Background: Cryptosporidium is a globally distributed zoonotic protozoan parasite in humans and animals. Infection is widespread in dairy cattle, especially in calves, resulting in neonatal enteritis, production losses and high mortality. However, the occurrence of Cryptosporidium spp. in pre- and post-weaned calves in Yunnan Province remains unclear. Methods: We collected 498 fecal samples from Holstein calves on 10 different farms in four regions of Yunnan Province. Nested PCR and DNA sequencing were used to determine the infection, species and genotypes of Cryptosporidium spp. in these animals. Results: The overall occurrence of Cryptosporidium spp. in Holstein calves was 32.9% (164/498), and the prevalence in pre- and post-weaned calves was 33.5% (106/316) and 31.9% (58/182), respectively. Four Cryptosporidium species were identified in these animals, namely C. bovis (n = 119), C. parvum (n = 23), C. ryanae (n = 20) and C. andersoni (n = 2). Based on sequencing analysis of the 60 kDa glycoprotein gene of C. bovis, C. parvum and C. ryanae, six subtypes of C. bovis (XXVIe, XXVIb, XXVIf, XXVIa XXVIc and XXVId), two subtypes of C. parvum (IIdA19G1 and IIdA18G1) and four subtypes of C. ryanae (XXIf, XXId, XXIe and XXIg) were identified. Conclusions: These results provide essential information to understand the infection rate, species diversity and genetic structure of Cryptosporidium spp. populations in Holstein pre-weaned and post-weaned calves in Yunnan Province. Further, the presence of IIdA18G1 and IIdA19G1 in C. parvum implies significant animal and public health concerns, which requires greater attention and more preventive measures.

Cryptosporidium spp. are protozoan pathogens that are widespread within mammals. In recent years, extensive molecular epidemiology studies on Cryptosporidium in dairy cattle have been conducted in Yunnan and worldwide. However, the infection status of these pathogens in beef cattle in Yunnan remains unclear. To examined the occurrence of Cryptosporidium spp. in beef cattle in Yunnan Province, China, we collected 735 fecal samples from six breeds of beef cattle in five regions of Yunnan. Nested PCR and DNA sequencing revealed the infection, species, and genotypes of Cryptosporidium spp. in these animals. The occurrence of Cryptosporidium spp. in Simmental cattle, Brahman cattle, Aberdeen Angus cattle, Yunnan Yellow cattle, Dulong cattle, and Hereford cattle was 32.9% (137/416), 3.8% (4/106), 24.4% (20/82), 3.8% (3/79), 3.2% (1/31), and 0% (0/21), respectively, with an overall rate of 22.4% (165/735). Regarding the regions, the occurrence of Cryptosporidium spp. in Boshan City, Kunming City, Lincang City, Dehong City and Xishuangbanna City was 41.8%, 28.6%, 19.4%, 6.7%, and 3.8%, respectively. In terms of age, the infection rates of Cryptosporidium spp. in pre-weaned, post-weaned, juvenile, and adult cattle were 62.1%, 52.6%, 42.7%, and 7.7%, respectively. According to sex, male cattle were more susceptible to Cryptosporidium infection (28.0%) than females (15.7%). Four Cryptosporidium species were identified in beef cattle: C. andersoni (n = 146), C. bovis (n = 11), C. ryanae (n = 7), and C. occultus (n = 1). Multilocus sequence typing analysis at the MS1, MS2, MS3, and MS16 gene loci revealed four subtype families of C. andersoni (A4A4A4A1, A5A4A4A1, A4A4A2A1, A1A4A4A1). Additionally, sequencing analysis of the 60-kDa glycoprotein gene identified three subtype families of C. bovis (XXVIc, XXVId, XXVIe) and one subtype family of C. ryanae (XXIb). These findings document the occurrence of Cryptosporidium spp. in beef cattle in Yunnan Province for the first time, providing reference data on the distribution, infection rate, species diversity, and genetic structure of these pathogens in China. To effectively reduce the prevalence of Cryptosporidium spp. in beef cattle in Yunnan, the implementation of proper sanitation management, rigorous rodent control, and farmer education programs is crucial. These integrated measures are critical for maintaining herd health, reducing economic losses, and ensuring meat safety across the province.

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.

Enterocytozoon bieneusi and Giardia duodenalis are major zoonotic pathogens that often cause diarrhea in immunocompromised humans or animals. Beef cattle are important reservoirs for these two pathogens. Yunnan Province is a major region for beef cattle farming, and its suitable climatic conditions facilitate the transmission of the pathogens. However, research on the prevalence and distribution of E. bieneusi and G. duodenalis in beef cattle in Yunnan remains understudied. This study collected 529 fecal samples from seven beef cattle breeds in four regions in Yunnan Province for an epidemiological survey. Nested PCR combined with sequencing was used to detect E. bieneusi and G. duodenalis, and the sequencing results were analyzed to determine genotypes or assemblage types. Our results demonstrate that the prevalence rates of E. bieneusi and G. duodenalis were 3.0% (16/529) and 3.6% (19/529), respectively. The study identified four genotypes of E. bieneusi, including I (n = 8), J (n = 4), BEB8 (n = 3), and BEB4 (n = 1). Both assemblages E (n = 18) and A (n = 1) were identified among G. duodenalis-positive animals. Phylogenetic analysis revealed that the E. bieneusi genotypes detected in this study belong to Group 2. In conclusion, these findings indicate that although the overall prevalence is relatively low compared to other regions, the presence of zoonotic Group 2 genotypes and assemblage A highlights the potential risk of cross-species transmission. Moreover, the results provide foundational data to support the development of region-specific surveillance and control strategies for bovine giardiasis and microsporidiosis, and emphasize the importance of the One Health approach in managing parasitic infections in livestock populations.