Occurrence and genetic characterization of Enterocytozoon bieneusi in pet dogs in Yunnan Province, China

Enterocytozoon bieneusi is the most common microsporidian species in humans and can affect over 200 animal species. Considering possible increasing risk of human E. bieneusi infection due to close contact with pet dogs and identification of zoonotic E. bieneusi genotypes, 589 fresh fecal specimens of pet dogs were collected from Yunnan Province, China to determine the occurrence of E. bieneusi, characterize dog-derived E. bieneusi isolates, and assess their zoonotic potential at the genotype level. Enterocytozoon bieneusi was identified and genotyped by PCR and sequencing of the internal transcribed spacer (ITS) region of the ribosomal RNA (rRNA) gene. Twenty-nine specimens (4.9%) were positive. A statistical difference was observed in occurrence rates of E. bieneusi in pet dogs among 11 sampling sites by Fisher’s exact test. Fifteen genotypes were identified and all of them phylogenetically belonged to zoonotic group 1, including four known genotypes (EbpC, D, Peru 8, and Henan-III) and 11 novel genotypes. Genotype Henan-III was reported in dogs for the first time. The finding of known genotypes found previously in humans and novel genotypes falling into zoonotic group 1 indicates that dogs may play a role in the transmission of E. bieneusi to humans in the investigated areas.


Introduction
Microsporidia are obligate intracellular parasitic fungi, and they have the ability to infect both humans and animals.Within microsporidia, there are at least 1,700 recognized species belonging to approximately 220 genera, with 17 species having been found in humans [10].Molecular epidemiological data have confirmed four major microsporidial species infecting humans, including Enterocytozoon bieneusi, Encephalitozoon cuniculi, Encephalitozoon intestinalis, and Encephalitozoon hellem [32].Among them, E. bieneusi has been regarded as the most prevalent, accounting for more than 90% of human microsporidiosis cases [32].According to the largest metaanalysis of microsporidia globally, the overall prevalence of E. bieneusi infection in humans was 7.9% [28].Immunocompromized/immunodeficient individuals are reported to be the most susceptible population, such as HIV-positive patients, with the prevalence of E. bieneusi infection ranging from 1.2% to 100% [40].Enterocytozoon bieneusi primarily invades the epithelial cells of the small intestine, and mainly causes disease characterized by diarrhea, and due to lack of effective drugs available, life-threatening diarrhea often occurs in immunocompromized patients [16].Enterocytozoon bieneusi is also infrequently identified in other epithelial cells of patients with AIDS, i.e., in the biliary tree, gallbladder, nonparenchymal liver cells, pancreatic duct, and tracheal, bronchial, and nasal epithelia [37].In addition to humans, E. bieneusi has also been detected in at least 210 animal species, suggesting its zoonotic potential [15].The findings of the same E. bieneusi genotypes in both humans and animals support the presumption of zoonotic potential.Meanwhile, in many epidemiological studies of E. bieneusi in animals, a high occurrence rate and a large percentage of zoonotic genotypes were observed, such as 93.7% and 100% in pigs in the Czech Republic [29], 37.6% and 100% in cattle in China [39], and 9.6% and 100% in dogs in Spain [9], implying a risk of zoonotic transmission.In fact, early in 2007 in Peru, Cama et al. reported zoonotic transmission of E. bieneusi based on the finding that an unusual genotype (Peru 16) was identified in seven guinea pigs and a 2-year-old child in the same household [2].
Enterocytozoon bieneusi transmission to humans is usually via the fecal-oral route through ingesting water and food contaminated by infective spores or direct contact with infected individuals (humans and animals) [27].The epidemiological roles of water and food in the transmission of microsporidiosis caused by E. bieneusi are now well recognized.Three outbreaks related to E. bieneusi have been documented worldwide: one waterborne outbreak in France; and two foodborne outbreaks in Sweden and Denmark [4,6,23].Due to clinical importance and public health significance, in the United States, the National Institute of Allergy and Infectious Diseases (NIAID) classified E. bieneusi as a Category B Priority Pathogen [8], and the Environmental Protection Agency (EPA) listed this microsporidian in its Contaminant Candidate List of Waterborne Organisms (https://www.epa.gov/ground-water-and-drinkingwater/national-primary-drinking-water-regulations).
Enterocytozoon bieneusi is now known as a species complex.By analyzing sequences of the internal transcribed spacer (ITS) region of the ribosomal RNA (rRNA) gene with a high degree of genetic polymorphism, 819 genotypes had been identified by June 2021: 126 exclusively in humans, 614 exclusively in animals, 58 in both humans and animals, and 21 only in environmental samples [15].These genotypes have been divided into 11 different phylogenetic groups and an outlier with a varying degree of host specificity and zoonotic potential [17].The genotypes of groups 1 and 2 present a broad range of hosts including humans, suggesting low host specificity and large potential for zoonotic or cross-species transmission; in contrast, the genotypes of groups 3-11 and the outlier appear to be more host-specific, indicating limited zoonotic potential [17].
In China, E. bieneusi as an emerging enteric pathogen was first reported in both humans and animals in 2011 [39].To date, epidemiological studies of E. bieneusi in dogs have been carried out in 12 provinces and municipalities (Table 1).However, in southwestern China's Yunnan Province, few data are available regarding E. bieneusi infection in dogs.Currently, there has been only one epidemiological study reporting occurrence and genotyping of E. bieneusi in dogs in Yunnan Province [36].Considering that close human contact with pet dogs and identification of zoonotic genotypes in dogs can increase the risk of E. bieneusi infection, the present study was performed to determine the occurrence rate, genetic characterization, and zoonotic potential of E. bieneusi at the genotype level in pet dogs in Yunnan Province.

Ethics statement
The objective and procedure of the present study were reviewed and approved by the Research Ethics Committee and the Animal Ethics Committee of Hangzhou Medical College and the National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, China (IPD-2020-15).All work with animals was strictly performed in accordance with the Regulations for the Administration of Affairs Concerning Experimental Animals.Beginning work on the study, we contacted the pet dog owners and obtained their permission to have their animal feces involved in our study.All the animal fecal specimens were collected only after defecation without disturbing them.

Fecal specimen collection
During a two-month period from April to June, 2021, 589 fresh fecal specimens (approximately 15 g) were collected from pet dogs (one specimen each) from 11 sampling sites distributing in eight cities/autonomous prefectures in Yunnan Province (Table 2).Each dog fecal specimen was collected immediately after defecation using disposable latex gloves and placed in clean sealed bags with their unique identification numbers on them.Meanwhile, ages of dogs and sampling sites were recorded.All the animals involved in the present study had no clinical signs of diarrhea at the time of sampling.The collected fecal specimens were shipped to our laboratory in a cooler with ice packs.If DNA extraction could be finished within 48 h, the fecal specimens were kept at 4 °C.If not, the fecal specimens were stored at À20 °C.

DNA extraction
Genomic DNA was extracted from 180 to 200 mg of fecal specimens using a QIAamp DNA Stool Mini Kit (QIAGEN, Hilden, Germany), following the manufacturer's instructions.To obtain a high yield of DNA, the lysis temperature was increased to 95 °C.DNA was eluted in 200 lL of ATE and stored at À20 °C before it was used for PCR analysis.

PCR amplification
All the DNA preparations were analyzed for the presence of E. bieneusi by nested PCR amplification of a 410 bp fragment including the ITS region (243 bp) of the rRNA gene, as previously described [24].Each specimen was subjected to at least two PCR reactions.A positive control (DNA of a humanderived genotype Type IV) and a negative control (nucleasefree water) were included in each PCR test.The secondary PCR products were analyzed by 1.5% agarose gel and visualized under UV by staining the gel with GelStrain (TransGen Biotech., Beijing, China).

Nucleotide sequencing and analyzing
All expected size secondary PCR products were directly sequenced on an ABI PRISM 3730XL DNA Analyzer by Comate Bioscience Company Limited (Jilin, China), using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Carlsbad, CA, USA).Accuracy of nucleotide sequences could be guaranteed by sequencing in both directions and by sequencing another two new PCR products for some of the DNA preparations yielding new nucleotide sequences.
The raw sequences obtained in the present study were spliced together with Clustal X 1.81 (http://www.clustal.org/)and aligned with sequences deposited in the GenBank database by BLAST analysis to determine E. bieneusi genotypes.All the genotypes were identified only based on 243 bp of the ITS region of the rRNA gene of E. bieneusi, according to the established nomenclature system [31].If the sequences obtained in the present study were identical to the previous published sequences, they were determined as known genotypes and given the first published genotype names.In contrast, if the sequences had single nucleotide substitutions, deletions or insertions compared to the sequences of the known genotypes, they were considered novel genotypes and would be given their names.

Phylogenetic analysis
To analyze the genetic relationship between E. bieneusi genotypes identified in the present study and those described in previous studies, a neighbor-joining (NJ) tree of the ITS sequences was constructed using Molecular & Evolution Genetic Analysis software version 11.0 (MEGA 11.0) (http:// www.megasoftware.net/) based on the evolutionary distances calculated by the Kimura-2-parameter model.Bootstrap analysis with 1,000 replicates was used to determine support for the clades.

Statistical analysis
Fisher's exact test implemented in Statistical Package for the Social Sciences (SPSS) version 23.0 software was used to compare differences in occurrence rates of E. bieneusi in animals from different sampling sites.A p value of < 0.05 was considered statistically significant.
There was an absence of E. bieneusi in Chuxiong, Deqin, and Shilin.A statistical difference was observed in occurrence rates of E. bieneusi in pet dogs from different sampling sites (p = 0.002) (Table 2).

Geographical distribution of E. bieneusi genotypes
Enterocytozoon bieneusi was found in dogs from eight sampling sites.Four known genotypes (EbpC, D, Peru 8, and Henan-III) were found at six, three, one and one sites, respectively with genotypes EbpC, D, and Peru 8 appearing at one site  (Shangri-La).The 11 novel genotypes were from five areas, with 5 appearing in Zhaoyang (Table 2).Genotype EbpC was observed to have the widest geographical distribution.

Phylogenetic relationship of E. bieneusi genotypes
In a phylogenetic analysis of a neighbor-joining tree of the ITS sequences of the rRNA gene of E. bieneusi, 15 genotypes (four known genotypes and 11 novel genotype) obtained in the present study all fell into group 1, considered to be the zoonotic group (Figure 1).

Discussion
It is already known that dogs can provide many services to humans.Recently, it has been evidenced that a dog companion may be good for our physical health, since contact with dogs can lower blood pressure, ameliorate depression, and even produce a survival benefit after myocardial infarction [12].Pet popularity has been increasing over the past few years.According to the Chinese Pet Industry White Paper, the number of pet dogs has increased rapidly to have reached 62 million up to 2019 (https://www.chyxx.com/industry/202006/874331.html).However, dogs can act as reservoir hosts for many zoonotic pathogens, and they can transmit the diseases to humans.Thus, dog health is not only a veterinary issue, but also a public health issue.In the investigated area (Yunnan Province), dogs were reported to carry at least 40 pathogens, with 24 of them being zoonotic [11].A recent epidemiological investigation reported E. bieneusi in dogs in Yunnan Province in 2021 for the first time [36].
In the present study, 4.9% (29/589) of pet dogs were infected with E. bieneusi.The occurrence rate was lower than the global average occurrence (8.4%) of E. bieneusi in pet dogs and was only slightly higher than those reported in pet dogs in two studies worldwide: 4.4% in Japan and 3.2% in Heilongjiang

T C T G A T G T A C T G T T G T C G C T G
The symbol "/" denotes base deletion.The symbol "-" denotes sequence identity to genotype EbpC (AF076042).1).This might be because the investigated stray dogs were usually housed at dog shelters and raised in a crowded house or enclosure, inevitably leading to occurrence of cross-transmission/infection of E. bieneusi between different individuals.Enterocytozoon bieneusi is a causative agent of opportunistic infection.The health status of hosts seems to be the key factor related to E. bieneusi infection, and it has been confirmed to be closely associated with host age.In a study of molecular detection of E. bieneusi in pet dogs in Japan, a significant difference in occurrence rates was found between two age groups (<1 year: 8.3% versus !1 year: 3.4%) [26].In another study of E. bieneusi in dogs in Australia, juvenile dogs were significantly associated with a higher occurrence rate of E. bieneusi than adult dogs [41].It is understandable that hosting inversely associates with occurrence rates of E. bieneusi, because the young animals usually have immature immune systems.In general, occurrence rates are complicated and difficult to compare.Due to a high degree of genetic polymorphism of the ITS region of the rRNA gene of E. bieneusi, sequence analysis of this region is currently considered the standard method for genotyping E. bieneusi isolates [31].Previous epidemiological studies of E. bieneusi in dogs have identified 54 genotypes, and most of them belonged to zoonotic group 1 (n = 29), group 2 (n = 7), and dog specific group 11 (n = 16).There were another two genotypes identified, with one (CD5) in group 3, and the other (VIC_dog1) in group 7 (Table 1).Among them, 14 of 54 genotypes have been identified in humans.Therefore, dogs infected with E. bieneusi might pose a threat to human health.In the present study, sequence analysis of the ITS region identified 15 genotypes in 29 E. bieneusi isolates, including 4 known genotypes (D, EbpC, Peru 8, and Henan-III) and 11 novel genotypes.
Genotypes D (syn.CEbC, NCF7, Peru 9, PigEBITS9, PtEb VI, SHW1, WL8) and EbpC (syn.CHG23, E, Peru 4, SC03, WL13, WL17) were the most common genotypes either in humans or in animals worldwide [15].To date, genotype D has been identified in 91 host species distributed in 40 countries, while genotype EbpC has been identified in 43 host species in 15 countries.They both showed a wide host range and geographical distribution of the two genotypes [40].In China, genotypes D and EbpC have been detected in humans and numerous wild, domestic, and companion animals from 26 provinces/municipalities [35].Meanwhile, human E. bieneusi infection cases caused by genotypes D and EbpC were ranked first and second, respectively [13].In addition, we also identified zoonotic genotypes Peru 8 (syn.CQR-1) and Henan-III (syn.BLC17), with genotype Henan-III being reported in dogs for the first time worldwide.In comparison with genotypes D and EbpC, the two genotypes seemed to have a relatively narrow host range and geographical distribution.Based on current epidemiological data of E. bieneusi summarized in a review article by Koehler et al. and the present genotyping data, to date, genotype Peru 8 has only been identified in 15 host species in 7 countries (China: 11 animal species), while genotype Henan-III has only been identified in 6 host species in 2 countries (China: 5 animal species) [7].In China, human microsporidiosis cases caused by genotypes Peru 8 (n = 1) and Henan-III (n = 1) were only found in HIV-positive patients on antiretroviral therapy in Henan Province [34].The finding of four zoonotic genotypes (D, EbpC, Peru 8, and Henan-III) in dogs indicates that the epidemiological role of dogs in the transmission of human microsporidiosis caused by E. bieneusi needs to be given more attention.Meanwhile, pet owners should also be made aware of the potential zoonotic transmission of microsporidiosis due to close contact with infected animals.
With accumulation of genotyping data of E. bieneusi, there has been a huge number of genotypes identified.By June 2021, 819 genotypes had been documented, with 465 in China [15], revealing the vast genetic variation of E. bieneusi in the ITS region of the rRNA gene.In the present study, 11 novel genotypes of E. bieneusi were obtained, and all of them fell into zoonotic group 1.Meanwhile, in a homology analysis, five novel genotypes had the largest similarity (99.6%) with two zoonotic genotypes (EbpC and Henan-IV), suggesting their zoonotic potential.

Conclusion
In conclusion, the present study demonstrated occurrence (4.9%, 29/589) and genetic characterization of E. bieneusi in the ITS region of the rRNA gene in pet dogs in Yunnan Province, China.The finding of four zoonotic genotypes in dogs indicates possible occurrence of dog-related zoonotic transmission of human microsporidiosis caused by E. bieneusi in the investigated area.Meanwhile, the observation of all the 11 novel genotypes falling into zoonotic group 1 implies broad zoonotic potential and public health significance.Since the investigated dogs in the present study were asymptomatic, the health condition of these animals was usually neglected.Once pet dogs are infected with E. bieneusi, they have more time and opportunity to continually release infective E. bieneusi spores with feces into the environment to spread the parasitic disease to humans.Furthermore, the high frequency of human contact with pet dogs increases the risk of E. bieneusi infection.Currently, due to no safe and effective drug treatment and vaccines prevention against microsporidiosis caused by E. bieneusi, it is particularly important to develop control strategies to intervene with and prevent human infection.Thus, advice should be given to pet owners, including adequate hygiene practices (adequate pet waste disposal and regular hand washing) and routine veterinary care.Health education should also be provided to pet owners to increase their risk awareness of the potential zoonotic transmission of E. bieneusi.

Figure 1 .
Figure 1.Phylogenetic relationships between genotypes of Enterocytozoon bieneusi identified in this study and known genotypes deposited in GenBank, as inferred by a neighbor-joining analysis of the ITS rRNA gene sequences based on genetic distances calculated by the Kimura-2parameter model.Numbers on the branches are percent bootstrapping values from 1,000 replicates.Each sequence in this figure is identified by its accession number, host origin, and genotype designation.The group terminology for the clusters is based on the work of Li et al. (2019).Before the genotype names, the circles and triangles filled in black indicate the known and novel genotypes identified in present study, respectively.

Table 1 .
Prevalence and genotype distribution of E. bieneusi in dogs worldwide.

Table 2 .
Prevalence and genotypes of E. bieneusi in pet dogs in Yunnan Province.

Table 3 .
Variation at 26 polymorphic sites within the ITS region of the rRNA gene of E. bieneusi isolates obtained in this study.

Table 4 .
Homology analysis of the novel E. bieneusi genotypes in the ITS region of the rRNA gene.Accession Nos.representing the published sequences with the largest similarity to the sequences obtained in the present study.6J.Jian et al.: Parasite 2024, 31, 27Province of northeast China (Table1).The low occurrence rate of E. bieneusi in pet dogs in the present study might be attributable to the fact that all the fecal specimens were collected from adult dogs without clinical signs of illness.In fact, occurrence rates of E. bieneusi in dogs are affected by many factors.