Abstract
Toxoplasma gondii is one of protozoan parasites resulting in zoonosis, which can infect nearly all of warm-blooded hosts, including humans and raccoon dogs (Nyctereutes procyonoides). However, related reports on prevalence and genetic characterization of T. gondii strains in raccoon dogs were few in China. The aim of this study was to survey the prevalence and genetic characterization of T. gondii strains in domestic raccoon dogs from Jilin, Liaoning, and Hebei provinces, northern China. During April 2016 to November 2017, a total of 337 tissue samples collected from domestic raccoon dogs were detected with B1 gene using a nested PCR. And the positive samples were genotyped at 11 genetic markers (SAG1, 5′-and 3′-SAG2, alternative SAG2, SAG3, BTUB, GRA6, L358, PK1, c22-8, c29-2, and Apico) using multilocus PCR-restriction fragment length polymorphism technology. Sixteen out of 337 sika deer (4.75%) were positive with B1 gene by nest PCR. Furthermore, four positive DNA samples were completely typed through further being genotyped, in which three samples were identified as ToxoDB Genotype #9, and one sample was confirmed as ToxoDB Genotype #10. The results of molecular detection not only revealed the existence of T. gondii in domestic raccoon dogs in Jilin, Liaoning, and Hebei for the first time, but also provided the information of genetic diversity. This study also indicated that ToxoDB Genotype #9 as a kind of potential reservoir for T. gondii transmission, may be main genotype in domestic raccoon dogs in China, posing a risk of infection in human health.
Introduction
Toxoplasmosis caused by Toxoplasma gondii is one of the most widespread zoonosis that can infect all warm-blooded animals and human beings globally, including raccoon dogs (Dubey and Jones 2008, Dubey 2010). Most of the people are with asymptomatic infection with T. gondii, accounting for one-third of the world population (Weiss and Dubey 2009, Dubey 2010, Zhang et al. 2019). Congenital infection with T. gondii can lead to spontaneous abortion, hydrocephaly, and mental retardation early in gestation while during the last trimester. And the asymptomatic infections or recurrent chorioretinitis usually occur, which can be fatal to immunocompromised patients, such as AIDS and people with cancer (Jones et al. 2001, Montoya and Liesenfeld 2004). And people may be infected by taking in tissue oocysts and bradyzoites from undercooked meat or water contaminated by oocysts excreted from infected cats (Montoya and Liesenfeld 2004, Zhou et al. 2011a). Relevant studies indicated that the genetic diversity of T. gondii strains was discovered in different hosts and the majority of T. gondii isolates were from Europe and North America that were part of the three closely related clonal lineages (identified as types I, II, and III), which were deemed to be caused by natural genetic crosses among similar parental types (Dubey et al. 2008). In addition, a fourth clonal lineage (type 12) was isolated in North America (Khan et al. 2011). Many atypical strain types have also been discovered in Central and South America, indicating that T. gondii population structure was complex and various (Lehmann et al. 2006, Shwab et al. 2014).
Raccoon dog as one of the most important economic animals provides fur and meat for humans. A total of 209,000,000 raccoon dogs were bred in China in 2016 (Zhang 2016). The fur and meat from domestic raccoon dogs are one of the main products, which are served as traditional clothing industry and medicine industry (Zhao et al. 2013). Especially in northern China, the number of domestic raccoon dogs raised constitutes a considerable part compared with other provinces and the meat of domestic raccoon dogs, resulting from consumption by local residents and transporting to other provinces of China. Previously, studies demonstrated that raccoon dogs can transmit many agents to other animals and humans, due to the close relationship among raccoon dogs, humans, and other animals (Zheng et al. 2017, Li et al. 2018, Son et al. 2019). Therefore, it is very important to determine the infectious status and genetic diversity of T. gondii in domestic raccoon dogs. Although the genotyping of T. gondii in domestic raccoon dogs has been reported only in Shandong province (Zhou et al. 2017), there are yet no reports about molecular detection and genetic typing of T. gondii in domestic raccoon dogs in other provinces in northern China.
Materials and Methods
Study sites and tissue collection
A total of 337 brain tissues were collected from domestic raccoon dogs in Jilin (n = 79; 122°–131°E, 41°–46°N), Liaoning (n = 123; 118°–125°E, 38°–43°N), and Hebei (n = 135; 113°–119°E, 36°–42°N) provinces during slaughter seasons. The period of collecting samples is from April 2016 to November 2017. Data of the raccoon dogs (regions, gender, and age) were acquired from owners. Tissues were stored at −20°C for further test. This study was approved by the Animal Ethics Committee of College of Animal Science and Technology, Jilin Agricultural University (20180512). The domestic raccoon dogs, from which the tissues were collected, were disposed based on good animal practices required by the Animal Ethics Procedures and Guidelines of the People's Republic of China.
DNA extraction and PCR detection
Genomic DNA was extracted from brain tissues of domestic raccoon dogs using the TIANamp Genomic DNA kit (TianGen™, Beijing, China). B1 gene of T. gondii was detected by means of a seminested PCR as described previously (Qin et al. 2015). The positive samples coming from brain tissues of domestic raccoon dogs were stored for further genetic characterization.
Genetic characterization of T. gondii
The positive samples with B1 gene were genotyped using multilocus PCR-restriction fragment length polymorphism with 11 genetic markers as described previously (Qin et al. 2014). In brief, genomic DNA showing positive for B1 gene were amplified by multiplex PCR with external primers, including 11 markers. Then the product of multiplex PCR was amplified using a thermal cycler (PTC 200; Bio-RAD). And nine reference strains were used as the positive controls in the process of experiment (Table 1). The PCR composed of 1 × PCR buffer, 0.2 mM of each primer, 200 μM dNTPs, 2 mM MgCl2, and 0.2 U of HotStart Taq DNA polymerase (TAKARA, Japan). The products were disposed with restriction enzymes for about 2 h, and then the 2.5% agarose gel were taken advantage of distinguishing single nucleotide polymorphisms with a gel imaging system (UVP GelDoc-It™, Cambridge, United Kingdom).
Genetic Characterization of Toxoplasma gondii Isolates from Raccoon Dogs in Jilin, Liaoning, and Hebei Provinces, China
u-1 and u-2 represent unique restriction fragment length polymorphism genotypes, respectively.
nd, not determined; WTD, white-tailed deer.
Statistical analysis
The differences in T. gondii prevalence of domestic raccoon dogs among different regions, gender, and age were analyzed using the binary logistic regression in SPSS (version 19; SPSS, Inc., IBM Corporation). If p < 0.05, the results were considered statistically significant. The odds ratios and their 95% confidence intervals (95% CI) based on likelihood ratio statistics were also calculated.
Results
Sixteen DNA samples from brain tissues of domestic raccoon dogs were positive for T. gondii B1 gene. The total positive rate of T. gondii infection in domestic raccoon dogs was 4.75% (95% CI 2.47–7.03) in northern China, including Jilin, Liaoning, and Hebei provinces. In addition, the prevalence of T. gondii infection in male and female domestic raccoon dogs were 3.48% (95% CI 0.93–6.04) and 6.62% (95% CI 2.39–10.85), respectively (Table 2). Owing to lower DNA concentration extracted from positive samples, only four positive samples presented complete genotyping locus. The results of genotyping of these isolates and nine references are summarized in Table 1, showing that three samples were identified as ToxoDB Genotype #9 in Jilin and Liaoning provinces, and one sample was identified as ToxoDB Genotype #10 in Hebei province.
Prevalence of Toxoplasma gondii Infection in Raccoon Dogs in Northern China
CI, confidence interval; OR, odds ratio.
Discussion
The results of this study indicated that T. gondii infection in domestic raccoon dogs was widely distributed in Jilin, Liaoning, and Hebei provinces with the infection rate 4.75%, which was lower than the 11.15% in domestic raccoon dogs in Shandong by seminested PCR (Zhou et al. 2017), 18.3% in mainland raccoon dogs in a zoo in Japan by latex fixation tests (Murasugi et al. 1996), and 60% in raccoon dogs in Poland by PCR (Gorecki et al. 2012), but higher than the 3.3% in raccoon dogs in suburban areas of Japan (Neagari et al. 1998). The differences in prevalence of T. gondii in raccoon dogs are probably due to differences in species, detection methods used, living conditions, and climatic conditions.
It is interesting that genotype (ToxoDB #9) was identified in Jilin and Liaoning provinces at the same time, whereas genotype (ToxoDB #10) was only identified in Hebei province, which suggests that the genotype (ToxoDB #9) may be predominantly prevalent in China. As far as we know, the genotype (ToxoDB #10) was identified in Hebei province for the first time. In conclusion, the genetic diversity of T. gondii strains in domestic raccoon dogs may be relatively low in northeastern China, although further studies should be conducted to draw a more accurate conclusion by collecting more samples from many provinces in China. Genotype #9 had been identified in several animals in China, such as raccoon dogs in Shandong province (Zhou et al. 2017), cats in Beijing, Guangdong, and Anhui provinces (Dubey et al. 2007, Chen et al. 2011, Qian et al. 2012), pigs in Henan, Anhui, Yunnan, and Guangdong provinces (Zhou et al. 2009, 2010, 2011b, Wang et al. 2012), Reed Vole in Jilin province (Zhang et al. 2014), bats in Guangxi and Yunnan provinces (Jiang et al. 2014), wild waterfowls in Jilin province (Zhang et al. 2015), horses in Xinjiang (Ren et al. 2019), and humans (Wang et al. 2013). And it was also reported in Vietnam and Sri Lanka, which indicated a widespread distribution in Eastern Asia (Shwab et al. 2014). By contrast, Genotype #10 had been identified in raccoon dogs in Guangdong province (Zhou et al. 2017), bats in Jilin, Guangdong, Yunnan, and Guangxi provinces (Jiang et al. 2014, Qin et al. 2014), Based on earlier reports, ToxoDB #9 was a predominant lineage prevalent in mainland China.
Raccoon dogs studied by us are economic animals domesticated in many provinces in China, which was taken full advantage of in clothing industry, foodservice industry, and medicine industry, including fur, meat, and gallbladder (juice). In addition, the products from domestic raccoon dogs are transported to many parts of the country from domestic raccoon dog farms in surveyed areas. Therefore, the significance of T. gondii infection in domestic raccoon dogs should be taken more attention, because it can lead to T. gondii infection in humans and other animals by consumption of undercooked or raw products from infected domestic raccoon dogs. Although the meat from domestic raccoon dogs only represents a small part of meat products compared with the consumption of pigs, cattle, sheep, and poultry, it should not be neglected as a potential risk of T. gondii infection in human health.
In conclusion, this study has determined the prevalence and genetic characterization of T. gondii strains in domestic raccoon dogs in Jilin, Liaoning, and Hebei provinces. Only two genotypes (ToxoDB #9 and ToxoDB#10) may be revealed, and the genotype (ToxoDB #9) was the major lineage in mainland China. As far as we know, this is the first report of genotyping of T. gondii from raccoon dogs in Jilin, Liaoning, and Hebei provinces. These findings will provide basic prevalence information about T. gondii infection in raccoon dogs, and have implications for better understanding of the genetic characterization of T. gondii in China.
Footnotes
Author Disclosure Statement
No conflicting financial interests exist.
Funding Information
Project support was provided by National Key Research and Development Program of China (2016YFC1200200) and Self-listed Scientific Research Projects from General Station of Forest and Grassland Pest Management, National Forestry, and Grassland Administration.