Genetic diversity of Cryptosporidium spp., Encephalitozoon spp. and Enterocytozoon bieneusi in feral and captive pigeons in Central Europe.

Cryptosporidium spp., Enterocytozoon bieneusi and Encephalitozoon spp. are the most common protistan parasites of vertebrates. The results show that pigeon populations in Central Europe are parasitised by different species of Cryptosporidium and genotypes of microsporidia of the genera Enterocytozoon and Encephalitozoon. A total of 634 and 306 faecal samples of captive and feral pigeons (Columba livia f. domestica) from 44 locations in the Czech Republic, Slovakia and Poland were analysed for the presence of parasites by microscopy and PCR/sequence analysis of small subunit ribosomal RNA (18S rDNA), 60 kDa glycoprotein (gp60) and internal transcribed spacer (ITS) of SSU rDNA. Phylogenetic analyses revealed the presence of C. meleagridis, C. baileyi, C. parvum, C. andersoni, C. muris, C. galli and C. ornithophilus, E. hellem genotype 1A and 2B, E. cuniculi genotype I and II and E. bieneusi genotype Peru 6, CHN-F1, D, Peru 8, Type IV, ZY37, E, CHN4, SCF2 and WR4. Captive pigeons were significantly more frequently parasitised with screened parasite than feral pigeons. Cryptosporidium meleagridis IIIa and a new subtype IIIl have been described, the oocysts of which are not infectious to immunodeficient mice, whereas chickens are susceptible. This investigation demonstrates that pigeons can be hosts to numerous species, genotypes and subtypes of the studied parasites. Consequently, they represent a potential source of infection for both livestock and humans.

Host-specific Cryptosporidium, Giardia and Enterocytozoon bieneusi in shelter dogs from central Europe.

Cryptosporidium spp., Giardia intestinalis and microsporidia are unicellular opportunistic pathogens that can cause gastrointestinal infections in both animals and humans. Since companion animals may serve as a source of infection, the aim of the present screening study was to analyse the prevalence of these intestinal protists in fecal samples collected from dogs living in 10 animal shelters in central Europe (101 dogs from Poland and 86 from the Czech Republic), combined with molecular subtyping of the detected organisms in order to assess their genetic diversity. Genus-specific polymerase chain reactions were performed to detect DNA of the tested species and to conduct molecular subtyping in collected samples, followed by statistical evaluation of the data obtained (using χ2 or Fisher's tests). The observed prevalence was 15.5, 10.2, 1 and 1% for G. intestinalis, Enterocytozoon bieneusi, Cryptosporidium spp. and Encephalitozoon cuniculi, respectively. Molecular evaluation has revealed the predominance of dog-specific genotypes (Cryptosporidium canis XXe1 subtype; G. intestinalis assemblages C and D; E. cuniculi genotype II; E. bieneusi genotypes D and PtEbIX), suggesting that shelter dogs do not pose a high risk of human transmission. Interestingly, the percentage distribution of the detected pathogens differed between both countries and individual shelters, suggesting that the risk of infection may be associated with conditions typical of a given location.

Microsporidia (Encephalitozoon cuniculi) in Patients with Degenerative Hip and Knee Disease, Czech Republic.

Total joint arthroplasty is a commonly used surgical procedure in orthopedics. Revision surgeries are required in >10% of patients mainly because of prosthetic joint infection caused by bacteria or aseptic implant loosening caused by chronic inflammation. Encephalitozoon cuniculi is a microsporidium, an obligate intracellular parasite, capable of exploiting migrating proinflammatory immune cells for dissemination within the host. We used molecular detection methods to evaluate the incidence of E. cuniculi among patients who had total hip or knee arthroplasty revision. Out of 49 patients, E. cuniculi genotypes I, II, or III were confirmed in joint samples from 3 men and 2 women who had implant loosening. Understanding the risks associated with the presence of microsporidia in periprosthetic joint infections is essential for proper management of arthroplasty. Furthermore, E. cuniculi should be considered a potential contributing cause of joint inflammation and arthrosis.

The effect of host admixture on wild house mouse gut microbiota is weak when accounting for spatial autocorrelation.

The question of how interactions between the gut microbiome and vertebrate hosts contribute to host adaptation and speciation is one of the major problems in current evolutionary research. Using bacteriome and mycobiome metabarcoding, we examined how these two components of the gut microbiota vary with the degree of host admixture in secondary contact between two house mouse subspecies (Mus musculus musculus and M. m. domesticus). We used a large data set collected at two replicates of the hybrid zone and model-based statistical analyses to ensure the robustness of our results. Assuming that the microbiota of wild hosts suffers from spatial autocorrelation, we directly compared the results of statistical models that were spatially naive with those that accounted for spatial autocorrelation. We showed that neglecting spatial autocorrelation can strongly affect the results and lead to misleading conclusions. The spatial analyses showed little difference between subspecies, both in microbiome composition and in individual bacterial lineages. Similarly, the degree of admixture had minimal effects on the gut bacteriome and mycobiome and was caused by changes in a few microbial lineages that correspond to the common symbionts of free-living house mice. In contrast to previous studies, these data do not support the hypothesis that the microbiota plays an important role in host reproductive isolation in this particular model system.

Dietary environmental factors shape the immune defense against Cryptosporidium infection.

Cryptosporidium is a leading cause of diarrheal-related deaths in children, especially in resource-poor settings. It also targets the immunocompromised, chronically infecting people living with HIV and primary immunodeficiencies. There is no vaccine or effective treatment. Although it is known from human cases and animal models that CD4+ T cells play a role in curbing Cryptosporidium, the role of CD8+ T cells remains to be defined. Using a Cryptosporidium tyzzeri mouse model, we show that gut-resident CD8+ intraepithelial lymphocytes (IELs) confer resistance to parasite growth. CD8+ IELs express and depend on the ligand-dependent transcription factor aryl hydrocarbon receptor (AHR). AHR deficiency reduces CD8+ IELs, decreases their cytotoxicity, and worsens infection. Transfer of CD8+ IELs rescues severely immunodeficient mice from death following Cryptosporidium challenge. Finally, dietary supplementation of the AHR pro-ligand indole-3-carbinol in newborn mice promotes resistance to infection. Therefore, common dietary metabolites augment the host immune response to cryptosporidiosis, protecting against disease.

European ground squirrels Spermophilus citellus (Linnaeus) do not share identical Cryptosporidium spp. with North American ground squirrels.

Cryptosporidium Tyzzer, 1910 is one of the most common protistan parasites of vertebrates. The results of this study provide the first data on Cryptosporidium diversity in the European ground squirrel Spermophilus citellus (Linnaeus). A total of 128 faecal samples of European ground squirrels from 39 localities in the Czech Republic were analysed for the presence of Cryptosporidium spp. by microscopy and PCR/sequence analysis of small subunit ribosomal RNA (SSU) and the actin gene. While the microscopical examination did not reveal the presence of any Cryptosporidium oocysts, eight samples from six localities were PCR-positive. Phylogenetic analyses revealed the presence of five different Cryptosporidium spp. isolates. Four isolates, designated as Cryptosporidium sp. isolate Sc01-04, detected in wild populations and never recorded before, clustered closely to Cryptosporidium genotypes that have previously been found in North American ground squirrels' species. Cryptosporidium sciurinum Prediger, Jezková, Holubová, Sak, Konecný, Rost, McEvoy, Rajský et Kvác, 2021 was found in an animal sanctuary. Because C. sciurinum had previously been detected in Eurasian red squirrels Sciurus vulgaris Linnaeus at the same facility, it can be concluded that this Cryptosporidium was transmitted from tree squirrels to ground squirrels within the animal sanctuary. The results indicate that populations of European and North American ground squirrels are parasitised by different Cryptosporidium spp. At the same time, this is the first description of the occurrence of C. sciurinum in ground squirrels.

Cryptosporidium mortiferum n. sp. (Apicomplexa: Cryptosporidiidae), the species causing lethal cryptosporidiosis in Eurasian red squirrels (Sciurus vulgaris).

BACKGROUND: Cryptosporidium spp. are globally distributed parasites that infect epithelial cells in the microvillus border of the gastrointestinal tract of all classes of vertebrates. Cryptosporidium chipmunk genotype I is a common parasite in North American tree squirrels. It was introduced into Europe with eastern gray squirrels and poses an infection risk to native European squirrel species, for which infection is fatal. In this study, the biology and genetic variability of different isolates of chipmunk genotype I were investigated. METHODS: The genetic diversity of Cryptosporidium chipmunk genotype I was analyzed by PCR/sequencing of the SSU rRNA, actin, HSP70, COWP, TRAP-C1 and gp60 genes. The biology of chipmunk genotype I, including oocyst size, localization of the life cycle stages and pathology, was examined by light and electron microscopy and histology. Infectivity to Eurasian red squirrels and eastern gray squirrels was verified experimentally. RESULTS: Phylogenic analyses at studied genes revealed that chipmunk genotype I is genetically distinct from other Cryptosporidium spp. No detectable infection occurred in chickens and guinea pigs experimentally inoculated with chipmunk genotype I, while in laboratory mice, ferrets, gerbils, Eurasian red squirrels and eastern gray squirrels, oocyst shedding began between 4 and 11 days post infection. While infection in mice, gerbils, ferrets and eastern gray squirrels was asymptomatic or had mild clinical signs, Eurasian red squirrels developed severe cryptosporidiosis that resulted in host death. The rapid onset of clinical signs characterized by severe diarrhea, apathy, loss of appetite and subsequent death of the individual may explain the sporadic occurrence of this Cryptosporidium in field studies and its concurrent spread in the population of native European squirrels. Oocysts obtained from a naturally infected human, the original inoculum, were 5.64 × 5.37 µm and did not differ in size from oocysts obtained from experimentally infected hosts. Cryptosporidium chipmunk genotype I infection was localized exclusively in the cecum and anterior part of the colon. CONCLUSIONS: Based on these differences in genetics, host specificity and pathogenicity, we propose the name Cryptosporidium mortiferum n. sp. for this parasite previously known as Cryptosporidium chipmunk genotype I.

Cryptosporidium equi n. sp. (Apicomplexa: Cryptosporidiidae): Biological and genetic characterisations.

The horse genotype is one of three common Cryptosporidium spp. in equine animals and has been identified in some human cases. The species status of Cryptosporidium horse genotype remains unclear due to the lack of extensive morphological, biological, and genetic data. In the present study, we have conducted biological and whole genome sequence analyses of an isolate of the genotype from hedgehogs and proposed to name it Cryptosporidium equi n. sp. to reflect its common occurrence in equine animals. Oocysts of C. equi measured 5.12 ± 0.36 µm × 4.46 ± 0.21 µm with a shape index of 1.15 ± 0.08 (n = 50). Cryptosporidium equi was infectious to 3-week-old four-toed hedgehogs (Atelerix albiventris) and mice, with a prepatent period of 2-9 days and a patent period of 30-40 days in hedgehogs. It was not infectious to rats and rabbits. Phylogenetic analyses of small subunit rRNA, 70 kDa heat shock protein, actin, 60 kDa glycoprotein and 100 other orthologous genes revealed that C. equi is genetically distinct from other known Cryptosporidium species and genotypes. The sequence identity between C. equi and Cryptosporidium parvum genomes is 97.9%. Compared with C. parvum, C. equi has lost two MEDLE genes and one insulinase-like protease gene and gained one SKSR gene. In addition, 60 genes have highly divergent sequences (sequence differences ≥ 5.0%), including those encoding mucin-like glycoproteins, insulinase-like peptidases, and MEDLE and SKSR proteins. The genetic uniqueness of C. equi supports its increasing host range and the naming of it as a valid Cryptosporidium species. This is the first known use of whole genome sequence data in delineating new Cryptosporidium species.

Follow-up investigation into Cryptosporidium prevalence and transmission in Western European dairy farms.

Cryptosporidium parvum is an enteric parasite and a major contributor to acute enteritis in calves worldwide, causing an important economic burden for farmers. This parasite poses a major public health threat through transmission between livestock and humans. Our previous pilot study in Western Europe revealed a high prevalence of Cryptosporidium in calves of dairy farms. In the sequel study herein, 936 faecal samples were collected from the same 51 dairy farms across Belgium, France, and the Netherlands. Following DNA extraction, Cryptosporidium screening was carried out using nested-PCR amplification targeting the SSU rRNA gene. All positive samples were sequenced, and phylogenetic analyses were used to identify the Cryptosporidium spp. present. The 60 kDa glycoprotein (gp60) gene was also sequenced to determine the C. parvum subtypes present. Prevalence of Cryptosporidium ranged from 23.3% to 25%, across the three countries surveyed. The parasite was found in most of the farms sampled, with 90.2% testing positive. Cryptosporidium parvum, C. bovis, C. ryanae and C. andersoni were all identified, with the former being the most predominant, representing 71.4% of all infections. Cryptosporidium parvum was associated with pre-weaned calves, while other species were associated with older animals. Subtyping of gp60 gene revealed nine subtypes, eight of which have previously been reported to cause clinical disease in humans. Similarly to the first study, vertical transmission was not a major contributor to Cryptosporidium spread. Our study highlights the need for further investigation into cryptosporidiosis transmission, and future studies will require a One Health approach to reduce the impact of this disease.

Calicophoron daubneyi (Dinnik, 1962) (Digenea) in beef and dairy cattle in the Czech Republic: prevalence and drug efficacy.

A total of 1,724 beef and 2,941 dairy cattle older than one year from 66 beef and 67 dairy farms in the Czech Republic were examined for the presence of rumen and liver fluke eggs in 2019-2022. Out of 227 positive animals, all were positive for paramphistome and five for fasciolid eggs. Molecular analysis of the ITS2 rDNA revealed the presence of Calicophoron daubneyi (Dinnik, 1962) and Fasciola hepatica Linnaeus, 1758. Faecal egg count (FEC) showed low infection intensity (12 EPG) in animals infected with F. hepatica and high variability in C. daubneyi infections (2-589 EPG). Efficacy of oxyclozanide, albendazole, ivermectin, and closantel against C. daubneyi infection was evaluated at eight beef cattle herds. Faecal samples were collected from all positive animals at 0 and 21days post-treatment. Based on FEC, albendazole, ivermectin and closantel reduced the number of C. daubneyi eggs shed by 0-9.9%, with no effect on the number of infected animals. The use of oxyclozanide on two beef farms showed 100% efficacy against C. daubneyi and F. hepatica. Follow-up examination 5-6 months after drug application showed reinfection of most animals with C. daubneyi, but the FEC was significantly lower. The finding of four dairy cows infected with C. daubneyi housed in a stable without pasture suggests the possibility of the infection being introduced through roughage.

Sympatric Recombination in Zoonotic Cryptosporidium Leads to Emergence of Populations with Modified Host Preference.

Genetic recombination plays a critical role in the emergence of pathogens with phenotypes such as drug resistance, virulence, and host adaptation. Here, we tested the hypothesis that recombination between sympatric ancestral populations leads to the emergence of divergent variants of the zoonotic parasite Cryptosporidium parvum with modified host ranges. Comparative genomic analyses of 101 isolates have identified seven subpopulations isolated by distance. They appear to be descendants of two ancestral populations, IIa in northwestern Europe and IId from southwestern Asia. Sympatric recombination in areas with both ancestral subtypes and subsequent selective sweeps have led to the emergence of new subpopulations with mosaic genomes and modified host preference. Subtelomeric genes could be involved in the adaptive selection of subpopulations, while copy number variations of genes encoding invasion-associated proteins are potentially associated with modified host ranges. These observations reveal ancestral origins of zoonotic C. parvum and suggest that pathogen import through modern animal farming might promote the emergence of divergent subpopulations of C. parvum with modified host preference.

Comparison of the Concentration of Encephalitozoon cuniculi Genotypes I and III in Inflammatory Foci Under Experimental Conditions.

Background: Microsporidia of the genus Encephalitozoon are usually associated with severe infections in immunodeficient hosts while, in immunocompetent ones, microsporidiosis produces minimal clinically apparent disease. Despite their microscopic size, microsporidia are capable of causing systemic infection within a few days. However, the mechanisms by which microsporidia reach target tissues during acute infection remain unclear. Out of four genotypes of Encephalitozoon cuniculi, only three are available for experimental studies, with E. cuniculi genotype II being the best characterized. Methods: In the present study, we tested the association between inflammation induction in immunocompetent and immunodeficient mice and the presence of spores of E. cuniculi genotypes I and III in selected organs using molecular methods and compared the results with previously published data on E. cuniculi genotype II. Results: We reported the positive connection between inflammation induction and the significant increase of E. cuniculi genotypes I and III occurrence in inflammatory foci in both immunocompetent BALB/c and immunodeficient severe combined immunodeficient (SCID) mice in the acute phase of infection. The induction of inflammation resulted in increased concentration of E. cuniculi of both genotypes in the site of inflammation, as previously reported for E. cuniculi genotype II. Moreover, our study extended the spectrum of differences among E. cuniculi genotypes by the variations in dispersal rate within host bodies after experimentally induced inflammation. Conclusion: The results imply possible involvement of immune cells serving as vehicles transporting E. cuniculi towards inflammation foci. The elucidation of possible connection with pro-inflammatory immune responses represents an important challenge with implications for human health and the development of therapeutic strategies.

Chronic Infections in Mammals Due to Microsporidia.

Microsporidia are pathogenic organism related to fungi. They cause infections in a wide variety of mammals as well as in avian, amphibian, and reptilian hosts. Many microsporidia species play an important role in the development of serious diseases that have significant implications in human and veterinary medicine. While microsporidia were originally considered to be opportunistic pathogens in humans, it is now understood that infections also occur in immune competent humans. Encephalitozoon cuniculi, Encephalitozoon intestinalis, and Enterocytozoon bieneusi are primarily mammalian pathogens. However, many other species of microsporidia that have some other primary host that is not a mammal have been reported to cause sporadic mammalian infections. Experimental models and observations in natural infections have demonstrated that microsporidia can cause a latent infection in mammalian hosts. This chapter reviews the published studies on mammalian microsporidiosis and the data on chronic infections due to these enigmatic pathogens.

Horse-Specific Cryptosporidium Genotype in Human with Crohn's Disease and Arthritis.

We identified an unusual subtype of a Cryptosporidium sp. horse genotype as the cause of cryptosporidiosis in a 13-year-old girl in Poland who was undergoing immunosuppressive treatment for juvenile rheumatoid arthritis and Crohn's disease. The same subtype was identified in a horse the girl had ridden.

High Occurrence of Zoonotic Subtypes of Cryptosporidiumparvum in Cypriot Dairy Farms.

Cryptosporidium parvum is one of the major causes of neonatal calf diarrhoea resulting in reduced farm productivity and compromised animal welfare worldwide. Livestock act as a major reservoir of this parasite, which can be transmitted to humans directly and/or indirectly, posing a public health risk. Research reports on the prevalence of Cryptosporidium in ruminants from east Mediterranean countries, including Cyprus, are limited. This study is the first to explore the occurrence of Cryptosporidium spp. in cattle up to 24 months old on the island of Cyprus. A total of 242 faecal samples were collected from 10 dairy cattle farms in Cyprus, all of which were screened for Cryptosporidium spp. using nested-PCR amplification targeting the small subunit of the ribosomal RNA (18S rRNA) gene. The 60 kDa glycoprotein (gp60) gene was also sequenced for the samples identified as Cryptosporidium parvum-positive to determine the subtypes present. The occurrence of Cryptosporidium was 43.8% (106/242) with at least one positive isolate in each farm sampled. Cryptosporidium bovis, Cryptosporidium ryanae and C. parvum were the only species identified, while the prevalence per farm ranged from 20-64%. Amongst these, the latter was the predominant species, representing 51.8% of all positive samples, followed by C. bovis (21.7%) and C. ryanae (31.1%). Five C. parvum subtypes were identified, four of which are zoonotic-IIaA14G1R1, IIaA15G1R1, IIaA15G2R1 and IIaA18G2R1. IIaA14G1R1 was the most abundant, representing 48.2% of all C. parvum positive samples, and was also the most widespread. This is the first report of zoonotic subtypes of C. parvum circulating in Cyprus. These results highlight the need for further research into the parasite focusing on its diversity, prevalence, host range and transmission dynamics on the island.

Encephalitozoon cuniculi and Extraintestinal Microsporidiosis in Bird Owners.

We identified Encephalitozoon cuniculi genotype II parasites as a cause of extraintestinal microsporidiosis in 2 owners of birds also infected with E. cuniculi. Patients experienced long-lasting nonspecific symptoms; the disease course was more progressive in a patient with diabetes. Our findings suggest direct bird-to-human transmission of this pathogen.

A productive immunocompetent mouse model of cryptosporidiosis with long oocyst shedding duration for immunological studies.

OBJECTIVES: Studies on the pathogenesis and immune responses of Cryptosporidium infection and development of drugs and vaccines use mostly immunocompromised mouse models. In this study, we establish an immunocompetent mouse model of cryptosporidiosis with high intensity and long duration of infection. METHODS: We have obtained a Cryptosporidium tyzzeri isolate from laboratory mice, and infect adult C57BL/6 J mice experimentally with the isolate for determinations of infectivity, infection patterns, pathological changes, and transcriptomic responses. RESULTS: The isolate has an ID50 of 5.2 oocysts, with oocyst shedding lasting at high levels for >2 months. The oocyst shedding is boosted by immunosuppression of animals and suppressed by paromomycin treatment. The isolate induces strong inflammatory and acquired immune responses, but down-regulates the expression of α-defensins in epithelium. Comparative genomics analysis has revealed significant sequence differences from other isolates in subtelomeric genes. The down-regulation of the expression of α-defensins may be responsible for the high-intensity and long-lasting infection in this animal model. CONCLUSIONS: The immunocompetent mouse model of cryptosporidiosis developed has the advantages of high oocyst shedding intensity and long oocyst shedding duration. It provides an effective mechanism for the propagation of Cryptosporidium, evaluations of potential therapeutics, and studies of pathogen biology and immune responses.

Cross-Border Investigations on the Prevalence and Transmission Dynamics of Cryptosporidium Species in Dairy Cattle Farms in Western Mainland Europe.

Cryptosporidium is an apicomplexan parasitic protist, which infects a wide range of hosts, causing cryptosporidiosis disease. In farms, the incidence of this disease is high in animals such as cows, leading to extensive economic loss in the livestock industry. Infected cows may also act as a major reservoir of Cryptosporidium spp., in particular C. parvum, the most common cause of cryptosporidiosis in these animals. This poses a risk to the trading of livestock, to other farms via breeding centres, and to human health. This study is a part of a global project aimed at strategies to tackle cryptosporidiosis. To reach this target, it was essential to determine whether prevalence was dependent on the studied countries or if the issue was borderless. Indeed, C. parvum occurrence was assessed across dairy farms in certain regions of Belgium, France, and the Netherlands. At the same time, the animal-to-animal transmission of the circulating C. parvum subtypes was studied. To accomplish this, we analysed 1084 faecal samples, corresponding to 57 dairy farms from all three countries. To this end, 18S rRNA and gp60 genes fragments were amplified, followed by DNA sequencing, which was subsequently used for detection and subtyping C. parvum. Bioinformatic and phylogenetic methods were integrated to analyse and characterise the obtained DNA sequences. Our results show 25.7%, 24.9% and 20.8% prevalence of Cryptosporidium spp. in Belgium, France, and the Netherlands respectively. Overall, 93% of the farms were Cryptosporidium positive. The gp60 subtyping demonstrated a significant number of the C. parvum positives belonged to the IIa allelic family, which has been also identified in humans. Therefore, this study highlights how prevalent C. parvum is in dairy farms and further suggests cattle as a possible carrier of zoonotic C. parvum subtypes, which could pose a threat to human health.

Enterocytozoon Bieneusi Infects Children With Inflammatory Bowel Disease Undergoing Immunosuppressive Treatment.

Objectives: Patients with inflammatory bowel disease (IBD) are susceptible to intestinal opportunistic infections due to both defective mucosal immunity and altered immune response resulting from immunosuppressive treatment. Microsporidia infecting the gastrointestinal tract and causing diarrhoea can potentially affect the course of IBD. Methods: Stool samples (90 IBD children and 121 healthy age-matched controls) were screened for Encephalitozoon spp. and Enterocytozoon bieneusi by microscopy and polymerase chain reaction followed by sequencing. Results: E. bieneusi genotype D was found in seven out of 90 (7.8%) IBD children. No children from the control group were infected, making the pathogen prevalence in the IBD group significant (P = 0.002). Furthermore, infection was confirmed only in patients receiving immunosuppressive treatment (P = 0.013). Conclusions: Children with IBD are at risk of intestinal E. bieneusi infection, especially when receiving immunosuppressive treatment. Therefore, microsporidia should be considered as a significant infectious agent in this group of patients.

Cryptosporidium sciurinum n. sp. (Apicomplexa: Cryptosporidiidae) in Eurasian Red Squirrels (Sciurus vulgaris).

Cryptosporidium spp. are common protozoan pathogens in mammals. The diversity and biology of Cryptosporidium in tree squirrels are not well studied. A total of 258 Eurasian red squirrels (Sciurus vulgaris) from 25 and 15 locations in the Czech Republic and Slovakia, respectively, were examined for Cryptosporidium spp. oocysts and specific DNA at the SSU, actin, HSP70, TRAP-C1, COWP, and gp60 loci. Out of 26 positive animals, only juveniles (9/12) were microscopically positive (18,000 to 72,000 OPG), and molecular analyses revealed the presence of Cryptosporidium sp. ferret genotype in all specimens. Oocysts obtained from naturally-infected squirrels measured 5.54-5.22 µm and were not infectious for laboratory mice (BALB/c and SCID), Mongolian gerbils, Guinea pigs, Southern multimammate mice, chickens, or budgerigars. None of naturally infected squirrels showed clinical signs of disease. The frequency of occurrence of the ferret genotype in squirrels did not vary statistically based on host age, gender or country of capture. Phylogenetic analysis of sequences from six loci revealed that Cryptosporidium sp. ferret genotype is genetically distinct from the currently accepted Cryptosporidium species. Morphological and biological data from this and previous studies support the establishment of Cryptosporidium sp. ferret genotype as a new species, Cryptosporidium sciurinum n. sp.