Molecular characterization and novel genotypes of Enterocytozoon bieneusi in pet snakes in Beijing, China

Little is known regarding the Enterocytozoon bieneusi genotypes in snakes worldwide. In the present study, a total of 273 fecal samples were collected from pet snakes in Beijing, China. They were then tested for the presence of E. bieneusi by PCR amplification of the internal transcribed spacer (ITS) gene. The overall infection rate of E. bieneusi was 4.4% (12/273), with the highest infection rate (20%, 1/5) of E. bieneusi was found in the Black rat snake (Pantherophis obsoletus), whereas no positive samples were detected from both Milk (0/22) and Coast garter snakes (0/2). Eight genotypes were identified, including four known genotypes: EbpA (n = 1), EbpC (n = 5), Henan-III (n = 1), and SHR1 (n = 1), and four novel genotypes: CRep-5 (n = 1), CRep-6 (n = 1), CRep-7 (n = 1), and CRep-8 (n = 1). Among them, EbpC (41.7%, 5/12) was the predominant genotype. Phylogenetic analysis showed that seven genotypes belonged to group 1, while genotype SHR1 belonged to group 2. Genotypes EbpA, EbpC, and Henan-III have been previously reported in humans. This suggests that pet snakes are a potential source of zoonotic microsporidiosis transmission in China.


Introduction
Enterocytozoon bieneusi, an opportunistic intestinal pathogen associated with microsporidiosis. The parasite is widespread among humans, domestic animals, and wildlife in China (Li et al., 2019a). Transmission occurs through direct or indirect contact with contaminated food and water from infected humans or animals (Decraene et al., 2012). Self-limiting diarrhea, malabsorption, and wasting are the main clinical signs of E. bieneusi infection in healthy individuals, although life-threatening diarrhea in immunocompromised patients, especially in HIV + persons, does occur (Wang et al., 2013).
Genotyping based on the internal transcribed spacer (ITS) region of the ribosomal RNA (rRNA) gene is a widely used approach in molecular epidemiology studies of E. bieneusi (Santin and Fayer, 2009;Deng et al., 2019). At present, over 500 genotypes have been classified into at least 11 groups (Li et al., 2019b). Among them, many of the common genotypes, such as D, EbpC, Type IV, Peru6, Peru8, and Peru11, are zoonotic, underscoring their importance in public health (Li et al., 2019c;Wang et al., 2018;Zhou et al., 2020).
Few studies have been carried out regarding the genetic characterization of E. bieneusi in snakes. In a previous study, six E. bieneusi genotypes, with two known (type IV and Henan V) and four novel genotypes (CRep-1 to CRep-4) were identified in captive snakes from the Guangxi province in China. These genotypes belong to the zoonotic group 1 (Karim et al., 2014a). The aim of the present study was to estimate the prevalence and molecular characterization of E. bieneusi from pet snakes in Beijing, China.

Ethics statement
Permission was obtained from the pet owners prior to collection of fecal samples. No specific permits were required for the described field studies. The pet owners were not engaged in any form of discussion about this study. The protocol was also reviewed and approved by the Ethics Committee of Tarim University.

Sample collection
A total of 273 fecal samples were collected from pet snakes belonging to 13 different owners. Snakes were kept in individual cages and the main food was mice. Fecal sample was collected individually from each snake using sterile disposable latex gloves and was transferred to individual plastic bags. Sex, breed, and owner were recorded. All fecal specimens were stored at 4°C until the time of processing.

DNA extraction and PCR amplification
Genomic DNA was extracted from fecal samples (approximately 200 mg) using E.Z.N.A. Stool DNA kits (Omega Biotek Inc., USA) according to the manufacturer's instructions. The primers and thermal cycling parameters used for PCR amplification were used as previously reported by Sulaiman et al. (2003). The outer forward primer was 5′-GATGGTCATAGGGATGAAGAGCTT-3′, and the outer reverse primer 5′-AATACAGGATCACTTGGATCCGT-3′ was used. The nested forward primer 5′-AGGGATGAAGAGCTTCGGCTCTG-3′ and nested reverse primer 5′-AATATCCCTAATACAGGATCACT′ was used for the second round of amplification. The 2×EasyTaq PCR SuperMix (TransGene Biotech Co., Beijing, China) was used for PCR amplification. Positive (DNA from dairy cattle-derived genotype I) and negative controls (distilled water) were included in all PCR tests. Amplicons were resolved by electrophoresis on 1% agarose gels (w/v) using GelRed™ (Biotium Inc., Hayward, CA, USA) staining.

Sequencing and phylogenetic analysis
Positive secondary PCR products were sequenced by GENEWIZ (Suzhou, China). All products were sequenced in both directions to ensure accurate sequencing results. ClustalX 2.1 (http://www.clustal. org/) was used to align the resulting DNA sequences. Sequences were aligned with reference sequences downloaded from the National Center for Biotechnology Information (https://www.ncbi.nlm.nih.gov/) to determine genotypes.
Phylogenetic trees were constructed in MrBayes (Version 3.2.6, http://mrbayes.sourceforge.net/) with Bayesian inference (BI). Bayesian inference was conducted with four independent Markov chains run for 1,000,000 metropolis-coupled MCMC generations. The analysis was performed until the potential scale reduction factor approached 1, and the average standard deviation of split frequencies was < 0.01. Phylograms were drawn using FigTree v.1.4 (http://tree. bio.ed.ac.uk/software/figtree).

Statistical analyses
Differences in rates of infection among different ages and species were compared using chi-square tests in the software SPSS version 22.0. Results with a P < 0.05 were considered statistically significant.

Results and discussion
In the 273 fecal samples, 12 positive samples of E. bieneusis (4.4%) were detected by PCR. A similar prevalence rate of 4.6% (11/240) has been reported in captive snakes in Guangxi province, China (Karim et al., 2014a). Furthermore, E. bieneusi infections were identified in pet snakes from 7/13 owners, with owner G having the highest infection rate (16.7%, 1/6) ( Table 1). There was no significant association between E. bieneusi prevalence snake sex, although prevalence in males (8.3%) was higher than in females (3.0%) (P > 0.05) ( Table 2). The highest infection rate was 20% (1/20) in black rat snakes (Elaphe obsoleta obsoleta). In contrast, E. bieneusi infection was not detected in either milk snakes (Lampropeltis triangulum) or coast garter snakes (Thamnophis sirtalis) (P > 0.05) ( Table 2). Many factors contribute to the prevalence of E. bieneusi, such as geographical regions, the host health status, seasonal variations, and the sample size (Deng et al., 2019;Hu et al., 2017). Due to the limited reports of E. bieneusi in snakes, more research is needed to investigate whether there are differences in prevalence in snakes produced by different breeding methods or geographical distribution.
Genotype EbpC (41.7%, 5/12) was the preponderant genotype in the present study, and the remaining seven genotypes accounted for 1 sample each (Table 1). Furthermore, EbpA and EbpC were the most common genotypes identified in both humans and animals worldwide. Genotype EbpA has been reported in humans, pigs, rats, and bamboo rats (Fiuza et al., 2015;Li et al., 2019cLi et al., , 2020Wang et al., 2019). Genotype EbpC has been reported in humans, nonhuman primates, pigs, deer, goat, cattle, rats, and in environmental samples (Karim et al., 2014b;Li et al., 2019aLi et al., , 2020Udonsom et al., 2019;Xie et al., 2019). Genotype Henan-III, has been identified in humans, pigs and whooper swans (Li et al., 2014(Li et al., , 2019aWang et al., 2013Wang et al., , 2020, whereas SHR1 has been reported in experimental rats . As pets come in close contact with humans, and pet snakes can harbor the zoonotic genotypes, the potential for snake owners to become infected with E. bieneusi is a real concern. Phylogenetic analysis showed that genotypes EbpA, EbpC, Henan-III and CRep-5 to CRep-8 clustered with group 1, while SHR1 belongs to group 2 (Fig. 1). Due to the low host specificity, groups 1 and 2 are considered to be zoonoses. In contrast, genotypes within groups three through 11 exhibit a tightly restricted host range (Li et al., 2019a). With more and more genotypes beingn identified, it is likely that additional groups will be added to E. bieneusi. Moreover, zoonotic E. bieneusi genotypes (e.g., D, EbpA, and EbpC) have been reported in rodents, including experimental mice and wild rats . These animals make up the majority of the snake diet, especially those raised in captivity. Whether the source of E. bieneusi infection of snakes is foodborne or not has yet to be investigated.
In conclusion, E. bieneusi was low infection rate in pet snakes in Beijing in China. Of the 12 identified genotypes, seven are zoonotic, with genotype EbpC the predominant genotype. This indicates the potential of pet snakes as the possible vectors for the transmission of E. bieneusi to humans.