The protocols used in the present study underwent a meticulous review process and were ultimately approved by the Research Ethics Committee of Wenzhou Medical University (approval number SCILLSC-2021-01).

Between November 2023 and February 2024, a cumulative total of 486 wild rodents were collected, with 229 rodents originating from Harqin Banner in Inner Mongolia and 257 rodents originating from Jianping County in Liaoning Province, China (Figure 1 and Table 1). Rodents were captured by utilizing cage traps baited with a mixture of peanut and sunflower seeds. For each designated capture location, approximately 50 cage traps were methodically placed in a straight line, ensuring a uniform spacing of 5 meters between each trap and effectively establishing transects. At 4:00 PM, the transects were positioned and retrieved the following morning at 8:00 AM. Each rodent captured was euthanized humanely via CO₂ asphyxiation and promptly transported to the laboratory within 48 h, ensuring its safety in sealed containers containing ice. A fecal sample weighing 0.5 grams was collected from the rectum of each rodent.

Exclusively designated for DNA extraction, 0.2 grams of each fecal sample was processed, while the remaining portion was preserved as a backup and stored at a chilled temperature of −80°C. Using the QIAamp DNA Mini Stool Kit (Qiagen, Germany), genomic DNA was extracted from each processed sample. During the extraction process, the lysis temperature was increased to 95°C, while all other steps were performed strictly according to the manufacturer’s guidelines. Subsequently, the DNA was reconstituted in 200 μL of AE elution buffer, provided with the kit, and was subsequently stored at −20°C prior to PCR analysis.

To identify the rodent species, the vertebrate cytochrome b (cytb) gene (421 bp) was amplified via PCR from the fecal DNA. The primer sequences were 5’-TACCATGAGGACAAATATCATTCTG-3′ and 5’-CCTCCTAGTTTGTTAGGGATTGATCG-3′, and PCR conditions were as follows: 35 cycles of denaturation at 94°C for 30 s, annealing at 51°C for 30 s, and extension at 72°C for 30 s. Initial denaturation was performed at 94°C for 5 min, followed by a final extension at 72°C for 5 min. These conditions followed the previously described protocol by Verma and Singh (10).

All DNA samples were subjected to nested PCR utilizing primers previously developed by Xiao et al. in 1999 to amplify an 830 bp fragment of the partial small subunit ribosomal RNA (SSU rRNA) gene of Cryptosporidium (9). The primers used for the primary PCR were 5’-TTCTAGAGCTAATACATGCG-3′ and 5’-CCCTAATCCTTCGAAACAGGA-3′, while the primers used for the secondary PCR were 5′-GGAAGGGTTGTATTTATTAGATAAAG-3′ and 5’-AAGGAGTAAGGAACAACCTCCA-3′. Both PCR amplification steps were conducted under identical conditions, commencing with initial denaturation at 94°C for 3 min. This was followed by 35 cycles of denaturation at 94°C for 45 s, annealing at 55°C for 45 s, extension at 72°C for 1 min, and a final extension at 72°C for 7 min. TaKaRa Taq DNA Polymerase was used for PCR amplification, with positive controls using DNA from chickens infected with C. baileyi and negative controls using deionized water without DNA templates. PCR products were analyzed via gel electrophoresis on a 1.5% agarose gel in TAE buffer, with GelRed (Biotium Inc., Fremont, California, United States) serving as the staining agent.

The PCR products of the expected size were purified using a DNA gel purification kit from Sangon Biotech (Shanghai, China). These purified products were then sequenced using the Sanger sequencing method by Sangon Biotech (Shanghai) Co., Ltd., on an ABI Prism 3,730 XL DNA analyzer. Sequencing was performed with the same primers used for the secondary PCR and was facilitated by a BigDyeTerminator v3.1 cycle sequencing kit (Applied Biosystems, Carlsbad, CA, United States). To guarantee the precision of the nucleotide sequence, sequencing was carried out from both ends of the product, and further PCR products were sequenced whenever mutations were identified. After acquiring the sequences, they were carefully edited using DNASTAR Lasergene version 7.1.0 and aligned with reference sequences retrieved from the National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/) using the basic local alignment search tool (BLAST) and ClustalX 2.0 software (http://www.clustal.org/) to accurately identify the Cryptosporidium species.

The SSU rRNA sequences of Cryptosporidium spp. obtained in this study were combined with reference sequences to construct a phylogenetic tree using Mega 7.0 software. The Tamura–Nei model-based Maximum Likelihood method was chosen to analyze the phylogenetic relationships. To ensure the reliability of the evolutionary tree, a bootstrap analysis was conducted with 1,000 replicates. The reference sequences necessary for tree construction were retrieved from GenBank and previous research studies.

Statistical analysis was performed utilizing SPSS version 22.0 (SPSS Inc., United States). The chi-square test was utilized to determine the disparities in the occurrence of Cryptosporidium spp. across diverse regions and rodent species. A p value less than 0.05 was considered to indicate statistical significance.

The nucleotide sequences of Cryptosporidium obtained in this study have been deposited in the GenBank database under accession numbers PP527771 to PP527783.

In this study, PCR and sequencing analysis of the cytb gene revealed the presence of four rodent species: Apodemus agrarius (n = 89), Cricetulus barabensis (n = 96), Mus musculus (n = 106) and Rattus norvegicus (n = 195). No additional data were gathered for these wild rodents (Table 1).

Nested PCR was performed on 486 fecal samples to assess the presence of Cryptosporidium species by analyzing the SSU rRNA gene. The results revealed that 36 samples were positive for this parasite, yielding an average infection rate of 7.4%, with 7.0% (16/229) in Liaoning (Jianping) and 7.8% (20/257) in Inner Mongolia (Harqin Banner) (Table 1). Statistical analysis did not indicate any significant differences in Cryptosporidium prevalence between the two regions (χ² = 0.11, df = 1, p = 0.74). Regarding rodent species variation, the highest infection rate of Cryptosporidium was observed for R. norvegicus (9.2%; 18/195), followed by M. musculus (6.6%; 7/106), A. agrarius (5.6%; 5/89), and C. barabensis (6.3%; 6/96). The difference in the infection rate of Cryptosporidium among the rodent species groups was not statistically significant (χ² = 1.65, df = 3, p = 0.65).

Five species of Cryptosporidium, namely, C. ubiquitum (n = 8), C. occultus (n = 5), C. muris (n = 2), C. viatorum (n = 1), and C. ratti (n = 1), as well as two genotypes—Cryptosporidium Rat genotype III (n = 10) and Cryptosporidium Rat genotype IV (n = 9)—have been identified through sequencing the PCR products of 36 Cryptosporidium-positive samples (Table 1).

In Liaoning, Cryptosporidium Rat genotype III emerged as the dominant species, accounted for 31.3% (5/16) of the positive samples, followed by C. occultus and Cryptosporidium Rat genotype IV each comprising 25.0% (4/16). The remaining three species, C. muris, C. ubiquitum, and C. viatorum, contributed equally with a share of 3.3% (1/16) each. On the other hand, in Inner Mongolia, C. ubiquitum emerged as the predominant species, accounting for 35.0% (7/20) of the positive samples. Cryptosporidium Rat genotype III and Cryptosporidium Rat genotype IV followed closely, each comprising 25.0% (5/20) of the positive samples. The remaining species, C. muris, C. occultus, and C. ratti, contributed 5.0% (1/20) each (Table 1).

Among the different rodent species, R. norvegicus carried a diverse range of species/genotypes of Cryptosporidium, including C. ubiquitum, C. ratti, C. occultus, Cryptosporidium Rat genotype III and Cryptosporidium Rat genotype IV. In contrast, C. barabensis was limited to carrying only C. occultus and Cryptosporidium Rat genotype IV. The remaining two rodent species each harbored three species/genotypes: C. ubiquitum, C. viatorum and Cryptosporidium Rat genotype III in A. agrarius and C. ubiquitum, C. muris and Cryptosporidium Rat genotype III in M. musculus (Table 1).

Among the nine SSU rRNA sequences belonging to Cryptosporidium Rat genotype IV, six sequences were found to be identical to each other, sharing perfect 100% similarity with the Cryptosporidium genotype W19 variant sequence (AY737581) previously isolated from water samples in the United States (US). The three remaining sequences of Cryptosporidium rat genotype IV were identical to each other and had not been previously described. They exhibited a remarkable similarity of 99.87% to the Cryptosporidium genotype W19 variant sequence (AY737582), which was also detected in US waters. The sole difference among them was a single nucleotide substitution, specifically from A to G, at position 441 (Table 2).

Ten SSU rRNA sequences of Cryptosporidium Rat genotype III revealed five types, with one type represented in five samples sharing an identical sequence (JX294367) with Cryptosporidium Rat genotype III from wild black rats in northern Australia. The second type represented two samples were novel, exhibiting 99.01% similarity (12 nucleotide differences, including 9 substitutions and 3 insertions) with JX294363, which was found in wild black rats from Australia. The remaining three types were each found in a single sample and were previously undescribed, differing from the Cryptosporidium Rat genotype III sequence (JX294367) of wild black rats from northern Australia by three (T to C at position 482 and T delete at positions 439 and 440), seven (T to G at position 93, G to A at position 398, T to C at position 438, T delete at positions 439 and 440, T to C at position 481, G to A at position 599), and six (T to C at position 481, G to A at position 559, T to C at position 593, T to A at position 683, G to A at position 739, and G to T at position 755) nucleotides (Table 2).

The present study identified eight sequences of C. ubiquitum that were consistent with each other and exhibited 100% similarity to the C. ubiquitum sequence (MW043441) isolated from cattle in Bangladesh. The two C. muris sequences were also identical and exhibited 100% similarity with MW090931, which was isolated from wastewater and sewage in Guangzhou, China (Table 2).

Among the five sequences of C. occultus obtained in this study, two exhibited 100% homology with MG699179, which was identified in Meriones unguiculatus from the Czech Republic. The remaining three sequences of C. occultus were homologous to each other and were novel, sharing 99.51% similarity with MG699179, differing by four nucleotides (Table 2).

The sequences of C. ratti and C. viatorum identified here were novel and differed by one nucleotide from MT504541 in R. norvegicus in the Czech Republic and from MK522269, which was found in Leopoldamys edwardsi from China (Table 2).

The phylogenetic analysis of the ssu rRNA sequences has confirmed that the sequences obtained in the present study, corresponding to C. viatorum, C. ubiquitum, C. occultus, Cryptosporidium Rat genotype IV, C. ratti, Cryptosporidium Rat genotype III, and C. muris, have clustered together with their respective reference sequences, forming distinct and clearly identifiable groups within the phylogenetic tree (Figure 2).