Molecular characterization of Cryptosporidium spp., Enterocytozoon bieneusi and Giardia duodenalis in laboratory rodents in China

Cryptosporidium spp., Enterocytozoon bieneusi and Giardia duodenalis are significant zoonotic intestinal pathogens that can cause gastrointestinal symptoms such as diarrhea and induce a host immune response. A total of 1237 fecal samples were collected from laboratory rodents (rats, mice and guinea pigs) from four different locations in China to investigate the infection rates and molecular characterization of these pathogens on experimental animals. Genomic DNA was extracted from each sample, and PCR amplifications were done. Overall, the Cryptosporidium spp. infection rate was 3.8% (47/1237). Four known Cryptosporidium species were identified, namely C. parvum, C. muris, C. tyzzeri and C. homai, the three former being zoonotic species. The overall E. bieneusi infection rate was 3.0% (37/1237). Seven known E. bieneusi genotypes, namely S7, BEB6, J, Henan-IV, CHG10, D and WL6, were detected by sequence analysis. Among these, genotypes D, Henan-IV and CHG10 have a high zoonotic risk. Giardia duodenalis was not detected at any of the three loci (SSU rRNA, bg and gdh) after PCR amplification. This study provides basic data for these pathogens in laboratory rodents in China and lays the foundation for their prevention and control in laboratory animals.


Introduction
Cryptosporidium spp., Enterocytozoon bieneusi, and Giardia duodenalis are three common intestinal pathogens found both in humans and in a large number of animals throughout the world [27]. These three pathogens can cause clinical symptoms such as weight loss, malnutrition and diarrhea, can induce an immune response in the infected host [2,34], and can even lead to the death of the host [6]. These pathogens mainly spread via the fecal-oral route and can also be transmitted through contaminated food or water [11].
Enterocytozoon bieneusi belongs to the phylum Microsporidia [21]. More than 500 E. bieneusi genotypes have been identified in humans and animals by polymorphism analysis based on the ribosomal internal transcribed spacer (ITS) [20], and they have been placed into 11 distinct groups (Groups 1-11) via phylogenetic analysis. More than 90% of the genotypes belong to Group 1 or 2 with Group 1 genotypes being highly zoonotic. To date, more than 80 genotypes have been identified in rodents (including companion, laboratory and wild rodents), among them BEB6, PigEbITS7, Henan-II, CHN4, Type IV, C, I, J, D, Peru11, Peru8, EbpA, EbpC, CZ3 and S6 can infect humans at the same time [9,12,28]. Scientists around the world have been studying the mechanism of infection and treatment of this pathogen [14]; however, the natural host, transmission route and the role of rodents in E. bieneusi transmission are still unclear.
Eight genetically distinct assemblages (A-H) of G. duodenalis have been defined, with zoonotic assemblages A and B found in both humans and animals. Host-adapted assemblages C and D are found primarily in dogs, E in ruminants, F in cats, G in rodents and H in seals [26,31]. Among these, assemblages A, B and G are commonly detected in wild rodents [21].
Laboratory rodents are widely used in medical, biological, pharmacy, animal husbandry, veterinary medical and scientific research [17]. Whenever laboratory rodents are infected with Cryptosporidium spp., E. bieneusi or G. duodenalis, their physiological, biochemical and immunological indicators are affected, and the infection seriously interferes with test results and can potentially cause disease in both humans and animals [14]. Therefore, the purpose of this study was to determine the infection status and genotype distribution of each of these three intestinal pathogens in laboratory rodents in China and assess the public health potential of Cryptosporidium spp., G. duodenalis and E. bieneusi in laboratory rodents.

Ethical standards
This study was conducted in accordance with the Chinese Experimental Animal Management Law of 1988. The research plan was approved by the Research Ethics Committee of Henan Agricultural University. Fecal samples of laboratory rodents were collected after the approval by the director of the Experimental Animal Center. No animals were injured during the fecal sample collection.

Sample animal and specimen collection
A total of 1237 fecal samples were collected between September 2019 and October 2020, from four medical experimental animal centers located in four different areas (300 from Zhengzhou, 150 from Kunming, 687 from Guangzhou and 100 from Shanghai) in China (Fig. 1). Sample distribution by animal host type was as follows: 118 samples from laboratory rats, 1027 samples from laboratory mice, and 92 samples from laboratory guinea pigs. These rodents belong to specific pathogen-free animals (SPF). The laboratory rodents were housed in individually ventilated cages (IVCs), each of which contained one to six animals. No obvious clinical symptoms were found when collecting feces. Approximately 2 g of fresh fecal samples were collected from each cage, placed in clean plastic zipper bags, marked with the pertinent information and shipped to the parasitology laboratory under cool conditions (4°C) for further detection. Once in the laboratory, the specimens were stored in 2.5% potassium dichromate (4°C) before undergoing molecular biology testing.

DNA extraction
Total genomic DNA was extracted from each fecal sample (approximately 200 mg) using an E.Z.N.A. Ò Stool DNA Kit (Omega Bio-Tek Inc., Norcross, GA, USA), according to the manufacturer's recommended protocol. The extracted DNA was stored at À20°C until PCR amplification.

PCR amplification
Cryptosporidium spp. was examined by nested PCR amplification of an~840 bp fragment of the small subunit (SSU) rRNA gene [32]. Enterocytozoon bieneusi was identified and genotyped based on the PCR amplification of an~389 bp fragment of the ITS of nuclear ribosomal DNA [7]. The presence of G. duodenalis was identified and genotyped by the PCR amplification of an~292 bp fragment of the SSU rRNA gene [3], an~510 bp fragment of the b-giardin (bg) gene [16], and glutamate dehydrogenase (gdh) gene (~520 bp) [8]. Both the positive and negative controls were included in each PCR amplification, and the PCR amplification was performed at least three times per sample.

Sequence and statistical analysis
The positive secondary PCR amplicons were sequenced bidirectionally by SinoGenoMax Biotechnology Co., Ltd. (Beijing, China). To determine the species and genotypes, the sequences obtained in this study were aligned with reference sequences downloaded from GenBank using Clustal X 2.1. All the nucleotide sequences have been submitted to GenBank at the National Center for Biotechnology Information (NCBI).
Geographical locations and ages of rodents for each intestinal pathogen were analyzed using the chi-squared (v 2 ) test. P-values were considered to be statistically significant when <0.05. To infer the phylogenetic relationships of the detected samples, neighbor-joining (NJ) trees were constructed with the MEGA X program, based on the evolutionary distance calculated with the Kimura 2-parameter model. The reliability of these trees was assessed via bootstrap analysis of 1000 replicates.

Nucleotide sequence accession numbers
The nucleotide sequences in this study have been submitted to the GenBank database (GenBank Accession No. OP102682-OP102686, OP103973-OP103978, OP104909).

Coinfection of enteric pathogens
Five samples were found to be positive for both Cryptosporidium and E. bieneusi, 0.4% (5/1237). Three C. ples had coinfection with E. bieneusi genotype BEB6, and two samples had coinfection with C. tyzzeri and E. bieneusi genotype Henan-IV.

Distribution of E. bieneusi genotypes
Thirty-seven specimens were positive for E. bieneusi, with a positive rate of 3.0%, and seven reported genotypes (namely, S7, BEB6, J, Henan-IV, CHG10, D and WL6) of E. bieneusi were identified, of which the infection rates of S7 and BEB6 were higher. Laboratory mice were infected with BEB6, Henan-IV, D, J, CHG10 and WL6, only S7 was found in laboratory guinea pigs, and J and BEB6 were found in laboratory rats. Genotypes D, Henan-IV and CHG10 were classified as Group 1 and had high zoonotic risk. Genotypes J and BEB6 were classified as Group 2 and had potential zoonotic risk. Genotypes WL6 were classified as Group 3, and Genotypes S7 were classified as Group 10 (Fig. 3, Table 1).

Discussion
This study examined Cryptosporidium, G. duodenalis and E. bieneusi infection rates in certain species of laboratory rodents in China. Cryptosporidium is a common pathogen that infects rodents worldwide. The overall prevalence of Cryptosporidium in rodents was found to be 17% in one systematic summary [29]. In the current study, the overall infection rate of Cryptosporidium was 3.8% (47/1237), which is higher than the previously reported infection rates of 1.9% (5/264) and 0.6% (2/355) in laboratory rodents [21,22]. According to a report, the infection rate of laboratory mice 1.7% (4/229) is lower than that of this study 4.3% (44/1027), and that of laboratory rats 4% (1/25) is higher than that of this study 0.8% (1/118) [22]. A report investigated the Cryptosporidium infection rate of laboratory rats in four regions in China, which was 0.6% (2/355), similar to the results of this study [21]. Many studies have reported Cryptosporidium infections in wild rodents all over the world. In previous studies, it is reported that the infection rate of wild rodents was 20.5% (3848/18,804) around the world, and it has been suggested that Cryptosporidium infection is more common in wild rodents than in laboratory rodents [36]. This may be because the organism is easily transmitted through environmental pollution under natural conditions.
In this study, four Cryptosporidium species were identified, among which C. parvum, C. muris and C. tyzzeri were zoonotic. These results are different from those reported in previous studies. In one study, only C. tyzzeri (formerly known as Mouse genotype I) was found in laboratory mice and rats [22]. In a study from Nigeria, C. andersoni and Cryptosporidium rat genotype II were identified in laboratory rats [4]. In another investigation, C. ubiquitum and an undetermined Cryptosporidium genotype were found in experimental rats in China. [19]. In Australia, C. homai was reported to be found in laboratory guinea pigs [35]. The results of this study show that Cryptosporidium infection in laboratory rodents presents a zoonotic risk and provide basic information for the prevention and control of Cryptosporidium in laboratory rodents. However, the positive samples for C. parvum failed to be sub-genotyped by the gp60 marker to determine the genotypes within C. parvum.
There are few reports about E. bieneusi infection in laboratory rodents, showing significant differences in infection rates. In the current study, the E. bieneusi infection rate was found to be 3.0% (37/1237). The E. bieneusi infection rate was found to be 37.9% (11/29) in laboratory prairie dogs in the USA [24]. In a study carried out in China on 291 laboratory rat fecal samples, PCR amplification showed that 4.8% (14/291) were infected with E. bieneusi, which is lower than in this study [18]. The results of this study show that more research is needed on E. bieneusi.
In this study, seven known genotypes were identified in 37 samples. Genotypes J and D are common and have been reported in many countries and many animals. S7 has been reported in humans and bovines in the Netherlands and China [30]. BEB6 has been reported in chinchilla in China and in other animals in the USA, Brazil and Peru. Henan-IV and CHG10 have only been reported in China. WL6 has been reported in muskrats in the USA [28]. Previous studies have identified several known and previously unknown E. bieneusi genotypes. Four known E. bieneusi genotypes (namely, EbpA, Table 1. Occurrence and genotypic distributions of Cryptosporidium spp. and Enterocytozoon bieneusi in certain laboratory rodents in China.

Items
No. samples examined Cryptosporidium spp.

Enterocytozoon bieneusi Cryptosporidium + E. bieneusi
Positive % (n) Species (n) Positive % (n) Genotypes (n) Positive % (n) Species (n) EbpC, S7 and N) and a newly discovered genotype (SHR1) were found in laboratory rats in three cities in China [18]. Genotypes D (n = 1), Henan-IV (4) and CHG10 (n = 1) were classified as Group 1 and had high zoonotic risk. Twelve E. bieneusi genotypes were found in four species of wild mice in southwestern Poland: two known genotypes, D and new gorilla1, and genotypes WR1-WR10 [23]. Seven genotypes (namely, EpbA, C, D, H, PigEBITS5, CZ3 and Peru8) have been identified in wild house mice in the Czech-German border area, while only one (Row) has been reported in an American laboratory [24,25]. Peru16 was found in guinea pig in Peru [9]. Two genotypes (D and Peru6) were found in wild rodents in Heilongjiang Province, and four (CHG14, BEB6, D and CHG2) were found in homologous rodents around dairy farms in Henan Province [37]. These studies identified different intestinal E. bieneusi genotypes in different animal hosts. In general, zoonotic genotypes, such as D, EbpA, EbpC, CHG9 and Pig EBITS7, were identified, indicating that intestinal E. bieneusi spreads between rodents and different animals.
Giardia duodenalis was not found in laboratory rodents in this investigation. However, a previous study reported a G. duodenalis infection rate of 9.3% (33/355) in laboratory rats [19]. The different detection rates in these surveys may be due to differences in animal species, age distribution, sample size, host health status, management level, and population density. Since data regarding G. duodenalis infection in rodents are limited, further epidemiological surveys involving various species of rodent hosts should be undertaken to better understand the genetic diversity, host specificity, and transmission modes of this parasite.
Laboratory animal centers usually have exceptional sanitary conditions and standards, with food and water sterilized prior to being given to animals [17]. The laboratory rodents in this study were fed purified water and pellet food that had been disinfected, and lived in an independent isolation environment; however, some laboratory rodents were still infected with Cryptosporidium and E. bieneusi. The laboratory rodents investigated in this test are SPF animals. According to the standard formulated by China (Gb/T 14922. , these three kinds of pathogens are not included in the test items; however, these pathogens are at risk of zoonotic diseases. There is no direct evidence of how these pathogens spread in laboratory rodents, and data on environmental samples from positive feeding facilities are lacking [1]. Therefore, environmental samples from feeding facilities should be further examined to understand possible transmission routes. Research also shows that laboratories should conduct parasite detection when using non-specific pathogen-free rodents, to improve the accuracy of experimental results and the safety of the experimenter. Laboratory rodents are usually asymptomatic after infection. However, the infection can cause bowel disorders even in rodents with no obvious symptoms [5], which can significantly impact experimental results. Frequent validation of purification for laboratory rodents and the environment is needed. In particular, attention should be paid to the disinfection and sterilization of feed, bedding and cages, as well as the sanitation of drinking

Conclusion
This study indicates that the infection rates with Cryptosporidium, E. bieneusi, and G. duodenalis were low in laboratory rodents in China. These animals can be co-infected with Cryptosporidium spp. and E. bieneusi. Most of the species found in this study are zoonotic. Therefore, improving the management level, strengthening the monitoring and control of parasite infection, improving the feeding conditions and environmental settings, and strictly establishing the health management system for laboratory animals are necessary. A public education program on the infection potential of the three pathogens should be implemented, including breeders and laboratory personnel as part of the "one health" approach to the prevention and control of infection.