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Cryptosporidiosis of calves is caused by the enteroprotozoan Cryptosporidium spp. The disease results in intense diarrhea of calves associated with substantial economic losses in dairy farming worldwide. The aim of this study was to determine calf, herd, and within-herd Cryptosporidium prevalence and identify Cryptosporidium species and subtypes in calves with diarrhea in intensive dairy herds in central Argentina. A total of 1073 fecal samples were collected from 54 randomly selected dairy herds. Cryptosporidium-oocysts were isolated and concentrated from fecal samples using formol-ether and detected by light microscopy with the modified Ziehl-Neelsen technique. Overall prevalence of oocyst-excreting calves was found to be 25.5% (274/1073) (95% C.I. 22.9; 28.1%). Of the herds studied, 89% (48/54) included at least one infected calf, whereas within-herd prevalence ranged from the absence of infection to 57% (20/35). A highly significant association was found between the presence of diarrhea and C. parvum infection (χ2 = 55.89, p < 0.001). For species determination, genomic DNA isolated from oocyst-positive fecal samples was subjected to PCR-RFLP of the 18S rRNA gene resulting exclusively in Cryptosporidium parvum identification. C. parvum isolates of calves displaying diarrhea and high rate of excretion of oocysts were subtyped by PCR amplification and direct sequencing of the 60 kDa glycoprotein (GP60) gene. Altogether five GP60 subtypes, designated IIaA18G1R1, IIaA20G1R1, IIaA21G1R1, IIaA22G1R1, and IIaA24G1R1 were identified. Interestingly, IIaA18G1R1 and IIaA20G1R1 were predominant in calves with diarrhea and high infection intensity. Notably, IIaA24G1R1 represents a novel, previously unrecognized C. parvum subtype. The subtype IIaA18G1R1, frequently found in this study, is strongly implicated in zoonotic transmission. These results suggest that calves might be an important source for human cryptosporidiosis in Argentina.

Cryptosporidium is a major cause of severe diarrhea. Although Cryptosporidium isolates exhibit significant differences in infectivity and virulence, the genetic determinants for these traits are not clear. In this study, we use classical genetics to cross two Cryptosporidium parvum isolates of different virulence and use bulk segregant analysis of whole-genome sequences from the progeny to identify quantitative trait loci (QTL) associated with Cryptosporidium infectivity and virulence. Of the 23 genes in three QTL, two have loss-of-function mutations in the low-virulence isolates, including the SKSR1 gene encoding a variant secretory protein. Deletion of the SKSR1 gene or expression of the frame-shifted sequence reduces the pathogenicity of the virulent isolate. SKSR1 is expressed in small granules and secreted into the parasite-host interface during invasion. These results demonstrate that SKSR1 is an important virulence factor in Cryptosporidium , and suggest that the extended SKSR protein family, encoded by clusters of subtelomeric genes, may contribute to pathogenesis. This study uses genetic crossing to identify the genes underlying the differences in virulence between two Cryptosporidium isolates. Candidate genes are validated using genetic editing, revealing that the small granule protein SKSR1 is a key virulence factor in Cryptosporidium .

Background Cryptosporidium spp. are worldwide protozoan parasites which include species that can lead to cryptosporidiosis in humans. Different animal species can serve as reservoirs and sources of dissemination of the disease, such as rodent species due their potential in transmitting zoonotic pathogens to humans and other animals. In the Canary Islands (Spain), Cryptosporidium parvum and Cryptosporidium hominis have been identified in patients with diarrhea. However, the occurrence of Cryptosporidium spp. in possible reservoirs in this archipelago remains unclear. Considering the zoonotic potential of these protozoans, the aim of the present study was to determine the presence of Cryptosporidium spp. in peridomestic wild rodents and the possible role of these mammals as a source of transmission of these protozoans in Canary Islands. Methods A total of 179 rodents belonging to Rattus rattus and Mus musculus domesticus from four Canary Islands, La Palma, El Hierro, Tenerife and Lanzarote, were analyzed. Feces were screened for Cryptosporidium spp. by nested PCR of the 18S ribosomal RNA fragment and the sequences used for phylogenetic analyses. Results Cryptosporidium spp. were found widely distributed with an overall prevalence of 12.30% in rodents (13.86% for R. rattus and 10.25% for M. m. domesticus ). The overall prevalence by island was 19.60% for Tenerife, 7.14% for La Palma, 5.71% for El Hierro and 0% for Lanzarote. Cryptosporidium tyzzeri , Cryptosporidium meleagridis , Cryptosporidium muris and Cryptosporidium sp. rat genotype I and II/III were successfully identified, in addition to two unidentified Cryptosporidium genotypes. Conclusions This study contributes to the knowledge of the biodiversity and distribution of Cryptosporidium spp. in wild rodents from the Canary Islands, highlighting the presence of three zoonotic species, C. tyzzeri , C. meleagridis and C. muris , being the first detection of these three species in wild rodents in the Canary Islands and the first report of C. meleagridis in R. rattus . Given the results obtained in our study, future studies in non-sampled areas are required to better understand the epidemiology of these protozoans in wild rodents in the archipelago.

Background Cryptosporidium hominis is a dominant species for human cryptosporidiosis. Within the species, IbA10G2 is the most virulent subtype responsible for all C. hominis –associated outbreaks in Europe and Australia, and is a dominant outbreak subtype in the United States. In recent yearsIaA28R4 is becoming a major new subtype in the United States. In this study, we sequenced the genomes of two field specimens from each of the two subtypes and conducted a comparative genomic analysis of the obtained sequences with those from the only fully sequenced Cryptosporidium parvum genome. Results Altogether, 8.59-9.05 Mb of Cryptosporidium sequences in 45–767 assembled contigs were obtained from the four specimens, representing 94.36-99.47% coverage of the expected genome. These genomes had complete synteny in gene organization and 96.86-97.0% and 99.72-99.83% nucleotide sequence similarities to the published genomes of C. parvum and C. hominis , respectively. Several major insertions and deletions were seen between C. hominis and C. parvum genomes, involving mostly members of multicopy gene families near telomeres. The four C. hominis genomes were highly similar to each other and divergent from the reference IaA25R3 genome in some highly polymorphic regions. Major sequence differences among the four specimens sequenced in this study were in the 5′ and 3′ ends of chromosome 6 and the gp60 region, largely the result of genetic recombination. Conclusions The sequence similarity among specimens of the two dominant outbreak subtypes and genetic recombination in chromosome 6, especially around the putative virulence determinant gp60 region, suggest that genetic recombination plays a potential role in the emergence of hyper-transmissible C. hominis subtypes. The high sequence conservation between C. parvum and C. hominis genomes and significant differences in copy numbers of MEDLE family secreted proteins and insulinase-like proteases indicate that telomeric gene duplications could potentially contribute to host expansion in C. parvum .

Cryptosporidium species cause acute gastroenteritis and diarrhoea worldwide. They are members of the Apicomplexa—protozoan pathogens that invade host cells by using a specialized apical complex and are usually transmitted by an invertebrate vector or intermediate host. In contrast to other Apicomplexans, Cryptosporidium is transmitted by ingestion of oocysts and completes its life cycle in a single host. No therapy is available, and control focuses on eliminating oocysts in water supplies 1 . Two species, C. hominis and C. parvum , which differ in host range, genotype and pathogenicity, are most relevant to humans 1 , 2 , 3 . C. hominis is restricted to humans, whereas C. parvum also infects other mammals 2 . Here we describe the eight-chromosome ∼9.2-million-base genome of C. hominis 2 . The complement of C. hominis protein-coding genes shows a striking concordance with the requirements imposed by the environmental niches the parasite inhabits. Energy metabolism is largely from glycolysis. Both aerobic and anaerobic metabolisms are available, the former requiring an alternative electron transport system in a simplified mitochondrion. Biosynthesis capabilities are limited, explaining an extensive array of transporters. Evidence of an apicoplast is absent, but genes associated with apical complex organelles are present. C. hominis and C. parvum exhibit very similar gene complements, and phenotypic differences between these parasites must be due to subtle sequence divergence.

The Cryptosporidium parasite is one of the leading causes of diarrheal morbidity and mortality in children, and adolescent infections are associated with chronic malnutrition. There are no vaccines available for protection and only one drug approved for treatment that has limited efficacy. A major barrier to developing new therapeutics is a lack of foundational knowledge of Cryptosporidium biology, including which parasite genes are essential for survival and virulence. Here, we iteratively improve the tools for genetically manipulating Cryptosporidium and develop a targeted CRISPR-based screening method to rapidly assess how the loss of individual parasite genes influence survival in vivo. Using this method, we examine the parasite’s pyrimidine salvage pathway and a set of leading Cryptosporidium vaccine candidates. From this latter group, using inducible knockout, we determined the parasite gene known as Cp23 to be essential for survival in vivo. Parasites deficient in Cp23 were able to replicate within and emerge from infected epithelial cells, yet unable to initiate gliding motility which is required for the reinfection of neighbouring cells. The targeted screening method presented here is highly versatile and will enable researchers to more rapidly expand the knowledge base for Cryptosporidium infection biology, paving the way for new therapeutics. Cryptosporidium is an important threat to public health, yet it lacks a robust genetic toolkit. Here, Watson et al. introduce a targeted CRISPR-based screening method to identify parasite genes that are essential for its survival within the intestine.

Background Little is known on the occurrence and identity of Cryptosporidium species in sheep and goats in Algeria. This study aimed at investigating the occurrence of Cryptosporidium species in lambs and goat kids younger than 4 weeks. Methods A total of 154 fecal samples (62 from lambs and 92 from kid goats) were collected from 13 sheep flocks in Médea, Algeria and 18 goat flocks across Algiers and Boumerdes. They were screened for Cryptosporidium spp. by nested-PCR analysis of a fragment of the small subunit ( SSU ) rRNA gene, followed by restriction fragment length polymorphism and sequence analyses to determine the Cryptosporidium species present. Cryptosporidium parvum and C. ubiquitum were further subtyped by sequence analysis of the 60 kDa glycoprotein gene. Results Cryptosporidium spp. were detected in 17 fecal samples (11.0%): 9 from lambs (14.5%) and 8 from goat kids (8.7%). The species identified included C. parvum in 3 lambs, C. xiaoi in 6 lambs and 6 goat kids, and C. ubiquitum in 2 goat kids. Cryptosporidium infections were detected mostly in animals during the first two weeks of life (7/8 for goat kids and 7/9 for lambs) and in association with diarrhea occurrence (7/17 or 41.2% goat kids and 7/10 or 70.0% lambs with diarrhea were positive for Cryptosporidium spp.). Subtyping of C. parvum and C. ubiquitum isolates identified the zoonotic IIaA13G2R1 and XIIa subtype families, respectively. Minor differences in the SSU rRNA gene sequences were observed between C. xiaoi from sheep and goats. Conclusions Results of this study indicate that three Cryptosporidium species occur in lambs and goat kids in Algeria, including zoonotic C. parvum and C. ubiquitum . They are associated with the occurrence of neonatal diarrhea.

PCR-based diagnostics has revealed the previously largely unknown transmission and infections in high-income countries. This study aimed to determine domestic and imported subtypes of species in Norway, evaluate their demographic distribution, and identify potential small outbreaks. -positive human faecal samples were obtained from six medical microbiology laboratories between February 2022 and January 2024, together with 22 -positive animal samples. Species and subtypes were identified by sequencing PCR products from gp60 and SSU rRNA genes. Most cryptosporidiosis cases occurred during late summer/early autumn, primarily in children and young adults. Of 550 human samples, 359 were successfully characterized molecularly (65%), revealing infection with 10 different species. occurred in 245 (68%) human isolates with IIa and IId being major allele families, with distinct regional distribution patterns of common subtypes. A kindergarten outbreak with 5 cases was due to IIaA14G1R1. was identified in 33 (9.2%) human cases of which 24 were known to be of domestic origin, making it the second most common species in human autochthonous cases in Norway. All isolates were of the same genotype; XIVaA20G2T1, including 13 cases from a suspected small outbreak in Trøndelag. occurred in 68 typed cases (19%), but mostly in infections acquired abroad, with allele families Ib and If occurring most often. In conclusion, this study of recent spp. and subtypes in Norway, highlights the predominance of and the emergence of among autochthonous cases.

Objective This study aimed to assess the prevalence of Cryptosporidium genotypes in the canal water bodies of Minya Al-Qamh District in Sharqia Governorate, Northern Egypt. Rural populations in Egypt lack access to clean water. They obtain their water supplies from different drains and canals, which are frequently exposed to contamination by human activities and untreated agricultural waste, introducing Cryptosporidium infection to unprotected waterways. A total of (72) canal water samples served for the molecular detection of Cryptosporidium species by PCR amplification and sequencing. Results Only one sample 1.4% (1/72) belonging to the village of Al-Aziziyyah was PCR-positive for Cryptosporidium contamination. Based on the ( COWP ) gene sequencing, this species was identified as Cryptosporidium parvum (C. parvum). The current work’s findings marked the first report on the molecular characterization of Cryptosporidium species in the canal water of Sharqia Province. Our results suggested that the source of C. parvum contamination could be of human and/or animal origin. Therefore, further studies are warranted to track the source of human infection and mitigate contamination for improved water quality.

Cryptosporidium is a ubiquitous enteric protozoan pathogen infecting humans, domestic animals, and wildlife worldwide. It is a waterborne pathogen with recognized zoonotic potential and a definite cause of diarrhea and nutritional disorders in institutional and community settings. One challenge facing the world's supply of clean drinking water is contamination from feces and soil. It has been established that small quantities of oocysts, the infective stage, can cause human disease. Also, their resistance to chlorination and other water treatment procedures has been demonstrated. Kpong, a community in the Lower Manya Krobo Municipality of the Eastern Region of Ghana, is one of the primary sources of water supply to Accra, the capital city of Ghana. Being able to determine the effectiveness of water treatment processes and identifying sources of contamination of this pathogen in our water bodies is thus of public health importance. The study aimed to conduct molecular epidemiology of Cryptosporidium spp. in the Lower Manya Krobo Municipality. A total of 230 samples, 180 fecal samples from cattle and 50 water samples (tap water and well water) were collected from the following communities: Kpong, Akwernor, Ablotsi, Nuaso, and Atua, all in the Lower Manya Krobo Municipality. Samples were screened for Cryptosporidium by microscopy and PCR. The 18S rRNA gene was amplified by nested polymerase chain reaction (PCR), and the final product was sequenced. The prevalence of Cryptosporidium from the fecal samples was estimated as 10% (18/180) by microscopy, while all 50 water samples were negative. However, PCR gave the prevalence of Cryptosporidium as 47.8% (86/180) for fecal samples and 20% (10/50) for water samples. Based on the 18S rRNA gene, three sequenced samples showed high homology to C. parvum species. The phylogenetic analysis confirmed this as these sequences clustered with C. parvum sequences from other countries. Cryptosporidium parvum was identified as the persistent species in the study communities. This outcome supports the evidence that domesticated animals serve as potential reservoirs of zoonotic transmission of cryptosporidiosis. The persistence of cryptosporidiosis in cattle indicates its presence in the human population. In addition, the presence of Cryptosporidium parvum in the wells makes it alarming and necessary to consider a holistic approach such as One Health Strategies to identify and control cases in humans.