Phylogenetic analysis
The phylogenetic analysis of the ITS region of E. bieneusidivided the identified genotypes into the following four groups: Group 1 (n = 13), Group 2 (n = 1), Group 12, and the novel Group 13 (n = 1)(Figure 1) .
DISCUSSION
This study is the first report on the identification of E. bieneusi in rodents in Hainan, the southernmost province of China. Currently, there are 14 studies reporting the presence of E. bieneusi in rodents from six different countries. These studies describe the rate of prevalence of E. bieneusi infection among the rodents in the range of 1.1-100.0% (Li et al., 2020; Gui et al., 2019; Deng et al., 2016, 2018; Qi et al., 2015; Wang et al., 2019; Yu et al., 2019; Zhao et al., 2018; Perec-Matysiak et al., 2015; Daniaová et al., 2015; Guo et al., 2014; Roelling et al., 2015; Sak et al., 2011; Cama et al., 2007) (Table 1 ). There were geographical location-based variation in the average rate of prevalence of E. bieneusi in rodents: 87.55% (7/8) in Peru (Cama et al., 2007), 38.9% (121/311) in Poland (Perec-Matysiak et al., 2015), 35.9% (52/145) in the United States (Guo et al., 2014; Roelling et al., 2015), 10.7% (31/289) at the Switzerland and Germany border (Sak et al., 2011), 12.2% (278/2272) in China (Li et al., 2020; Gui et al., 2019; Deng et al., 2016, 2018, 2020; Qi et al., 2015; Wang et al., 2019; Yu et al., 2019; Zhao et al., 2018), and 1.1% (3/280) in Slovakia (Danišová et al., 2015). These studies also reported species-based variation in the prevalence rate of E. bieneusi infection: 5.1% for bamboo rats, 87.5% for guinea-pigs, 48.3% for prairie dogs, 39.1% for voles, 24.3% for hamsters, 16.7-42.9% for squirrels, 4.0-7.9% for rats, 3.6-71.4% for chipmunks, and 1.1-87.5% for mice (Table 1) . Notably, except for China and the United States, only one study was performed in each of the other countries and thus further large-scale surveillance studies should be conducted to ascertain these findings. We also found a variation in the prevalence rate of E. bieneusiinfection in our rodents. Thus, the rate of prevalence of E. bieneusi was 16.6% in the wild rats, 4.2% in the red-bellied squirrels, and 11.9% in the farmed rodents. Wild rats showed a significantly higher rate of E. bieneusi infection compared with farmed rats and squirrels.
Among the 16 identified E. bieneusi genotypes, genotypes D, Peru8, PigEbITS7, Type IV, Peru11, and EbpA are known human pathogens (Li et al., 2019). Genotype D was the predominant genotype which was found in 44.2% (42/95) of E. bieneusi isolates. This genotype was widely distributed and present in all sampled rodent species. It is also commonly found in human infections in ≥20 countries and has been isolated from ≥25 animal species and water samples (Li et al., 2019). Genotype PigEbITS7 in Asian house rats, brown rats, and Chinese white-bellied rats. This genotype was originally identified in pigs in Massachusetts, USA, and in immunocompromised patients in Ahvaz in Iran, and Gangxi and Henan in China (Buckholt et al., 2002; Liu et al.,2017; Wang et al., 2013). In addition to pigs and humans, genotype PigEbITS7 has been identified in monkeys and bamboo rats from China (Wei et al., 2019; Zhao et al., 2020). Genotypes Peru8, type IV, and Peru11 were detected in a single rat species but are known to be human and animal pathogens (Li et al., 2019). Thus, the identification of the above mentioned six genotypes in rodents indicated the transmission of parasites from pathogen-infected rodents to humans as well as other animals.
The remaining two known genotypes ESH-02 and S7 were found in Asian house rats and Asiatic brush-tailed porcupines, respectively. Genotype ESH-02 (also named Ind 1) was originally identified in wastewater treatment plant effluents in Shanghai, China (Ma et al., 2016), and also in renal transplant recipients and AIDS patients in India (Khanduja et al., 2017). There are no published reports of the presence of this genotype in any animal species. This study confirmed for the first time that genotype ESH-02 can infect rats, suggesting its zoonotic potential. Genotype S7 (also named CHY1) was previously identified in an immunosuppressed patient in the Netherlands in 2009 (ten Hove et al., 2009), yark in Henan, China (Li et al., 2015), chipmunks and rabbits from Sichuan, China (Deng et al., 2018) and experimental rats in Henan, China (Li et al., 2020). We found that this genotype of E. bieneusi was also found in Asiatic brush-tailed porcupines. These finding indicated that genotype S7 has a wide range of animal reservoirs and potential for zoonotic transmission. Further studies should be conducted to explore additional animal reservoirs of these genotypes.
In this study, 13/16 (81.3%) genotypes and 95.8% (91/95) of theE. bieneusi isolates belonged to Group 1. The genotypes in this group has been identified in several hosts, such as humans, and possess a high potential for cross-species and zoonotic transmission of E. bieneusi (Li et al., 2019). Group 1 was deemed zoonotic based on the prevalence of genotypes such as Type IV, D, Peru11, EbpC, and Peru8 in several animal hosts (Li et al., 2019). The fact above suggesting that the E. bieneusi -infected rodents posed a serious threat to the local inhabitants. Meanwhile, the identification of genotype HNR-VII belonging to the novel Group 13, was a unique epidemiological feature ofE. bieneusi in rodents in Hainan Province of China.