Abstract
Toxoplasma gondii is an important zoonotic parasite infecting humans and various animals with a worldwide distribution. However, limited information is available on T. gondii infection in wild rats. The present study aimed to examine the prevalence and characterize the genotypes of T. gondii in wild rats in two regions of China. Brain tissues were collected from 111 Edward's long-tailed rats (Leopoldamys edwardsi) and 117 Bower's white-toothed rats (Berylmys bowersi) between November 2017 and January 2018. Genomic DNA was extracted and amplified by PCR targeting the T. gondii B1 gene. B1 gene-positive samples were genotyped at 10 genetic markers (SAG1, SAG2 [5′, 3′] and [alternative], SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico) using multilocus nested polymerase chain reaction/restriction fragment length polymorphism. Six (5.41%, 6/111) Edward's long-tailed rats from Chongqing Municipality were positive for T. gondii B1 gene, whereas no T. gondii infection was detected in Bower's white-toothed rats (n = 117) from Guangdong province. T. gondii prevalence in female and male rats was 1.77% (2/113) and 3.48 (4/115), respectively. Four of the six positive DNA samples were completely genotyped at 10 genetic loci and were identified as ToxoDB#20. The present study revealed the occurrence of T. gondii infection in Edward's long-tailed rats. These findings raised public health concerning about T. gondii infection in wild rats. These results provide reference data for understanding the distribution of T. gondii genotypes in wild rats in China.
Introduction
T
Humans get infected T. gondii mainly through ingesting tissue cysts in undercooked meat or oocysts in contaminated food or water, causing fetal developmental defects, abortion during pregnancy, and serious symptoms, even death in immunocompromised individuals (Pan et al., 2017; Wang et al., 2017). So far, Types II and III of T. gondii genotypes have been identified from wild rats in Saudi Arabia and Grenada (Choudhary et al., 2013; Elamin, 2014) as well as ToxoDB#9, ToxoDB#10, and ToxoDB#137 in China (Wang et al., 2013, 2018; Yan et al., 2014; Zhang et al., 2014). However, these results were still inadequate to understand the distribution of T. gondii genotypes in wild rats, which have a variety of species.
Wild rats play an important role in the transmission of T. gondii among homothermal animals (Dubey and Frenkel, 1998; Yin et al., 2010; Yan et al., 2014; Murata et al., 2018). Thus, it is of significance to investigate T. gondii infection in wild rats. This study was conducted to examine the prevalence and genotypes of T. gondii infection in two wild rat species, namely Edward's long-tailed rats (Leopoldamys edwardsi) and Bower's white-toothed rat (Berylmys bowersi) in China to provide reference data for the prevention and controlling of T. gondii infection in wild rats.
Materials and Methods
Ethics statement
This study was approved by the Animal Ethics Committee of Hunan Agricultural University (No. 43321503). The wild rats used for the present study were handled in accordance with good animal practices required by the Animal Ethics Procedures and Guidelines of the People's Republic of China.
Sample collection
Brain tissues were collected from 228 wild rats purchased from markets in Chongqing Municipality and Guangdong province between November 2017 and January 2018, and were transported to the laboratory and stored at −20°C. Of these, 111 wild rats were collected from Chongqing Municipality and 117 wild rats were collected from Guangdong province (Table 1). The specific identity of the examined wild rats was determined by PCR-based sequencing of the mitochondrial (mt) cox1 gene using an established method (Robins et al., 2007).
The Prevalence of Toxoplasma gondii Infection in Wild Rats in Chongqing Municipality and Guangdong Province, China
CI, confidence interval.
DNA extraction and PCR amplification
Genomic DNA was extracted from each brain tissue with 50 mg from wild rats using a commercially available DNA Extraction Kit (Wizard® SV Genomic DNA Purification System; Promega, USA) according to the manufacturer's instructions. A total of 228 DNA samples were amplified by a seminested PCR targeting the B1 gene of T. gondii, according to a previous study (Hill et al., 2006). Genotyping was performed using multilocus nested polymerase chain reaction/restriction fragment length polymorphism (Mn-PCR-RFLP) targeting nine nuclear loci (SAG1, SAG2 [5′, 3′] and [alternative], SAG3, BTUB, c22-8, GRA6, c29-2, L358, PK1) and one apicoplast locus (Apico), as described previously (Su et al., 2006, 2010; Zheng et al., 2016). Eight reference T. gondii strains, namely GT1, PTG, CTG, MAS, TgCgCa1, TgCatBr5, TgCatBr64, and TgRsCr1, were used as controls (Table 2). The products of second PCR were digested at suitable temperatures with different restriction enzymes for 1.5–2 h. The specific restriction enzyme fragments were visualized by agarose gel electrophoresis under ultraviolet light. The results were compared and matched according to genotypes deposited in ToxoDB (
Summary of Genotyping of Toxoplasma gondii in Wild Rats in Chongqing Municipality, China
u-1 and u-2 represent unique restriction fragment length polymorphism genotypes, respectively.
Observation of T. gondii cysts under microscope
Each T. gondii-positive brain tissue was diluted with phosphate-buffered saline 1:4, which was observed under a microscope to examine the presence of T. gondii cysts.
Statistical analyses
All statistical analyses were performed by SAS (version 9.1).
Results and Discussion
The mt cox1 sequences of the two wild rats had 99% identity to previously published sequences for L. edwardsi from China (GenBank accession no. KM434322.1) or B. bowersi from Vietnam (JN105105.1), respectively, and thus were identified as Edward's long-tailed rats (L. edwardsi) and Bower's white-toothed rat (B. bowersi), respectively.
While no T. gondii DNA was detected from all Bower's white-toothed rats (n = 117) from Guangdong province, possibly due to the small sample size, six (5.41%, 95% confidence interval [CI] 1.20–9.61) out of the 111 Edward's long-tailed rats from Chongqing Municipality were positive for T. gondii. The prevalence of T. gondii infection in female and male rats was 1.77% (2/113, 95% CI 0.00–4.20) and 3.48 (4/115, 95% CI 0.13–6.83), respectively (Table 1).
Four of these six positive DNA samples were completely genotyped at 10 genetic markers by Mn-PCR-RFLP and were identified as ToxoDB#20. The genotype of other two DNA samples cannot be identified, due to the low DNA concentration, and amplifying was not successful in some markers (Table 2).
T. gondii cysts were observed in each of the four positive brain tissues, with a diameter of about 30–90 μm (Supplementary Fig. S1).
The results of this study revealed a low (5.41%) T. gondii prevalence in L. edwardsi rats from Chongqing Municipality. ToxoDB#20 of T. gondii, which was reported previously in cats from Yunnan province, southwestern China (Tian et al., 2014), was identified in L. edwardsi rats in this study. It was also reported in other parts of Asia, such as in dogs in Sri Lanka (Dubey et al., 2007), sand cats in Qatar (Dubey et al., 2010), and sand cats in United Arab Emirates (Dubey et al., 2010). ToxoDB#20 has also been reported frequently in Africa, such as in feral cats in Egypt (Al-Kappany et al., 2010), stray dogs in Egypt (El Behairy et al., 2013), and feral cats in Ethiopia (Dubey et al., 2013). These reports indicated that ToxoDB#20 was widely distributed from Africa to Asia (Tian et al., 2014; Chaichan et al., 2017). Previous studies have reported that some T. gondii genotypes have been identified from wild rats, such as ToxoDB#9, ToxoDB#10, and ToxoDB#137 in China (Wang et al., 2013, 2018; Yan et al., 2014; Zhang et al., 2014); types II and III in Saudi Arabia (Elamin, 2014); and ToxoDB#2 in Grenada (Choudhary et al., 2013). To our knowledge, the present study was the first report about the T. gondii ToxoDB#20 in L. edwardsi rats.
Wild rats play an important role in the transmission of T. gondii because they can circulate between the wild and the residential areas. More importantly, rat meat is a popular foodstuff in southern China, and the meat in these investigation areas is transported to other provinces of China. So, the significance of rats in the prevalence of T. gondii should be paid more attention because they could serve as sources for T. gondii infection in humans. The rat meat only represents a small portion of foodstuff, but it should not be neglected as a potential source of human toxoplasmosis in these provinces and elsewhere in China.
Conclusion
The present study revealed a 5.41% prevalence of T. gondii infection in L. edwardsi rats from Chongqing Municipality, China, and identified ToxoDB#20 as the causative genotype. T. gondii cysts were observed in the brain tissue of positive L. edwardsi rats, which raised a public health concern, since more attention should be given to T. gondii infection in wild rats. These results provide reference data for understanding the distribution of T. gondii genotypes in wild rats in China.
Footnotes
Acknowledgments
This work was supported, in part, by the Scientific Research Fund of Hunan Provincial Education Department (Grant No. 16A102) and the Training Program for Excellent Young Innovators of Changsha (Grant No. KQ1802035).
Disclosure Statement
No competing financial interests exist.
Supplementary Material
Supplementary Figure S1
References
Supplementary Material
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