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All issues Volume 31 (2024) Parasite, 31 (2024) 75 Full HTML
Open Access
Issue
Parasite
Volume 31, 2024
Article Number 75
Number of page(s) 7
DOI https://doi.org/10.1051/parasite/2024073
Published online 04 December 2024

© S. Zhou et al., published by EDP Sciences, 2024

Licence Creative CommonsThis is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Introduction

Laboratory animals are widely used in academic and research institutions for experimental purposes. To date, at least 200 animal species – mainly rodent species, such as mice, rats, guinea pigs, and hamsters – have been used as laboratory animals [1]. Among them, mice and rats are used most commonly [27] because both of them share high genetic similarity with humans (on average, 85% of protein-coding regions are the same) and demonstrate a strong and fast reproductive capacity, easy inbred strain cultivability, and low breeding cost. According to the annual reports of the United States Department of Agriculture, mice and rats account for 99.3% (range = 97.3%–99.9%; median = 99.4%) of mammals used in laboratories annually [5]. To ensure the welfare of laboratory animals and obtain reliable experimental data, it is necessary to monitor their health status and well-being. In recent years, Cryptosporidium spp. were detected in laboratory mice and rats in several studies [2, 21, 24, 26, 40]. Cryptosporidium spp. are important intestinal protozoa infecting a broad variety of hosts, including humans [34]. The fact that some species/genotypes of Cryptosporidium spp. are found in both humans and animals reflects their zoonotic nature [32].

Cryptosporidium oocysts excreted by infected hosts are immediately infectious to other hosts. Human Cryptosporidium infections spread through fecal-oral transmission, either directly (person-to-person and animal-to-person transmission) or indirectly (waterborne and foodborne transmission) [31]. Due to the wide range of animal hosts and the huge number of animals, animal-to-person transmission has been attracting more and more concern. To date, more than 20 outbreaks related to contact with animals have been reported [22], mainly occurring among veterinarians and veterinary students as well as other people exposed to livestock and children visiting farms [4, 14, 18, 20, 30, 33]. In an academic research laboratory in the Unites States, one cryptosporidiosis outbreak occurred among workers caring for pre-weaned calves [12]. Cryptosporidium spp. primarily invade the epithelial cells of the small intestine of infected hosts and cause a disease mainly characterized by diarrhea. Most seriously, in patients with HIV/AIDS or immunocompromized individuals, diarrhea caused by Cryptosporidium spp. can become chronic or life-threatening [17].

Cryptosporidium is a complex genus, with extensive genetic variations. To date, at least 49 Cryptosporidium species and approximately 120 genotypes have been recognized based on the small-subunit ribosomal RNA (SSU rRNA) gene [15, 38], with 23 species and two genotypes being found in humans [9, 19]. Recently, it was noted that C. mortiferum, which is found in rodents (particularly, but not only, squirrels), has been commonly identified in human cases of Cryptosporidium infection in many countries of Scandinavia (Norway, Sweden, and Finland), raising concern about this emerging zoonotic species of importance [37, 38]. Since the first report of Cryptosporidium spp. in the peptic glands of tame mice in 1907 [39], epidemiologic studies have documented at least 25 Cryptosporidium species and 43 genotypes in 54 rodent species [41]. Limited studies have detected five Cryptosporidium species (C. parvum, C. ubiquitum, C. andersoni, C. muris, and C. tyzzeri) and one genotype (rat genotype II) in laboratory mice and rats [2, 21, 24, 26, 40]. Molecular epidemiologic data of human cases of cryptosporidiosis have confirmed C. parvum and C. ubiquitum as common species, while C. andersoni and C. muris are minor species, and C. tyzzeri is a rare species [9]. The establishment of subtyping tools targeting the 60-kDa glycoprotein (gp60) gene and the application of the gp60 nomenclature system have substantially enhanced our understanding of the transmission of Cryptosporidium spp., mainly tracing the source of infection and inferring the route of transmission more accurately [25, 32]. In the present study, we assessed the occurrence rate and genetic characterizations of Cryptosporidium spp. in laboratory mice and rats at both genotype and subtype levels.

Materials and methods

Ethics statement

Fecal specimens of laboratory mice and rats were obtained from three medical experimental animal centers (MEACs) after obtaining permission from the director of each MEAC. The animal care and experimental procedures complied with the Chinese Laboratory Animal Administration Act (2017), and no animals were harmed during specimen collection. The objectives and protocols of the present study were reviewed and approved by the Laboratory Animal Welfare & Ethics Committee (LAWEC) of the National Institute of Parasitic Diseases, Chinese Center for Disease Control and Prevention, China (reference no. IPD-2021-21).

Specimen collection

During a period of 5 months (April to August 2023), we collected fecal specimens from > 2,000 laboratory mice and rats from three MEACs, including MEAC1 and MEAC2 in Heilongjiang (China) and MEAC3 in Shanghai (China). In total, 506 combined fecal specimens (approximately 2 g per specimen from 110 to 130 mouse fecal pellets or 9 to 11 rat fecal pellets) were collected by arbitrarily selecting fresh fecal pellets from laboratory animal cages (no. of the mice per cage = 5–7; no. of the rats per cage = 3–5). Among them, 457 mouse fecal specimens (390 from Heilongjiang and 67 from Shanghai) and 49 rat fecal specimens (45 from Heilongjiang and four from Shanghai) were collected (Table 1). Gender, age, strain, and health status were not recorded during sampling.

Table 1

Molecular identification of Cryptosporidium spp. in laboratory mice and rats.

DNA extraction

Each fecal specimen was homogenized in distilled water. A QIAamp Fast DNA Stool Mini Kit (QIAGEN, Hilden, Germany) was used to extract genomic DNA from approximately 200 mg of each processed specimen, according to the manufacturer’s instructions. The DNA preparations were stored at −80 °C before polymerase chain reaction (PCR) analysis.

Genotyping and subtyping Cryptosporidium spp.

Cryptosporidium species were identified by nested PCR amplification and sequence analysis of the partial SSU rRNA gene (~830 bp) [16]. Cryptosporidium-positive specimens were further subtyped by nested PCR amplification and sequence analysis of the partial gp60 gene (~400 bp) [35]. Each DNA specimen was amplified in three PCR reactions. A negative control (DNase-free water) and a positive control (C. baileyi DNA for the SSU rRNA gene or C. hominis DNA for the gp60 gene) were included in each PCR test. 2 × TransTaq®–T PCR SuperMix (+dye) (TransGen Biotech Co., Beijing, China) was used for all the PCR reactions. All the secondary PCR products were analysed electrophoretically in 1.5% agarose gel dyed with GelStrain (TransGen Biotech, Beijing, China) and observed, photographed, and recorded on a Gel Doc™ XR + Imaging System (Bio-Rad, Hercules, CA, USA).

Sequencing and analysing

All secondary PCR products of the expected size were sequenced with their respective secondary PCR primers at Shanghai Saiheng Biotechnology Co. Ltd. (Shanghai, China) by an ABI 3730XL Genetic Analyzer (Applied Biosystems, Foster City, CA, USA). The accuracy of the sequencing data was confirmed by bidirectional sequencing. Each individual sequence chromatogram was examined using the software Chromas 2.6.6 (https://technelysium.com.au/wp/chromas). For high-quality chromatograms, the forward and reverse sequences were manually assembled and aligned with each other to create a contig using the software MEGA 7 (http://www.megasoftware.net/). The obtained contigs were used to determine Cryptosporidium species and subtypes by comparison with reference sequences retrieved from GenBank databases (http://www.ncbi.nlm.nih.gov). For low-quality chromatograms, such as double peaks and gaps, specimens were re-amplified under optimised PCR conditions, including increasing annealing temperatures, adjusting primer concentrations and diluting DNA template.

Nucleotide sequence accession numbers

The novel nucleotide sequences obtained in the present study were deposited in the GenBank database under the following accession numbers: PP124619 to PP124630 (SSU rRNA gene) and PP115548 to PP115564 (gp60 gene).

Results

Occurrence of Cryptosporidium spp.

At the SSU rRNA locus, 88 (17.4%) of 506 combined fecal specimens were successfully amplified. 76 specimens had high-quality DNA chromatograms; however, only 75 were determined to be infected with Cryptosporidium spp. based on sequence analysis. Among the remaining 12 specimens with low-quality DNA chromatograms, only nine were confirmed to be Cryptosporidium infections by optimising PCR conditions. In the end, a total of 84 combined fecal specimens were positive for Cryptosporidium spp., with an average occurrence rate of 16.6% (84/506) (Fig. S1). For mice, 18.2% (83/457) were Cryptosporidium-positive. Cryptosporidium spp. were detected from all three MEACs; in particular, the occurrence rates were 22.5% (55/244), 14.4% (21/146), and 10.5% (7/67) at MEAC1, MEAC2, and MEAC3, respectively. In contrast, Cryptosporidium spp. were detected in only one rat fecal specimen from MEAC1 (Table 1).

Cryptosporidium genotyping and subtyping

At the SSU rRNA locus, sequence analysis identified two Cryptosporidium species in 84 Cryptosporidium-positive specimens: C. parvum (n = 40), C. tyzzeri (n = 33), and C. parvum + C. tyzzeri (n = 11) (Table 1). Additionally, C. parvum and C. tyzzeri were found in both MEAC1 and MEAC2, with co-infection of the two species (MEAC1: 3.0%, 8/268; MEAC2: 1.8%, 3/167). In contrast, only C. tyzzeri was found in MEAC3. The C. parvum + C. tyzzeri-positive specimens accounted for 14.3% (8/56, 3/21) of the Cryptosporidium-positive specimens in both MEAC1 and MEAC2. In the case of C. parvum, 15 representative sequences were obtained out of 51 specimens (40 C. parvum and 11 C. parvum + C. tyzzeri-positive specimens). Among them, three sequences from 38 specimens had been published previously, while the remaining 12 from 13 specimens had not been described previously (PP124619 to PP124630). In the case of C. tyzzeri, all 44 sequences (33 C. tyzzeri and 11 C. parvum + C. tyzzeri-positive specimens) were identical to each other. Detailed results of the homology analysis of the SSU rRNA gene sequences of Cryptosporidium-positive specimens are shown in Table S1.

At the gp60 locus, 71.4% (60/84) of the Cryptosporidium-positive specimens were successfully amplified and subtyped, with 42.5% (17/40), 97.0% (32/33), and 100% (11/11) of C. parvum, C. tyzzeri, and C. parvum + C. tyzzeri specimens, respectively (Table 2). Five C. parvum subtypes were identified out of 12 unique sequences by analyzing 23 gp60 gene sequences (17 C. parvum and six C. parvum + C. tyzzeri-positive specimens): IIaA17G2R1 (n = 18), IIaA16G2R1 (n = 2), IIaA14G2R1 (n = 1), IIaA17G1R1 (n = 1), and IIaA18G2R1 (n = 1). Two C. tyzzeri subtypes were identified out of eight distinct sequences by analyzing 40 gp60 gene sequences (32 C. tyzzeri and eight C. parvum + C. tyzzeri-positive specimens): IXaA6R1 (n = 34) and IXbA8 (n = 6). Detailed results of homology analysis of the gp60 gene sequences of Cryptosporidium-positive specimens are shown in Table S2.

Table 2

Subtyping of Cryptosporidium spp. in laboratory mice and rats at the gp60 locus.

Discussion

Cryptosporidium spp. are common intestinal protozoa infecting rodents. However, only a few studies are available on Cryptosporidium infections in laboratory rodents. In the present study, the overall occurrence rate of Cryptosporidium spp. in laboratory mice and rats was 16.6%, which was higher than those reported in three Chinese studies (1.7%–4.3% in mice and 0.6%–4.0% in rats) and one Nigerian study (1.5% in rats), but lower than that in mice in a Turkish study (100%) [2, 21, 24, 26, 40]. In our study, the higher Cryptosporidium occurrence rate might be attributable to sample pooling from each cage, as a single positive specimen could represent as many as seven animals, not all of which are necessarily shedding oocysts.

In the present study, based on sequence analysis of the SSU rRNA gene, C. parvum and C. tyzzeri were identified in mice fecal specimens, while C. parvum was detected in one rat fecal specimen. Two and four molecular studies of Cryptosporidium spp. have been conducted in laboratory mice and rats, respectively [2, 21, 24, 40]. All relevant studies to date, including the present study, have identified three and five Cryptosporidium species/genotypes in mice and rats, respectively: C. tyzzeri (n = 68), C. parvum (n = 48), and C. muris (n = 4) as well as C. tyzzeri + C. parvum (n = 11) in mice (n = 131) [24, 40]; C. tyzzeri (n = 2), C. parvum (n = 1), C. ubiquitum (n = 1), C. andersoni (n = 1), and rat genotype II (n = 1) in rats (n = 6) [2, 21, 24, 40]. All these Cryptosporidium species, except rat genotype II, have also been found in humans [41], suggesting a potential risk of cryptosporidiosis transmission from laboratory mice and rats to humans. In this study, co-infection of C. parvum and C. tyzzeri was observed in MEAC1 and MEAC2 and accounted for 14.3% of the Cryptosporidium-positive specimens in both MEAC1 and MEAC2. Only C. tyzzeri was found in MEAC3. The reasons behind the results remained unclear, which might be related to the number of investigated specimens.

At the gp60 locus, only 71.4% of the Cryptosporidium-positive specimens were successfully amplified. Meanwhile, C. tyzzeri was observed to have a higher PCR amplification rate than C. parvum. This might be related to the genetic variations within Cryptosporidium species. In the present study, C. parvum showed more intra-genetic variations than C. tyzzeri. We obtained more representative sequences of C. parvum than those of C. tyzzeri at the SSU rRNA and the gp60 loci. A similar finding was reported in a genotyping and subtyping study of Cryptosporidium spp. in snake fecal specimens conducted in Brazil, with a PCR amplification rate of 75.0% for C. tyzzeri and 42.9% for C. parvum at the gp60 locus [23]. The lower amplification rate of C. parvum-positive specimens at the gp60 locus might be related to the low number of oocysts in some fecal specimens.

Cryptosporidium parvum is one of the two most common species reported in human cases of cryptosporidiosis, and it was found to be the dominant Cryptosporidium species in rodents (39.2%; 1801/4589) [41]. Meanwhile, sequence analysis of the gp60 gene has identified 16 subtypes in six subtype families (IIa, IIc, IId, IIi, IIo, and IIp) in rodent-derived C. parvum isolates [7–8, 36, 41]. In the present study, five subtypes belonged to the subtype family IIa, and they all came from mice. Subtype IIaA17G2R1 has also been found in urban rats in Malaysia [36]. The other four subtypes (IIaA14G2R1, IIaA16G2R1, IIaA17G1R1, and IIaA18G2R1) were identified in rodents for the first time. Notably, all the above five subtypes have also been documented in humans [3, 13], implying the zoonotic potential for individuals in close contact with laboratory mice, particularly breeders, researchers, and workers.

Cryptosporidium tyzzeri, Cryptosporidium mouse genotype I previously, was formally described as a new species in 2012 [29]. Of note, C. tyzzeri was originally detected in laboratory mice and is mostly detected in domestic mice and small rodents, such as wood mice, brown rats, and bank voles [41]. With the accumulation of epidemiologic data on Cryptosporidium spp., it was also found in humans and some other non-specific animal hosts, such as pandas, black leopards, horses, and snakes [41]. Based on sequence analysis of the gp60 gene, to date, there have been five subtypes identified, and they belonged to three subtype families (IXa–IXc) in rodent-derived C. tyzzeri isolates, including IXaA6R1, IXaA6R2, IXaA8, IXbA6, and IXcA6 [6, 10, 24]. The present study identified two subtypes (IXaA6R1 and IXbA8) of C. tyzzeri. Interestingly, C. tyzzeri subtypes have also been identified in some sporadic human cases of Cryptosporidium infection: a child with gastrointestinal symptoms in Kuwait (subtype IXaA6R2) [35], a symptomatic 25-year-old woman in the Czech Republic (subtype IXaA8) [28], and three human patients in New Zealand (subtype family IXb) [11]. Due to limited data on gp60 sequences of C. tyzzeri, the zoonotic potential of the subtypes IXaA6R1 and IXbA8 needs further investigation.

In this study, we found Cryptosporidium spp. in laboratory mice and rats. Since breeding and experimental work conducted with these animals was performed in Chinese MEACs according to two Chinese national standards: Laboratory animal – Environment and housing facilities (GB 14925-2023) and Laboratories – General Requirements for biosafety (GB 19489-2008), we speculated that Cryptosporidium infections in these animals might be related to rodent chows obtained commercially. The chows are disinfected before leaving the factory. However, they might be contaminated with Cryptosporidium oocysts during storage (they are actually assumed to be free from contaminants when used). Currently, Cryptosporidium spp. contamination sources and transmission routes in these laboratory animals remain unclear. Thus, it is also necessary to investigate Cryptosporidium spp. in environmental specimens. To uphold the quality of laboratory animals and enhance the accuracy of experimental outcomes, the detection of Cryptosporidium spp. should be routinely implemented in laboratory animals. These animals positive for Cryptosporidium spp., even if asymptomatic, should be removed to mitigate any potential influence of Cryptosporidium infections on the experimental results.

Conclusion

We demonstrated the occurrence (16.6%) of Cryptosporidium spp. in laboratory mice and rats, revealing the presence of C. parvum and C. tyzzeri. Five C. parvum subtypes and two C. tyzzeri subtypes were detected, and five of them were detected in rodents for the first time. All identified C. parvum subtypes may pose a zoonotic threat to individuals in close contact with laboratory mice and their feces. As a result, individuals should exercise caution and implement appropriate safety measures when handling them.

Funding

This study was supported by the National Key Research and Development Program of China (2021YFC2300902), the Three-Year Initiative Plan for Strengthening Public Health System Construction in Shanghai (2023-2025) Key Discipline Project (GWVI-11.1-12), and the National Natural Science Foundation of China (82273693).

Conflicts of interest

The authors declare that there are no competing interests among them.

Supplementary material

Table S1. Homology analysis of the SSU rRNA gene sequences of Cryptosporidium-positive specimens. Access here

Table S2. Homology analysis of the gp60 gene sequences of Cryptosporidium-positive specimens. Access here

thumbnail Figure S1.

Flow chart demonstrating the molecular identification of Cryptosporidium spp.

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Cite this article as: Zhou S, Hu X, Li H, Yuan Z, Li Z, Liu A, Jiang Y & Cao J. 2024. Molecular identification and subtyping of Cryptosporidium spp. in laboratory mice and rats. Parasite 31, 75. https://doi.org/10.1051/parasite/2024073.

All Tables

Table 1

Molecular identification of Cryptosporidium spp. in laboratory mice and rats.

In the text
Table 2

Subtyping of Cryptosporidium spp. in laboratory mice and rats at the gp60 locus.

In the text

All Figures

thumbnail Figure S1.

Flow chart demonstrating the molecular identification of Cryptosporidium spp.
In the text

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