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PubMed2026

Detection and genomic characterization of cryptosporidium parvum virus 1 (CSpV1): A potential biomarker for Cryptosporidium parvum detection in bovine calves.

Cryptosporidium parvum is a ubiquitous enteric parasite that infects a diverse range of vertebrate species. The detection of C. parvum can be confounded when oocysts are intermittently shed below an assay's limit of detection, yielding a false-negative result. We therefore investigated the utility of Cryptosporidium parvum virus 1 (CSpV1), a putative symbiont of Cryptosporidium parvum, as a surrogate target for detecting the parasite in bovine calves. Using real-time polymerase chain reaction (qPCR), we tested 422 samples for Cryptosporidium spp., C. parvum-associated targets, and CSpV1. Among the 189 samples positive for at least one target, CSpV1 was detected in 24 (12.70%) samples without concurrent detection of Cryptosporidium. Additionally, we analyzed CSpV1 genomic sequences to ascertain its value as an epidemiological biomarker. Evaluation of dsRNA1 amino acid sequences identified country-associated patterns, suggesting potential utility for geographic distribution analyses. These findings suggest that CSpV1 may serve as a biological signature of C. parvum and support further investigation into its usefulness as an adjunct molecular target.

Biomarker↗

PubMed2024

Phylogenomic reconstruction of Cryptosporidium spp. captured directly from clinical samples reveals extensive genetic diversity.

Cryptosporidium is a leading cause of severe diarrhea and mortality in young children and infants in Africa and southern Asia. More than twenty Cryptosporidium species infect humans, of which C. parvum and C. hominis are the major agents causing moderate to severe diarrhea. Relatively few genetic markers are typically applied to genotype and/or diagnose Cryptosporidium. Most infections produce limited oocysts making it difficult to perform whole genome sequencing (WGS) directly from stool samples. Hence, there is an immediate need to apply WGS strategies to 1) develop high-resolution genetic markers to genotype these parasites more precisely, 2) to investigate endemic regions and detect the prevalence of different genotypes, and the role of mixed infections in generating genetic diversity, and 3) to investigate zoonotic transmission and evolution. To understand Cryptosporidium global population genetic structure, we applied Capture Enrichment Sequencing (CES-Seq) using 74,973 RNA-based 120 nucleotide baits that cover ~92% of the genome of C. parvum. CES-Seq is sensitive and successfully sequenced Cryptosporidium genomic DNA diluted up to 0.005% in human stool DNA. It also resolved mixed strain infections and captured new species of Cryptosporidium directly from clinical/field samples to promote genome-wide phylogenomic analyses and prospective GWAS studies.

Cryptosporidium↗

PubMed2026

Epidemiological overview of Cryptosporidium infection in Capra hircus from southern Khyber Pakhtunkhwa, Pakistan.

Objectives: Cryptosporidium is a protozoan parasite that commonly infects livestock, including Capra hircus, causing diarrhea, dehydration, and sometimes mortality. These infections lead to substantial economic losses in the farming industry. This study aimed to determine the prevalence and associated risk factors, such as region, season, age, and sex, of Cryptosporidium infection in C. hircus in southern Khyber Pakhtunkhwa, Pakistan. Materials and Methods: A total of 360 fecal samples were collected from C. hircus from the Kohat, Bannu, and Lakki Marwat Districts of Khyber Pakhtunkhwa, Pakistan. Fecal smears were stained using the modified Ziehl-Neelsen method, and statistical analysis, including chi-square tests, was employed to assess the association between Cryptosporidium infection prevalence and various factors. Results: The overall Cryptosporidium infection prevalence was 20.55% (74/360). District-wise analysis revealed the highest prevalence in Kohat (23.33%), followed by Bannu (20%) and Lakki Marwat (18.33%). Monthly examination identified August as the peak prevalence month (36.67%), displaying non-significant monthly variations (p = 0.075). Seasonal disparity indicated a significantly higher prevalence in the summer (30%) compared to other seasons (p = 0.008). Age-wise prevalence demonstrated susceptibility in kids aged ≤ 15 days (≤ 33.93%), with significant overall age-based differences (p = 0.001). Female kids exhibited a slightly higher prevalence at 22.22% compared to males (19.19%), with no significant gender-based difference (p = 0.479). Conclusions: This study highlights the influence of geographic, seasonal, and demographic factors on Cryptosporidium prevalence in goat populations. The findings emphasize the importance of implementing targeted preventive measures. Recommended strategies include farmer education programs, seasonal deworming schedules, early screening for infection, and strict hygiene and water sanitation practices to reduce transmission risks effectively.

Cryptosporidiosis↗

PubMed2024

WHOLE GENOME TARGETED ENRICHMENT AND SEQUENCING OF HUMAN-INFECTING CRYPTOSPORIDIUM spp.

Cryptosporidium spp. are protozoan parasites that cause severe illness in vulnerable human populations. Obtaining pure Cryptosporidium DNA from clinical and environmental samples is challenging because the oocysts shed in contaminated feces are limited in quantity, difficult to purify efficiently, may derive from multiple species, and yield limited DNA (<40 fg/oocyst). Here, we develop and validate a set of 100,000 RNA baits (CryptoCap_100k) based on six human-infecting Cryptosporidium spp. (C. cuniculus, C. hominis, C. meleagridis, C. parvum, C. tyzzeri, and C. viatorum) to enrich Cryptosporidium spp. DNA from a wide array of samples. We demonstrate that CryptoCap_100k increases the percentage of reads mapping to target Cryptosporidium references in a wide variety of scenarios, increasing the depth and breadth of genome coverage, facilitating increased accuracy of detecting and analyzing species within a given sample, while simultaneously decreasing costs, thereby opening new opportunities to understand the complex biology of these important pathogens.

Journal Article↗

PubMed2026

Genome-targeted enrichment and sequencing of human-infecting Cryptosporidium spp.

Cryptosporidium spp. are parasites that cause severe illness in vulnerable human populations. Obtaining pure and sufficient Cryptosporidium DNA from clinical and environmental samples is a challenging task. Oocysts shed in available fecal samples can be limited in quantity, require purification (biased towards dominant strains), and yield limited DNA ( < 40 fg/oocyst). Here, we use updated genomic sequences from a broad diversity of Cryptosporidium species that have been found to infect humans (C. cuniculus, C. hominis, C. meleagridis, C. parvum, C. tyzzeri, and C. viatorum) to develop and validate a set of 100,000 RNA baits (CryptoCap_100k) with the aim of enriching Cryptosporidium DNA from varied samples. Compared to unenriched libraries, CryptoCap_100k increases the percentage of reads mapping to target genome sequences, increases the depth and breadth of genome coverage, and facilitates analyses of genetic variants in many samples, while decreasing overall costs.

Cryptosporidium↗

PubMed2025

GENOME TARGETED ENRICHMENT AND SEQUENCING OF HUMAN-INFECTING CRYPTOSPORIDIUM spp.

Cryptosporidium spp. are parasites that cause severe illness in vulnerable human populations. Obtaining pure and sufficient Cryptosporidium DNA from clinical and environmental samples is a challenging task. Oocysts shed in available fecal samples can be limited in quantity, require purification (biased towards dominant strains), and yield limited DNA (<40 fg/oocyst). Here, we use updated genomic sequences from a broad diversity of human-infecting Cryptosporidium species ( C. cuniculus , C. hominis , C. meleagridis , C. parvum , C. tyzzeri , and C. viatorum ) to develop and validate a set of 100,000 RNA baits (CryptoCap_100k) with the aim of enriching Cryptosporidium spp. DNA from varied samples. Compared to unenriched libraries, CryptoCap_100k increases the percentage of reads mapping to target genome sequences, increases the depth and breadth of genome coverage and the reliability of detecting species and mixed infections within a sample, and allows assessment of genetic variation via SNP calling, while decreasing costs.

Journal Article↗

PubMed2025

Genetic crosses reveal genomic loci responsible for virulence in Cryptosporidium parvum infection.

The relationship between parasite genotype and pathogenesis is largely unknown for Cryptosporidium, a leading cause of diarrheal disease in children. An array of parasites with similar genomes produces varied disease outcomes in different hosts. Here, we isolate and characterize Cryptosporidium parvum strains that show marked differences in virulence and persistence in mice. Taking advantage of the sexual life cycle of this eukaryotic pathogen, we use genetic crosses to discover the underlying chromosomal loci. Whole-genome sequencing and bulk segregant analysis of infection-selected progeny mapped three loci on chromosomes 2, 6, and 7 associated with the ability to colonize and persist in mice and the positions of drug resistance genes. The chromosome 6 locus encodes the hyper-polymorphic surface glycoprotein GP60. Reverse genetic studies in both parental strains demonstrate that GP60 controls parasite burden and virulence, but not persistence, and reveal the dominance of the less virulent allele, suggesting it restricts virulence.

Cryptosporidium parvum↗

PubMed1979

Cryptosporidium cuniculus in the rabbit (Oryctolagus cuniculus).

Cryptosporidium cuniculus was identified by light and electron microscopy in two apparently healthy rabbits. Organisms were firmly attached at the brush borders of intestinal epithelial cells. Cellular alterations were minimal, consisting of elongation and shortening of microvilli adjacent to the attachment sites of organisms. Various developmental stages of cryptosporidium were identified by electron microscopy.

Animals↗

PubMed2026

m6A RNA methylation modulates IFN-γ-stimulated intestinal epithelial cell-intrinsic antiparasitic defense.

N6-methyladenosine (m6A) RNA methylation is one of the most prevalent reversible post-transcriptional RNA modifications and has been recognized as a crucial regulator of host immune responses. Intestinal epithelial cells (IECs) constitute an important component of gastrointestinal mucosal immunity. Interferons (IFNs) play a central role in maintaining intestinal homeostasis, and m6A methylation status influences IFN-mediated cell-intrinsic defense. In this study, we investigated the potential role of m6A RNA modifications in IFN-γ-stimulated IEC-intrinsic defense. We observed significant alterations in the topology of the m6A mRNA methylome in murine IECs following IFN-γ stimulation. A subset of IFN-γ-stimulated immune gene transcripts exhibited increased m6A RNA methylation, including several members of the immunity-related GTPase family M (IRGM) genes. In addition, IFN-γ-responsive long non-coding RNAs may modulate the m6A methylation levels of multiple IFN-γ-stimulated immune transcripts. Enhanced m6A methylation of the Irgm2/3 transcripts was associated with strengthened cell-intrinsic defense against infection by the protozoan parasite Cryptosporidium. Notably, Cryptosporidium infection altered the host m6A mRNA methylome in IECs, thereby counteracting the IFN-γ-mediated defense response. Although the RNA levels of Irgm2/3 genes were upregulated, their m6A RNA methylation levels and protein expression were reduced in infected cells. This effect was associated with host delivery of dsRNAs derived from Cryptosporidium parvum virus 1, a virus harbored in the parasite. Collectively, our findings suggest that m6A methylation of RNA transcripts enhances IFN-γ-mediated IEC-intrinsic antiparasitic defense, while Cryptosporidium has evolved mechanisms to evade this response by suppressing m6A RNA methylation of IFN-γ-stimulated immune genes.

Animals↗

PubMed1979

Spontaneous cryptosporidiosis in an adult female rabbit.

An asymptomatic adult female rabbit had intestinal cryptosporidiosis. The ileum had blunted villi, a decrease in villus-crypt ratio and a mild edema in the lamina propria. Transmission electron microscopy showed the parasite to be a Cryptosporidium similar to those reported in mouse, guinea pig, lamb, calf, horse, monkey and man. This organism is referred to as Cryptosporidium cuniculus. Scanning electron microscopy on ileal mucosa showed altered intestinal microvilli in the attachment of the cryptosporidia. It is postulated that the organism was enveloped by the microvilli of the ileal epithelial cells which then fused and formed a continuous double membrane around the parasite.

Animals↗

PubMed2025

Advances in diagnosis of diseases causing diarrhea in newborn calves.

Diarrhea in newborn calves is a serious global health problem. It poses challenges for animal industry, veterinarians and researchers due to the rapid onset of dehydration. Mixed infections make treatment complicated, and many young calves suffer high rates of illness and death from this condition. Numerous enteropathogens are associated with diarrhea in newborn calves, encompassing viruses, bacteria, parasites, and protozoa. Their occurrence differs by region, yet the most prevalent infections include E. coli, Salmonella species, Clostridium perfringens, Clostridium difficile, Rotavirus, Coronavirus, Cryptosporidium, Toxocara, Giardia and Eimeria. This review outlines the diagnostic techniques for diseases that lead to diarrhea in newborn calves. Diagnosis is based on clinical manifestations; however, the laboratory identification of etiological items is the only valid way for detecting the illness's aetiology and initiating treatment protocols. Classic methods such as bacterial culturing, fecal flotation, direct microscopy, and virus isolation help us understand pathogens better. Immunological assays like ELISA and immunochromatography are fast, accurate, affordable, and useful for on-farm detection. They help identify specific antigens or antibodies efficiently. Molecular methods including PCR (standard, multiplex, real time and digital), LAMP assays, DNA microarrays and whole-genome sequencing allow highly accurate and sensitive detection. They can identify pathogens effectively, even at very low levels. Nanotechnology-based assays introduce a novel level of sensitivity and specificity, often yielding quick results with minimal sample volumes. In conclusion, accurate and rapid diagnosis using advanced techniques is critical for managing and preventing diseases that lead to diarrhea in newborn calves.

Animals↗

PubMed1979

Cryptosporidial enteritis in a patient with congenital hypogammaglobulinemia.

Cryptosporidium is a potentially pathogenic coccidial protoxoan known to inhabit the striated border of interestinal epithelium in several animal species. Recently two cases of human intestinal cryptosporidiosis have been described. The purpose of this report is to document a third case of chronic debilitating cryptosporidial enteritis in a young boy with congenital hypogammaglobulinemia. The endogenous protozoan life cycle and morphologic features are examined as studied by light and electron microscopy.

Agammaglobulinemia↗

PubMed1978

Cryptosporidiosis in immunodeficient Arabian foals.

Five of six immunodeficient Arabian foals that died of adenoviral infection were found to be infected with an intestinal coccidian of the genus Cryptosporidium. Various developmental stages of the organism were found in the microvillous border of the intestinal mucosa. The foals had diarrhea but it was not possible to separate the effects of the cryptosporidial infection from those of the concomitant adenoviral enteritis.

Animals↗

PubMed1978

Bovine cryptosporidiosis: a transmission and scanning electron microscopic study of some stages in the life cycle and of the host-parasite relationship.

Transmission and scanning electron microscopy of the ileal mucosae from 12 calves infected with Cryptosporidium sp. showed cryptosporidia free in the lumen and attached to epithelium. The attached parasites were interpreted to be extracellular and adherent to the microvillous border of epithelial cells. Stages of the organism included crescentic-free merozoites, trophozoites, schizonts, gametes and oocysts. Attached parasites were detected chiefly at villous tips and all stages were present on a single villus. Attachment sites were characterized by absence or disintegration of microvilli, disorganization of the terminal web and development of a specialized attachment zone. There were increased numbers of lysosomes and irregularities in the nuclear membrane of parasitized epithelial cells. It was concluded that cryptosporidia exist in bovine intestine as extracellular parasites and cause epithelial changes that in turn probably cause or contribute to diarrheal disease.

Animals↗