Background: Cryptosporidium parvum is a zoonotic parasitic protozoan that can infect a variety of animals and humans and is transmitted between hosts via oocysts. The oocyst wall provides strong protection against hostile environmental factors; however, research is limited concerning the oocyst wall at the proteomic level. Methods: A comprehensive analysis of the proteome of oocyst wall of C. parvum was performed using label-free qualitative high-performance liquid chromatography (HPLC) fractionation and mass spectrometry-based qualitative proteomics technologies. Among the identified proteins, a surface protein (CpSP1) encoded by the C. parvum cgd7_5140 (Cpcgd7_5140) gene was predicted to be located on the surface of the oocyst wall. We preliminarily characterized the sequence and subcellular localization of CpSP1. Results: A total of 798 proteins were identified, accounting for about 20% of the CryptoDB proteome. By using bioinformatic analysis, functional annotation and subcellular localization of the identified proteins were examined for better understanding of the characteristics of the oocyst wall. To verify the localization of CpSP1, an indirect immunofluorescent antibody assay demonstrated that the protein was localized on the surface of the oocyst wall, illustrating the potential usage as a marker for C. parvum detection in vitro. Conclusion: The results provide a global framework about the proteomic composition of the Cryptosporidium oocyst wall, thereby providing a theoretical basis for further study of Cryptosporidium oocyst wall formation as well as the selection of targets for Cryptosporidium detection. Graphical

Cryptosporidiosis is a gastrointestinal disease in humans and animals caused by the protozoan parasite Cryptosporidium . Control and treatment of the disease is challenging due to the lack of sensitive diagnostic tools and effective chemotherapy.

Background: Cryptosporidium is an opportunistic pathogen that infects a wide variety of vertebrates. The aim of the present study was to characterize Cryptosporidium spp. isolates from Bactrian camels and to foster further understanding of the biological characteristics of the pathogen. Methods: Fecal specimens were collected from two 4-year-old Bactrian camels resident at the Kaifeng City Zoo in China and examined for Cryptosporidium. Fecal specimens were screened using the floatation method, and then genomic DNA was extracted from the oocysts and identified by nested-PCR amplification of the small subunit ribosomal RNA (SSU rRNA) gene, the actin gene and the Cryptosporidium oocyst wall-protein (COWP) gene. Subtype analysis was performed based on four minisatellite (MS) loci (MS1, MS2, MS3 and MS16) that were aligned and phylogenetically analyzed to determine the species and subtype of Cryptosporidium. We then established a BALB/c mice infection model and further verified the results through clinical status, pattern of oocyst excretion and histological examination. Results: Cryptosporidium oocyst isolates from the two Bactrian camels had an average (± standard deviation) size of 7.49 ± 0.13 × 5.70 ± 0.10 μm (n = 50). The sequencing and phylogenetic analysis confirmed the species as C. muris. Multilocus sequence typing analysis indicated that the subtypes were M13, M4, M1 and M5. Following the inoculation of BALB/c mice, we found that the prepatent period and number of oocysts per gram increased with increasing infective dose. Oocysts were first detected in the feces of BALB/c mice at 7–8 days post-infection (dpi), with levels peaking twice thereafter, at 15–16 dpi and 19–20 dpi. Histology and scanning electron microscopy studies showed that the stomach contained gastric pits filled with Cryptosporidium that adhered to the surface of gastric mucosa gland epithelial cells, causing the latter to deform, swell and become disordered. Conclusions: The findings of this study indicated that oocysts isolated from Bactrian camels were from C. muris. This is the first report of C. muris isolated from camels in China. More epidemiological data are needed to understand the prevalence and transmission of C. muris in camels in different geographic areas. Graphical

Cryptosporidium spp. are common protist pathogens, and the growing popularity of pet rodents raises concerns about their potential role in zoonotic parasites transmission. However, epidemiological data on Cryptosporidium spp. in pet rodents in Yunnan Province is scarce. To examine the occurrence of Cryptosporidium spp. in pet rodents in Yunnan, we collected 762 fecal samples from four rodent species across four cities. Nested PCR and DNA sequencing were used to characterize the species and subtypes of Cryptosporidium. The occurrence of Cryptosporidium spp. in guinea pigs (Cavia porcellus), Siberian dwarf hamsters (Phodopus sungorus), Syrian hamsters (Mesocricetus auratus) and fancy rats (Rattus norvegicus domestica) was 18.7% (80/426), 17.3% (36/207), 12.5% (15/120), 0% (0/9), respectively, with an overall rate of 17.2% (131/762). According to regions, the positivity rate of Cryptosporidium spp. in Zhaotong city, Kunming city, Yuxi city and Qujing city was 21.0%, 17.9%, 16.8% and 10.5%, respectively. In terms of sampling location, the occurrence of Cryptosporidium spp. in pet markets, farms and shops was 19.5%, 18.6% and 0%, respectively. Sequence analysis of the small subunit ribosomal RNA (SSU rRNA) gene identified six Cryptosporidium species/genotypes: Cryptosporidium homai (n = 52), Cryptosporidium wrairi (n = 30), Cryptosporidium sp. hamster genotype (n = 25), Cryptosporidium andersoni (n = 20), Cryptosporidium parvum (n = 5), and Cryptosporidium muris (n = 1). Further subtyping of C. andersoni isolates using multilocus sequence typing (MLST) revealed a single subtype, with all isolates identified as A3A4A2A2. All five C. parvum isolates were identified as subtype IIdA15G1 based on the gp60 gene. Our findings demonstrated the presence of the zoonotic C. parvum IIdA15G1 subtype in pet rodents, suggesting that these animals, particularly hamsters, may serve as reservoirs for human-pathogenic Cryptosporidium species. These results underscore the need for improved biosecurity and husbandry practices in the pet rodent trade to mitigate public health risks.