First detection and genotyping of Enterocytozoon bieneusi in pet fancy rats (Rattus norvegicus) and guinea pigs (Cavia porcellus) in China

Enterocytozoon bieneusi, an obligate intracellular microsporidian parasite, can infect humans and a wide variety of animals worldwide. However, information on the prevalence and molecular characterization of E. bieneusi in pet rats and guinea pigs is lacking. In this study, 325 fecal samples were collected from 152 pet fancy rats and 173 pet guinea pigs purchased from pet shops in Henan and Shandong provinces. The prevalence of E. bieneusi was 11.2% (17/152) in pet fancy rats and 20.2% (35/173) in pet guinea pigs. Genotypes D (n = 12), Peru11 (n = 3), S7 (n = 1) and SCC-2 (n = 1) were identified in pet fancy rats, and genotype S7 (n = 30) and a novel genotype PGP (n = 5) were identified in pet guinea pigs. The ITS sequence and its phylogenetic analysis showed that the novel genotype PGP was distinctly different; it exhibited less than 50% similarity to the reference sequences, and did not cluster with any of the known E. bieneusi genotype groups, forming a unique branch between groups 6 and 7. These data suggest that this is a new E. bieneusi genotype group. This is the first report of E. bieneusi infection in pet fancy rats and pet guinea pigs worldwide. The identification of zoonotic genotypes D, Peru11, and S7 suggests that pet fancy rats and guinea pigs can be potential sources of human microsporidiosis.


Introduction
Enterocytozoon bieneusi, a unicellular and obligate intracellular pathogen, has an extensive host range and has been identified in humans, livestock, companion animals, and wildlife, as well as in wastewater [17,23]. Enterocytozoon bieneusi infection can cause self-limiting diarrhea, malabsorption, and wasting in immunocompetent hosts and life-threatening diarrhea in immunocompromized individuals [10]. Humans and animals can acquire infection via fecal-oral transmission of spores from infected individuals through direct contact or by consumption of contaminated food or water [17].
Genotyping based on the internal transcribed spacer (ITS) region of the rRNA gene has identified 11 major phylogenetic groups and more than 470 genotypes of E. bieneusi from various hosts [17]. To date, more than 60 E. bieneusi genotypes have been identified in rodents worldwide [6,8,12,17,24,26,30,34]. For rats (Rattus spp.), only three surveys have focused on the molecular characterization of E. bieneusi in wild rats in Iran and China, and six genotypes (D, M, Peru6, CD6, BEB6, and CHG2) have been identified [24,30,34]. Only one published article has reported genotype peru16 from household guinea pigs in Peru [3].
Fancy rats, Rattus norvegicus forma domestica, are rodents belonging to the order Rodentia and family Muridae. Fancy rats have been bred as pets at least since the late 19th century; they are considered to be intelligent, playful, and trainable animals (http://www.afrma.org/). In recent years, fancy rats have become a very popular pet in China. Pet rodents can be hosts to several zoonotic pathogens, including viruses, bacteria, and parasites [20]; zoonotic transmission of E. bieneusi to a child from household guinea pigs has been reported [3]. However, no literature is available about the prevalence and genetic characteristics of E. bieneusi in pet rats and pet guinea pigs. Therefore, the aim of the present study was to determine the prevalence and genotypes of E. bieneusi in these animals and to assess its zoonotic potential.

Ethics statement
The research protocol was reviewed and approved by the Research Ethics Committee of Henan University of Science and Technology.

Sample collection
Between September 2018 and October 2019, 152 pet fancy rats and 173 pet guinea pigs were purchased from six pet shops in Luoyang, Henan and Weifang, Shandong, China (Tables 1  and 2). Upon arrival in the laboratory, each animal was immediately placed into a single clean plastic box for collection of fresh feces. A single sample was collected from each animal.
All the specimens were refrigerated at 4°C and DNA was extracted within one week. Only young pet fancy rats (4-10 week-old) and 1-8-month-old pet guinea pigs were available in these pet shops. All pet fancy rats and guinea pigs examined in this study were asymptomatic at the time of sample collection, and information on region, age, and sex of these animals was recorded.

DNA extraction
Each specimen was washed with distilled water by centrifugation for 10 min at 3000 Âg at room temperature. Before DNA extraction, 200 mg of each fecal sample was added to a 2 mL microcentrifuge tube containing 200 mg of glass beads, and were vortexed at maximum speed until the fecal samples were completely homogenized. Genomic DNA was extracted using an E.Z.N.A. Stool DNA Kit (Omega Bio-tek Inc., Table 1. Prevalence and genotypes of Enterocytozoon bieneusi in pet fancy rats (Rattus norvegicus) in Henan and Shandong provinces, China.

Characteristics
No  Norcross, GA, USA), according to the manufacturer's instructions. The extracted DNA was kept at À20°C before being used in PCR analysis.

Sequencing and phylogenetic analysis
Two-directional sequencing of positive PCR products was done by Sangon Biotech Co. Ltd., (Shanghai, China). The obtained nucleotide sequences were aligned with available sequences in GenBank, using ClustalX 2.1 (http://www. clustal.org/) [15]. Genotypes of E. bieneusi were determined based on~243 bp of the ITS region, according to the established nomenclature system [22]. A neighbor-joining tree was generated using MEGA7 software (http://www.megasoftware. net/) [14]. The evolutionary distances were computed using the maximum composite likelihood method, and the reliability of branches in the tree was assessed by bootstrap analysis using 1000 replicates.

Statistical analysis
Chi-square analysis was performed to assess the correlation between the prevalence of E. bieneusi and the age, sex, and region of pet fancy rats and guinea pigs using SPSS, version 17.0 (Statistical Package for the Social Sciences).

Nucleotide sequence accession numbers
Unique ITS nucleotide sequences of E. bieneusi obtained from pet fancy rats and guinea pigs in this study were deposited in the GenBank database under accession numbers MN550998-MN551001 and MN998614-MN998615, respectively.

Results and discussion
In the present study, E. bieneusi was detected by PCR in 17 (11.2%) of 152 pet fancy rats and 35 (20.2%) of 173 pet guinea pigs. To our knowledge, this is the first report of E. bieneusi infection in pet rats and pet guinea pigs worldwide. To date, there have been three studies focusing on E. bieneusi infection in wild rats in Iran and China [24,30,34] (Table 3). In this study, the prevalence of E. bieneusi in pet fancy rats was slightly higher than that (4.0%-8.9%) in wild rats in the above-mentioned reports. The prevalence of E. bieneusi in pet guinea pigs in this study was higher than that (14.9%, 10/67) in household guinea pigs in Peru [3], and also higher than other pet rodents, such as pet chinchillas (3.6%), pet squirrels (16.7%) and chipmunks (17.6%) [7,8,21].
In both pet fancy rats and guinea pigs, although the prevalences of E. bieneusi in younger animals and those from Weifang, Shandong were higher than those in older animals and animals from Luoyang, Henan (Tables 1 and 2), the differences in prevalence in both species between different regions, ages and sex groups were not significant (p > 0.05). This finding was consistent with the observations reported in a previous study on pet red-bellied tree squirrels in China [7].
In the present study, genotype Peru11, a zoonotic genotype, was identified in pet fancy rats for the first time. This genotype has been found previously in humans in Peru, China and Thailand, non-human primates in Kenya and China, raccoons, voles and cottontails in the United States, chickens in Brazil, cats in Spain, and minks and water in China [4,5,17,33]. Genotype SCC-2 was reported previously in pet chipmunks and squirrels in China [6,8], and was found in pet fancy rats for the first time. Genotype S7 (synonyms: CHY1) was originally reported in a patient in the Netherlands [25], and recently identified in a yak and pet chipmunks in China [8,16]. This genotype was identified in a fancy rat; moreover, it was predominant in pet guinea pigs in this study, suggesting that guinea pig might be an important reservoir host of genotype S7. More studies are needed to understand the host range and public health importance of genotypes S7 and SCC-2.
In the phylogenetic tree of the E. bieneusi ITS region (Fig. 1), genotypes D and Peru11 were clustered into group 1 with strong zoonotic potential [17], and genotype S7 was clustered into group 10. Genotype SCC-2 belonged to a group which includes several chipmunk and squirrel-derived genotypes such as SCC-1-3 and RS01. Sequence and phylogenetic analysis showed that the novel genotype PGP was distinctly different. Genotype PGP exhibited less than 50% sequence similarity to the reference sequences from the known E. bieneusi genotype groups and outliers, i.e., 45.5% similarity to genotype CM18 in group 7, and less than 30% as compared with those in group 11 and outliers. The novel genotype PGP identified in pet guinea pigs did not cluster with any of the known E. bieneusi genotype groups, and formed a unique branch which was located at an intermediate position between groups 6 and 7 (Fig. 1). These data suggest that the genetic variability of E. bieneusi is broad, and indicate the presence of a new E. bieneusi genotype group; similar observations have been reported in previous studies [1,13,19]. Further studies on more samples collected from different regions should be conducted to understand the genetic diversity of E. bieneusi from rodents in China.

Conclusions
This is the first report of E. bieneusi infection in pet fancy rats and pet guinea pigs. Five genotypes (D, Peru11, S7, SCC-2, and a novel genotype PGP) were identified in this study, and genotypes D and S7 were the dominant genotypes in pet fancy rats and guinea pigs, respectively. Rats (Rattus norvegicus) are a new host of E. bieneusi genotypes Peru11, S7, and SCC-2, and guinea pigs might be an important reservoir host of genotype S7. The identification of three zoonotic genotypes (D, Peru11, and S7) suggests that pet fancy rats and guinea pigs may be the sources of E. bieneusi infection in humans. Therefore, pet owners, especially children, should be educated to take precautions to reduce the transmission risk.