of T. gondii in wild rodents in China, highlighting their potential role as reservoirs for diverse strains and emphasizing the need for ongoing surveillance and research to address the public health implications of toxoplasmosis.
Conclusion
This study reveals a low prevalence of T. gondii (0.59%) in wild rodents, highlighting the potential role of wild rodents as reservoirs for diverse parasite strains, including T. gondii genotypes already described in human strains. The identification of ToxoDB #9 and a novel genotype underscores the genetic diversity of T. gondii in Yunnan province. Our findings underscore the necessity for continuous surveillance and further research to elucidate the ecological factors driving transmission and to mitigate public health risks associated with toxoplasmosis.
Footnotes
Authors’ Contributions
G-R.B. and H-T.W.: Methodology, software, writing—original draft. Q.-Y.H.: Data curation, methodology, writing—review and editing. S.-Y.Q.: Data curation, resources, visualization, writing—review and editing. Z.-Q.G.: Visualization, writing—review and editing. Y-X.S. and J.-H.L.: Resources, writing—review and editing. Y.Q. and Q.Z.: Resources, supervision, writing—review and editing. H.M.: Conceptualization, resources, funding acquisition, supervision, writing—review and editing.
Ethics Statement
The investigated wild rodents were handled in accordance with good animal practices required by the Animal Ethics Committee of Qingdao Agricultural University (Approval No. QAU-AEW-20210701001).
Author Disclosure Statement
No competing financial interests exist.
Funding Information
The study was supported by the Shandong Provincial Natural Science Foundation (No. ZR2022QC047) and Doctoral Startup Fund of Qingdao Agricultural University (No. 663-1120013).