Metabolic needs and capabilities among Cryptosporidium species.

Development and validation of qPCR assay targeting COWP conserved region for sensitive detection and quantification of Cryptosporidium infections.

Cryptosporidium spp. is an important pathogen responsible for severe diarrheal illness, especially in children, and is transmitted by various modes. The present work is aimed at categorizing Cryptosporidium spp. infection and determining associated risk factors by ML on a known dataset of diarrhea among children. For classification, we used random forest and bagging CART trees. Model discrimination was measured by accuracy, balanced accuracy, sensitivity, specificity, positive predictive value, negative predictive value, and F1-score. Then, a 5-fold cross-validation method was used to verify the reliability of the model. Importance values were also calculated to identify the most important risk factors for infection. The bagged CART model emerged as the best among the models applied, with slightly better classification. For this model, performance metrics were: accuracy (87.2%), balanced accuracy (56.3%), sensitivity (97.2%), specificity (15.4%), positive predictive value (89.3%), negative predictive value (42.9%), F1-score (93.0%). As shown by the variable importance analysis, the strongest risk factor was the number of people in the household (people ≥ 5), which represented a higher risk of infection in crowded housings. Sources of water also came up as an important environmental factor; plain tap water and pipe-line water appeared to be major causes of transmission. Such results indicate that waterborne transmission is the main route of Cryptosporidium spp. These findings underscore the importance of water quality improvements, including efforts to address water disinfection, particularly in areas with household crowding and inadequate sanitation access.

Background: One of the common dietary practices in Iran is the consumption of raw vegetables, providing a significant range of vitamins and essential substances for the body. However, many human intestinal parasites are transmitted through water, soil, and vegetables including Cryptosporidium species. This study investigated the intestinal parasites of ready-to-eat fresh raw vegetables collected from Delis in Yazd, Iran, during 2023-2024. Methods: Following collection from food sales centers, the vegetables were transported to the laboratory. Samples were immersed in 1.5 L of a detergent solution containing 0.1% sodium dodecyl sulfate and 0.1% Tween 100 for 15 min under agitation. The samples were then centrifuged for 15 min at 1500 revolutions per min (rpm), after which the vegetable material was removed. Subsequently, 4% formaldehyde was added to the supernatant and centrifuged at 1500 revolutions per min (rpm) for 10 min. The obtained sediment was tested with Lugol dye and without Lugol under a microscope. Data analysis was performed using SPSS version 26. Results: The results indicated that lettuce was the most frequently sampled vegetable, comprising 32% of the total, while white cabbage had the lowest (9%). Furthermore, 51% of the vegetables were free from contamination with intestinal parasites, and 49% were contaminated. Based on Pearson's chi-square test, the average level of intestinal parasite infection was not significantly different among the vegetable types (p=0.382). Conclusion: Given the year-round consumption of vegetables, it is essential to implement strict hygienic controls and provide necessary public training on proper methods for consumption, cultivation, and harvesting.

Genetically tractable rodent models for Cryptosporidium hominis and C. parvum infections: identifying differences in infection sites between host species.

Detection of Cryptosporidium spp. and Giardia duodenalis DNA in wild deer in Scotland.

ABSTRACTCryptosporidium spp. and Giardia duodenalis are significant intestinal protozoan parasites affecting humans and animals worldwide. These infections are transmitted through the faecal–oral route, by contaminated water, food or close contact with infected hosts. The zoonotic risk of transmission from reptiles is considered low; yet, limited research exists regarding Cryptosporidium and Giardia infections in these animals. This study investigated the presence of Cryptosporidium spp. and G. duodenalis in reptile faecal samples from a breeding farm in the Chonburi Province, Eastern Thailand. The test population included 363 iguanas, 50 tortoises, 79 bearded dragons and nine snakes. DNA extraction from faecal samples was performed, followed by nested PCR for Cryptosporidium and G. duodenalis, and all positive samples were sequenced for molecular characterisation. Cryptosporidium and G. duodenalis were detected in 13 and 25 out of 501 faecal samples, respectively. Specifically, seven of 363 iguana samples and six of 79 bearded dragon samples tested positive for Cryptosporidium. Cryptosporidium avium was found in 11 samples, and one sample revealed Cryptosporidium serpentis; the remaining sample could not be identified at the species level. For G. duodenalis, 15 of 363 iguanas tested positive, along with six of 79 bearded dragons, two of 50 tortoises and two of nine snakes. Only G. duodenalis assemblage B was identified in positive samples. This study provides epidemiological information on the infection rate and distribution of species of Cryptosporidium and Giardia in a breeding reptile farm in Chonburi Province, Eastern Thailand, and the findings are discussed in the context of the scientific literature.SummaryThis study represents one of the largest surveys on Cryptosporidium and Giardia duodenalis in reptiles.Cryptosporidium avium, typically associated with avian hosts, was the predominant Cryptosporidium species identified in iguanas and bearded dragons.Giardia duodenalis assemblage B, known for its zoonotic potential, was the primary assemblage found in iguanas.

BackgroundCryptosporidium infection occurs in humans, domestic animals, and wildlife. To date, at least 49 species and 120 genotypes have been identified. Hitherto, molecular identification of Cryptosporidium species in wildlife has seldom been reported in China.MethodsDuring 2014–2025, a total of 1855 small intestinal or fecal specimens were collected from 1500 mammals, 121 reptiles, and 234 birds in Xinjiang Uygur Autonomous Region (XUAR) and Inner Mongolia Autonomous Region (IMAR), northern China. The identification of each animal species was based on morphological characteristics and mitochondrial gene amplification. Detection of Cryptosporidium species was performed by amplifying part of the small subunit (SSU) ribosomal RNA (rRNA) gene. The 60 kDa glycoprotein (GP60) gene was used to confirm their species and subtypes.ResultsThe samples were collected from 39 mammalian, 6 reptilian, and 30 avian species. In these samples, the average rate of infection with Cryptosporidium species was 8.09% (150/1855). In total, 18 known Cryptosporidium species and genotypes were identified, including Cryptosporidium hominis, Cryptosporidium ubiquitum, Cryptosporidium muris, Cryptosporidium canis, Cryptosporidium felis, Cryptosporidium equi, Cryptosporidium proventriculi, Cryptosporidium ryanae, Cryptosporidium rubeyi, chipmunk genotype V, vole genotype III, vole genotype V, muskrat genotype I, bat genotype IV, yak genotype, deer genotype, goose genotype I, and one unnamed Cryptosporidium sp. In addition, a novel genotype, here designated as Cryptosporidium Mongolian pika genotype, was identified in the Mongolian pika (Ochotona pallasi).ConclusionsInvestigation of Cryptosporidium infection was carried out by screening 75 animal species. Overall, 19 Cryptosporidium species and genotypes were detected, including a novel genotype in Mongolian pika and first-time diagnosis of this infection in several rodent species (e.g., red-cheeked ground squirrels, great gerbils, northern mole voles, and Libyan jirds).Graphical Supplementary InformationThe online version contains supplementary material available at 10.1186/s13071-025-07152-9.

Toxoplasma gondii and Cryptosporidium species are apicomplexan parasites of significant medical and veterinary importance. Although current therapeutic options for toxoplasmosis and cryptosporidiosis demonstrate notable efficacy, their clinical efficacy is often limited by suboptimal efficacy and frequent adverse effects. Moreover, therapeutic alternatives remain limited or nonexistent, particularly for cryptosporidiosis, for which nitazoxanide is currently the only approved medication to treat diarrhea in adults and children older than 1 year of age. To identify alternative therapeutic options for addressing these health challenges, we performed a phenotypic screening of an FDA-approved drug repurposing library against Toxoplasma. This screening identifies LY2090314 as a potent inhibitor of T. gondii and Cryptosporidium growth in mammalian cells. Through a target deconvolution strategy combining forward genetics, transcriptome sequencing, and computational mutation analysis, we elucidate the parasiticidal mechanism of LY2090314 and demonstrate that TgGSK3 kinase is its primary molecular target. We also report the first X-ray crystal structure of LY2090314 bound to TgGSK3, resolved at 2.1 Å, which reveals an interaction mode characteristic of type I ATP-competitive inhibitors. Furthermore, interactome analysis uncovers functional connections between TgGSK3 and key cytoskeletal and signaling regulators, providing insights into compound’s effects. Collectively, these findings validate TgGSK3 as a promising therapeutic target for toxoplasmosis and offer mechanistic insights into apicomplexan GSK3 biology.

Cryptosporidium and Giardia are significant causes of gastrointestinal diseases in humans and animals worldwide, especially in pigs. This study investigated the molecular occurrence, genetic diversity, and zoonotic potential of Cryptosporidium spp. and Giardia duodenalis in domestic pigs in the Chattogram Hill Tracts (CHT), Bangladesh. A total of 408 fecal samples were collected from Rangamati, Khagrachari, and Bandarban districts and examined using nested PCR, targeting the SSU rRNA gene for Cryptosporidium and the β-giardin (bg), glutamate dehydrogenase (gdh), and triosephosphate isomerase (tpi) genes for G. duodenalis. Nucleotide sequencing and phylogenetic analysis were conducted for molecular characterization. The prevalence of Cryptosporidium was 3.43%, and G. duodenalis was 23.52%. The prevalence varied by several factors, although the variations were not significant for Cryptosporidium. Age, anthelmintic administration, and diarrheal status of the pigs had a significant influence on the occurrence of Giardia. Among the 14 Cryptosporidium and 96 Giardia isolates, two Cryptosporidium species (C. suis and C. scrofarum) and three G. duodenalis assemblages (A, B, and E) were found, among which assemblage A (56.25%) was the most prevalent. Additionally, 13 novel genotypic variations were identified at the bg, gdh, and tpi genes of G. duodenalis. Pigs in the CHT region harbor Cryptosporidium and Giardia with notable genotypic diversity and zoonotic potential. Addressing this issue requires further research within a One Health framework, focusing on integrated surveillance and the development of evidence-based policies for pig farming to reduce transmission and safeguard animal and public health.

Detection and genetic diversity of Cryptosporidium in yaks in Xinjiang, China

BackgroundCryptosporidium is a globally prevalent parasite that infects the gastrointestinal tract of a large number of vertebrates. The Cryptosporidium sp. lizard genotype is found almost exclusively in geckos but has also been detected in a corn snake. In this study, the biology and genetic variability of the lizard genotype were investigated.MethodsThe genetic variability of the Cryptosporidium sp. lizard genotype was analysed by polymerase chain reaction (PCR) and Sanger sequencing of the SSU rRNA, actin, HSP70 and COWP genes. Its biological characteristics, including oocyst size, tissue tropism and associated pathology, were investigated using light microscopy, electron microscopy and histological techniques. Experimental infections were performed to assess host specificity and infectivity.ResultsPhylogenetic analysis of the target genes confirmed that the Cryptosporidium sp. lizard genotype is genetically distinct from other Cryptosporidium species. Experimental infections showed that chickens, mice and corn snakes were not susceptible to this genotype, as no infection was detected. In contrast, leopard geckos began to excrete oocysts between 6 and 8 days post-infection, although they showed no clinical symptoms. Excretion of oocysts continued for over 200 days. Oocysts isolated from naturally infected leopard and smooth knob-tailed geckos were morphometrically identical to those from experimentally infected leopard geckos and measured 5.89 × 4.79 μm. The parasite mainly colonised the stomach, where the highest intensity of infection was observed, but was also found throughout the intestine and in the lungs.ConclusionsDue to the distinct genetic differences, host range and tissue tropism, we propose the name Cryptosporidium geckonae n. sp. for this organism, which was previously referred to as Cryptosporidium sp. lizard genotype.Graphical

BackgroundThis study presents findings from a 15-year longitudinal surveillance program (2009–2024) monitoring Cryptosporidium and Giardia in protected drinking water catchments in Melbourne and environs in the State of Victoria, Australia. As one of the few major cities worldwide sourcing largely unfiltered water from forested catchments, Melbourne presents a unique opportunity to assess the occurrence and prevalence of protozoan parasites in a minimally disturbed ecosystem.MethodsA total of 14,960 animal faecal samples were analysed using polymerase chain reaction (PCR)-based sequencing, including 8695 samples collected over the past 9 years.ResultsCryptosporidium was detected in 3.15% of samples and Giardia in 0.16%. A total of 12 recognised Cryptosporidium species and genotypes were identified, nine of which have known zoonotic potential, as well as two sub-assemblages (AI and AIII) of Giardia duodenalis, including four novel assemblage AI variants. Parasite diversity was the highest in eastern grey kangaroos, which hosted at least 18 Cryptosporidium variants. Temporal analyses revealed significant inter-annual variation, with peak prevalence during the 2023 La Niña year and seasonal differences by host group. Notably, C. ubiquitum, C. muris and C. occultus were recorded for the first time in these catchments. In spite of the low prevalence of high-risk species such as C. parvum and the absence of C. hominis, the detection of emerging and previously uncharacterised genotypes emphasises the importance of sustained surveillance.ConclusionsThese findings have broad implications for managing zoonotic risk in unfiltered water systems worldwide. Advances in metagenomics and high-throughput sequencing platforms will be critical for enhancing future pathogen monitoring and catchment management strategies in the context of increasing climate and environmental pressures.Graphical Supplementary InformationThe online version contains supplementary material available at 10.1186/s13071-025-07048-8.

Cryptosporidium is a globally endemic parasite genus with over 40 recognized species. While C. hominis and C. parvum are responsible for most human infections, human cases involving other species have also been reported. Furthermore, there is increasing evidence of simultaneous infections with multiple species. Therefore, we devised a new means to identify various species of Cryptosporidium in mixed infections by sequencing a 431 bp amplicon of the 18S rRNA gene encompassing two variable regions. Using the DADA2 pipeline, amplicons were first identified to a genus using the SILVA 132 reference database; then Cryptosporidium amplicons to a species using a custom database. This approach demonstrated sensitivity, successfully detecting and accurately identifying as little as 0.001 ng of C. parvum DNA in a complex stool background. Notably, we differentiated mixed infections and demonstrated the ability to identify potentially novel species of Cryptosporidium both in situ and in vitro. Using this method, we identified Cryptosporidium parvum in Egyptian rabbits with three samples showing minor mixed infections. By contrast, no mixed infections were detected in Egyptian children, who were primarily infected with C. hominis. Thus, this pipeline provides a sensitive tool for Cryptosporidium species-level identification, allowing for the detection and accurate identification of minor variants and mixed infections.IMPORTANCECryptosporidium is a eukaryotic parasite and a leading global cause of waterborne diarrhea, with over 40 recognized species infecting livestock, wildlife, and people. While we have effective tools for detecting Cryptosporidium in clinical and agricultural water samples, there is still a need for a method that can efficiently identify known species as well as infections with multiple Cryptosporidium species, which are increasingly being reported. In this study, we utilized sequencing of a specific region to develop a sensitive and accurate identification workflow for Cryptosporidium species based on high-throughput sequencing. This method can distinguish between all 40 recognized species and accurately detect mixed infections. Our approach provides a sensitive and reliable means to identify Cryptosporidium species in complex clinical and agricultural samples. This has important implications for clinical diagnostics, biosurveillance, and understanding disease transmission, ultimately benefiting clinicians and produce growers.

Molecular characterization of Cryptosporidium spp. and Giardia duodenalis infections and impact on growth performance of Churra Galega Mirandesa lambs.

Unraveling the Cryptosporidium Threat: Epidemiology, Genetic Diversity, and Prevalence in HIV-Positive Patients in Tehran, Iran.

Background: Calf diarrhea caused by infectious agents is one of the most significant and frequently encountered health problems in cattle breeding worldwide. Among these infectious agents, Cryptosporidium stands out as an important protozoan parasite, particularly affecting neonatal calves and young animals with immature or weakened immune systems. These early-life infections can lead to severe dehydration, weight loss, and even death if not properly managed. Cryptosporidium spp. is a zoonotic coccidian parasite known to play a substantial role in both human and animal health, especially in regions where hygiene and sanitation practices are inadequate. Its oocysts are highly resistant to environmental factors and common disinfectants, making control and prevention difficult. Importantly, due to its zoonotic nature, it poses a considerable threat to public health. This study was conducted to investigate the prevalence of cryptosporidiosis in buffaloes and cattle raised in the Van region using various diagnostic methods, and to evaluate its potential public health significance. Materials, Methods & Results: The fecal materials of the study were collected from 100 buffaloes and 200 cattle from different farms in Van province. The samples brought to the laboratory were stained with the Kinyoun Acid Fast method and DNA extraction was performed from each sample. Smear preparations were prepared from fresh fecal samples. The smear preparations were fixed in pure methanol for 1 min and allowed to dry, then stained in Kinyoun Carbol-Fuxin for 5 min, and then the slides were immersed in 50% ethyl alcohol, shaken, and immediately washed in tap water. The slides were placed in a decolorizing solution containing 1% sulfuric acid for 2 min and washed in tap water, then kept in a flask containing methylene blue for 1 min and washed again in tap water and left to dry. After drying, immersion oil was dripped and examined under a microscope at a magnification of 100x. DNA extraction was performed in all samples using a GeneMATRIX Stool DNA Purification Kit. Nested PCR analysis; Primers were used to amplify the SSU rRNA gene region of the obtained DNAs. In the PCR stage, 5’-TTCTAGAGCTAATA CATGCG-3’ and 5’-CCCATTTCCTTCGAAACAGGA-3’ primers were used to amplify the 1325 bp gene region. In the nested PCR stage, primers 5’- GGAAGGGTTG TATTTATTTATTAGATAAAG-3’ and 5’-AAGGAGTA AGGAACAACCTCCA-3’ were used to amplify the 826-864 bp gene region DNA extraction. The PCR products obtained were stained with RedSafe™ Nucleic Acid Staining Solution and images were obtained on 1.5% agarose gel. Positive PCR products were subjected to bidirectional sequencing at a commercial company (BM Labosis, Ankara, Turkey). As a result of microscopic and PCR analyses, 5.50% and 9.50% positivity were detected in cattle, 3% and 6% positivity in buffaloes, respectively. The infection prevalence was highest in cattle and buffaloes in the 0-6 month age group, 17.31% and 15.38%, respectively. In addition, zoonotic C. parvum was isolated from cattle calves. Discussion: In conclusion buffaloes and cattle, with their large fecal volumes, can easily contaminate the environment and serve as significant reservoirs for this pathogen, which is transmitted through water and food. Therefore, it is crucial to identify the Cryptosporidium species present in these animals. For this, epidemiological studies should be conducted and expanded with molecular methods. Keywords: calf, Cryptosporidium spp., protozoan, sequence analysis, zoonotic coccidian, neonatal enteritis, Van region.

Detection and genotyping of Cryptosporidium species in diarrheic patients in Assiut, Egypt: A parasite of potential zoonotic transmission

The transformation of a Cryptosporidium reference microbiology service to tackle the One Health challenge.

Molecular characterization and phylogenetic analysis of cryptosporidium spp. in pediatric acute gastroenteritis: Epidemiological insights from northeastern Iran