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.