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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 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.

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.

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.

Cryptosporidium species can infect humans and more than 260 animal species, including 54 rodent species. However, data on the occurrence and genetic characterizations of Cryptosporidium spp. in laboratory rodents are limited. The present study aimed to determine the occurrence rate and genetic characterizations of Cryptosporidium spp. in laboratory mice and rats. We collected 506 fresh combined fecal pellet specimens (457 from mice and 49 from rats) of more than 2,000 laboratory rodents in Heilongjiang Province and Shanghai City, China. Cryptosporidium spp. were identified and subtyped by DNA sequencing of the SSU rRNA and the gp60 genes, respectively. By sequence analysis of the SSU rRNA gene, the occurrence rate of Cryptosporidium spp. was 16.6% (84/506) in combined fecal specimens, with 18.2% (83/457) for mice and 2.0% (1/49) for rats. Cryptosporidium parvum ( n  = 39), C. tyzzeri ( n  = 33), and C. parvum  +  C. tyzzeri ( n  = 11) were identified in mice. Cryptosporidium parvum was only detected in one rat fecal specimen. At the gp60 locus, 71.4% (60/84) of the Cryptosporidium -positive specimens were successfully amplified, and they all came from mice. We identified five C. parvum subtypes (IIaA14G2R1, IIaA16G2R1, IIaA17G1R1, IIaA17G2R1, and IIaA18G2R1) and two C. tyzzeri subtypes (IXaA6R1 and IXbA8). Based on the identification in laboratory mice of C. parvum subtypes that have been reported previously in humans, the mice infected with this species may threaten human health, especially for people who have contact with the animals and their feces. Les espèces de Cryptosporidium peuvent infecter les humains et plus de 260 espèces animales, dont 54 espèces de rongeurs. Cependant, les données sur la présence et les caractérisations génétiques de Cryptosporidium spp. chez les rongeurs de laboratoire sont limitées. La présente étude visait à déterminer le taux de présence et les caractérisations génétiques de Cryptosporidium spp. chez des souris et des rats de laboratoire. Nous avons collecté 506 échantillons de boulettes fécales fraîches combinées (457 de souris et 49 de rats) de plus de 2 000 rongeurs de laboratoire dans la province du Heilongjiang et la ville de Shanghai, en Chine. Les Cryptosporidium spp. ont été identifiés et sous-typés par séquençage de l’ADN des gènes SSU rRNA et gp60 , respectivement. Par analyse de séquence du gène SSU rRNA, le taux de présence de Cryptosporidium spp. était de 16,6% (84/506) dans les échantillons fécaux combinés, avec 18,2 % (83/457) pour les souris et 2,0 % (1/49) pour les rats. Cryptosporidium parvum ( n  = 39), C. tyzzeri ( n  = 33) et C. parvum  +  C. tyzzeri ( n  = 11) ont été identifiés chez la souris. Cryptosporidium parvum n’a été détecté que dans un échantillon fécal de rat. Au locus gp60 , 71,4 % (60/84) des échantillons positifs à Cryptosporidium ont été amplifiés avec succès, et ils provenaient tous de souris. Nous avons identifié cinq sous-types de C. parvum (IIaA14G2R1, IIaA16G2R1, IIaA17G1R1, IIaA17G2R1 et IIaA18G2R1) et deux sous-types de C. tyzzeri (IXaA6R1 et IXbA8). Sur la base de l’identification, chez des souris de laboratoire, de sous-types de C. parvum qui ont déjà été signalés chez l’homme, les souris infectées par cette espèce peuvent menacer la santé humaine, en particulier pour les personnes qui sont en contact avec les animaux et leurs excréments.

This study was conducted to molecularly identify and classify spp. in fecal samples (n=150) from patients with diarrhea received at the microbiology laboratory of a private hospital in Denizli. In this study, the positivity of spp. in fecal samples was investigated using direct microscopy, Kinyoun's acid-fast staining method, and Nested polymerase chain reaction (PCR) techniques. Positive PCR products were sequenced. In the examined fecal samples of patients with diarrhea, no parasites were detected through direct microscopic examination. Using the Kinyoun acid-fast staining method, spp. was identified in 2.7% (n=4) of the samples, while Nested PCR detected it in 4.67% (n=7) of the samples. The four positive samples were sequenced using primers that amplify the 18S gene region. The sequencing results identified the isolates as . Cryptosporidiosis is an important public health issue as it is a zoonotic disease caused by the parasite that can be transmitted from animals to humans. This study focuses on the molecular characterization of species detected in human fecal samples, which is significant for understanding which specific strains or species are involved in human infections. According to the findings, it is recommended that control measures be implemented to reduce the risk of exposure to in both humans and animals in Türkiye.

Background Whole genome sequencing (WGS) of Cryptosporidium spp. has previously relied on propagation of the parasite in animals to generate enough oocysts from which to extract DNA of sufficient quantity and purity for analysis. We have developed and validated a method for preparation of genomic Cryptosporidium DNA suitable for WGS directly from human stool samples and used it to generate 10 high-quality whole Cryptosporidium genome assemblies. Our method uses a combination of salt flotation, immunomagnetic separation (IMS), and surface sterilisation of oocysts prior to DNA extraction, with subsequent use of the transposome-based Nextera XT kit to generate libraries for sequencing on Illumina platforms. IMS was found to be superior to caesium chloride density centrifugation for purification of oocysts from small volume stool samples and for reducing levels of contaminant DNA. Results The IMS-based method was used initially to sequence whole genomes of Cryptosporidium hominis gp60 subtype IbA10G2 and Cryptosporidium parvum gp60 subtype IIaA19G1R2 from small amounts of stool left over from diagnostic testing of clinical cases of cryptosporidiosis. The C. parvum isolate was sequenced to a mean depth of 51.8X with reads covering 100 % of the bases of the C. parvum Iowa II reference genome (Bioproject PRJNA 15586), while the C. hominis isolate was sequenced to a mean depth of 34.7X with reads covering 98 % of the bases of the C. hominis TU502 v1 reference genome (Bioproject PRJNA 15585). The method was then applied to a further 17 stools, successfully generating another eight new whole genome sequences, of which two were C. hominis ( gp60 subtypes IbA10G2 and IaA14R3) and six C. parvum ( gp60 subtypes IIaA15G2R1 from three samples, and one each of IIaA17G1R1, IIaA18G2R1, and IIdA22G1), demonstrating the utility of this method to sequence Cryptosporidium genomes directly from clinical samples. This development is especially important as it reduces the requirement to propagate Cryptosporidium oocysts in animal models prior to genome sequencing. Conclusion This represents the first report of high-quality whole genome sequencing of Cryptosporidium isolates prepared directly from human stool samples.

Introduction:Cryptosporidium is an intestinal parasite responsible for gastroenteritis. Conventional diagnosis of Cryptosporidium is made by microscopy. The most frequent molecular detection method for this parasite is polymerase chain reaction (PCR). The objective of the present study was to identify the novel DNA targets and development of PCR-based assays for the specific detection of two major human infecting species Cryptosporidium parvum and Cryptosporidium hominis. Methodology: Sensitive and specific SYBR green quantitative PCR (qPCR) and TaqMan qPCR assays were developed and validated at both diagnostic and analytical level using the new identified targets TU502HP-1 and TU502HP-2. Results: Assay validation results showed that the newly developed real-time PCR assays are 100% specific with a reliable limit of detection. Overall repeatability and reproducibility of these assays showed good quality results over intra- and inter-laboratory analysis. Conclusion: Novel target-based qPCR assays can be rapid an efficient tool for simultaneous detection of a C. parvum and C. hominis. These genes could also be utilized for the development of innovative DNA-based Point-of-Care test development.

Although microscopic examination of stool samples remains the reference method for the diagnosis of intestinal protozoal infections, these techniques are time-consuming and require operators who are experienced and well trained. Molecular biology seems to offer performances at least equivalent in terms of sensitivity and specificity for certain parasites. This study aimed to compare three multiplex PCR assays on 93 prospectively collected positive stools (prospective cohort) and a panel of 12 more Cryptosporidium -positive samples ( Cryptosporidium panel). On the prospective cohort, the sensitivity was 89%, 64% and 41% for Giardia sp. detection for BD Max TM , G-DiaPara TM and RIDA ® GENE, respectively and 75%, 100% and 100% for C. parvum/hominis detection. The sensitivity of the RIDA ® GENE assay for all Cryptosporidium species was 100%, and for D. fragilis 71%. All the techniques obtained the same results for E. histolytica detection, with one positive sample. All species in the Cryptosporidium panel were identified by the RIDA ® GENE PCR. The BD Max TM and G-DiaPara TM assays detected only C. parvum/hominis with the exception of one positive sample for C. meleagridis . No assay showed satisfactory results for all parasites simultaneously, and the DNA extraction seems to be the critical step. More studies are needed to standardize this procedure. Bien que l’examen microscopique des selles reste la méthode de référence pour le diagnostic des protozooses intestinales, ces techniques sont chronophages et demandent une grande expérience et des opérateurs entrainés. La biologie moléculaire semble offrir des performances au moins équivalentes en termes de sensibilité comme de spécificité pour certains parasites. Cette étude visait à comparer trois techniques de PCR multiplex sur une cohorte de 93 selles positives collectées prospectivement et un panel de 12 échantillons positifs à Cryptosporidium . Respectivement pour BD Max TM , G-DiaPara TM et RIDA ® GENE la sensibilité était de 89 %, 64 % et 41 % pour la détection de Giardia sp. et 75 %, 100 % et 100 % pour la détection de C. parvum/hominis . La sensibilité de la technique RIDA ® GENE pour l’ensemble des espèces de Cryptosporidium était de 100 % et de 71 % pour D. fragilis . Toutes les techniques ont obtenu les mêmes résultats pour la détection d’ E. histolytica (1 échantillon positif). Toutes les espèces de Cryptosporidium ont été détectées par la PCR RIDA ® GENE. Les techniques BD Max TM et G-DiaPara TM ont détecté seulement C. parvum/hominis en dehors d’un échantillon positif à C. meleagridis . Aucun essai n’a montré de résultats satisfaisants pour l’ensemble des parasites simultanément et l’extraction d’ADN semble être l’étape critique. Plus d’études sont nécessaires afin de standardiser cette procédure.

Background Cryptosporidium parvum is a zoonotic pathogen worldwide. Extensive genetic diversity and complex population structures exist in C. parvum in different geographical regions and hosts. Unlike the IIa subtype family, which is responsible for most zoonotic C. parvum infections in industrialized countries, IId is identified as the dominant subtype family in farm animals, rodents and humans in China. Thus far, the population genetic characteristics of IId subtypes in calves in China are not clear. Methods In the present study, 46 C. parvum isolates from dairy and beef cattle in six provinces and regions in China were characterized using sequence analysis of eight genetic loci, including msc6-7 , rpgr , msc6-5 , dz-hrgp , chom3t , hsp70 , mucin1 and gp60 . They belonged to three IId subtypes in the gp60 gene, including IIdA20G1 ( n = 17), IIdA19G1 ( n = 24) and IIdA15G1 ( n = 5). The data generated were analyzed for population genetic structures of C. parvum using DnaSP and LIAN and subpopulation structures using STRUCTURE, RAxML, Arlequin, GENALEX and Network. Results Seventeen multilocus genotypes were identified. The results of linkage disequilibrium analysis indicated the presence of an epidemic genetic structure in the C. parvum IId population. When isolates of various geographical areas were treated as individual subpopulations, maximum likelihood inference of phylogeny, pairwise genetic distance analysis, substructure analysis, principal components analysis and network analysis all provided evidence for geographical segregation of subpopulations in Heilongjiang, Hebei and Xinjiang. In contrast, isolates from Guangdong, Shanghai and Jiangsu were genetically similar to each other. Conclusions Data from the multilocus analysis have revealed a much higher genetic diversity of C. parvum than gp60 sequence analysis. Despite an epidemic population structure, there is an apparent geographical segregation in C. parvum subpopulations within China.