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.

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.

Raw Goat's Milk, Fresh and Soft Cheeses as a Potential Source of Encephalitozoon cuniculi.

The aim of this work was to evaluate the effect of pasteurization and coagulation during goat cheese production on the infectivity of Encephalitozoon cuniculi spores for immunodeficient (SCID, CD4-/-, and CD8-/-) and immunocompetent (BALB/c and C57BL/6) mice. Goat milk and fecal samples were screened for the presence and quantity of microsporidial DNA using molecular methods. Experimentally produced cheese from E. cuniculi-enriched goat milk or goat cheese purchased from retail producers was fed with experimental mice susceptible to E. cuniculi infection. The mice were sacrificed in the presumed acute phase of infection and samples of their tissues were subject to molecular detection of specific E. cuniculi DNA. Specific DNA of E. cuniculi genotype II was detected in feces and milk of three out of 99 goats kept on 6 farms in the Czech Republic. Under experimental conditions, spores of E. cuniculi genotype II remained viable in artificially enriched fresh cheese and were able to cause infection in laboratory mice. E. cuniculi genotype I and II DNA were detected in eight of the nine goat cheeses purchased from various producers/breeders in the Czech Republic and these cheeses were able to develop infection in both immunodeficient and immunocompetent mice. The results of these experiments showed that spores of E. cuniculi genotype I and II are able to remain viable after cheese processing and thus fresh and soft cheeses should be considered a potential source of microsporidia.

Cryptosporidium myocastoris n. sp. (Apicomplexa: Cryptosporidiidae), the Species Adapted to the Nutria (Myocastor coypus).

Cryptosporidium spp., common parasites of vertebrates, remain poorly studied in wildlife. This study describes the novel Cryptosporidium species adapted to nutrias (Myocastor coypus). A total of 150 faecal samples of feral nutria were collected from locations in the Czech Republic and Slovakia and examined for Cryptosporidium spp. oocysts and specific DNA at the SSU, actin, HSP70, and gp60 loci. Molecular analyses revealed the presence of C. parvum (n = 1), C. ubiquitum subtype family XIId (n = 5) and Cryptosporidium myocastoris n. sp. XXIIa (n = 2), and XXIIb (n = 3). Only nutrias positive for C. myocastoris shed microscopically detectable oocysts, which measured 4.8-5.2 × 4.7-5.0 µm, and oocysts were infectious for experimentally infected nutrias with a prepatent period of 5-6 days, although not for mice, gerbils, or chickens. The infection was localised in jejunum and ileum without observable macroscopic changes. The microvilli adjacent to attached stages responded by elongating. Clinical signs were not observed in naturally or experimentally infected nutrias. Phylogenetic analyses at SSU, actin, and HSP70 loci demonstrated that C. myocastoris n. sp. is distinct from other valid Cryptosporidium species.

The course of infection of Encephalitozoon cuniculi genotype I in mice possess combination of features reported in genotypes II and III.

Out of three genotypes of Encephalitozoon cuniculi (I-III) available for experimental studies, E. cuniculi genotype I remains the less characterized. This study describes for the first time individual phases of microsporidiosis caused by E. cuniculi genotype I and efficacy of albendazole treatment in immunocompetent BALB/c and C57Bl/6 mice and immunodeficient SCID, CD4-/- and CD8-/- mice using molecular detection and quantification methods. We demonstrate asymptomatic infection despite an intense dissemination of microsporidia into most organs within the first weeks post infection, followed by a chronic infection characterized by significant microsporidia persistence in immunocompetent, CD4-/- and CD8-/- mice and a lethal outcome for SCID mice. Albendazole application led to loss E. cuniculi genotype I infection in immunocompetent mouse strains, decreased spore burden by half in CD4-/- and CD8-/- mice, and prolongation of survival of SCID mice. These results showed Encephalitozoon cuniculi genotype I infection extend and albendazole sensitivity was comparable to E. cuniculi genotype II, but the infection onset speed and mortality rate was similar to E. cuniculi genotype III. These imply that differences in the course of infection and the response to treatment depend not only on immunological status of the host, but also on the genotype causing the infection.

Encephalitozoon cuniculi Genotype II Concentrates in Inflammation Foci.

BACKGROUND: Microsporidia of the genus Encephalitozoon are generally connected with severe infections with lethal outcome in immunodeficient hosts. In immunocompetent hosts, microsporidiosis typically establishes a balanced host-parasite relationship that produces minimal clinically overt disease. Although the alimentary tract represents one of the main primary target tissues, the mechanisms of reaching other tissues during systemic microsporidian infections remain unclear. METHODS: In the present study, we tested the relation between inflammation induction in immunocompetent and immunodeficient mice and the presence of spores of E. cuniculi genotype II in selected organs and in fecal specimens by using molecular and histology methods. RESULTS: We reported the positive connection between inflammation induction and the significant increase of E. cuniculi genotype II occurrence in inflammation foci in both immunocompetent BALB/c and immunodeficient severe combined immunodeficient (SCID) mice in the acute phase of infection and the re-activation of latent microsporidial infection following inflammation induction in immunocompetent mice. CONCLUSION: The results imply possible involvement of immune cells serving as vehicles transporting E. cuniculi genotype II purposefully across the whole host body towards inflammation. With increasing number of records of infections, it is necessary to reconsider microsporidia as agents responsible for various pathologies. The elucidation of possible connection with pro-inflammatory immune responses represents an important challenge with consequences for human health and development of therapeutic strategies.

A massive systematic infection of Encephalitozoon cuniculi genotype III in mice does not cause clinical signs.

Encephalitozoon cuniculi genotype III disseminated intensively into most of the organs in all strains of mice, followed by a chronic infection with massive microsporidia persistence in immunodeficient mice and a partial decrease in C57Bl/6 mice. Treatment with 0.2 mg Albendazole/mouse/day temporarily reduces the number of affected organs in immunocompetent C57Bl/6 mice, but not in CD4-/- and CD8-/- mice. The application of medication temporarily decreased the spore burden at least by one order of magnitude in all groups. These results demonstrate that the E. cuniculi genotype III infection had a progressive course and surprisingly, Albendazole treatment had only a minimal effect. The E. cuniculi genotype III spore burden in individual organs reached up to 108 or 109 in immunocompetent or immunodeficient mice, respectively; however, these mice did not demonstrate any obvious clinical signs of microsporidiosis, and the immunodeficient mice survived longer. Our findings clearly show that the survival of mice does not correspond to spore burden, which provides new insight into latent microsporidiosis from an epidemiological point of view.

Encephalitozoon cuniculi Genotype III Evinces a Resistance to Albendazole Treatment in both Immunodeficient and Immunocompetent Mice.

Of four genotypes of Encephalitozoon cuniculi, E. cuniculi genotype II is considered to represent a parasite that occurs in many host species in a latent asymptomatic form, whereas E. cuniculi genotype III seems to be more aggressive, and infections caused by this strain can lead to the death of even immunocompetent hosts. Although albendazole has been considered suitable for treatment of Encephalitozoon species, its failure in control of E. cuniculi genotype III infection has been reported. This study determined the effect of a 100× recommended daily dose of albendazole on an Encephalitozoon cuniculi genotype III course of infection in immunocompetent and immunodeficient mice and compared the results with those from experiments performed with a lower dose of albendazole and E. cuniculi genotype II. The administration of the regular dose of abendazole during the acute phase of infection reduced the number of affected organs in all strains of mice and absolute counts of spores in screened organs. However, the effect on genotype III was minor. Surprisingly, no substantial effect was recorded after the use of a 100× dose of albendazole, with larger reductions seen only in the number of affected organs and absolute counts of spores in all strains of mice, implying variations in albendazole resistance between these Encephalitozoon cuniculi genotypes. These results imply that differences in the course of infection and the response to treatment depend not only on the immunological status of the host but also on the genotype causing the infection. Understanding how microsporidia survive in hosts despite targeted antimicrosporidial treatment could significantly contribute to research related to human health.

Cryptosporidium proventriculi sp. n. (Apicomplexa: Cryptosporidiidae) in Psittaciformes birds.

Cryptosporidiosis is a common parasitic infection in birds that is caused by more than 25 Cryptosporidium species and genotypes. Many of the genotypes that cause avian cryptosporidiosis are poorly characterized. The genetic and biological characteristics of avian genotype III are described here and these data support the establishment of a new species, Cryptosporidium proventriculi. Faecal samples from the orders Passeriformes and Psittaciformes were screened for the presence of Cryptosporidium by microscopy and sequencing, and infections were detected in 10 of 98 Passeriformes and in 27 of 402 Psittaciformes. Cryptosporidium baileyi was detected in both orders. Cryptosporidium galli and avian genotype I were found in Passeriformes, and C. avium and C. proventriculi were found in Psittaciformes. Cryptosporidium proventriculi was infectious for cockatiels under experimental conditions, with a prepatent period of six days post-infection (DPI), but not for budgerigars, chickens or SCID mice. Experimentally infected cockatiels shed oocysts more than 30 DPI, with an infection intensity ranging from 4,000 to 60,000 oocysts per gram (OPG). Naturally infected cockatiels shed oocysts with an infection intensity ranging from 2,000 to 30,000 OPG. Cryptosporidium proventriculi infects the proventriculus and ventriculus, and oocysts measure 7.4 × 5.8 µm. None of the birds infected C. proventriculi developed clinical signs.

Diversity of Cryptosporidium spp. in Apodemus spp. in Europe.

The genetic diversity of Cryptosporidium spp. in Apodemus spp. (striped field mouse, yellow-necked mouse and wood mouse) from 16 European countries was examined by PCR/sequencing of isolates from 437 animals. Overall, 13.7% (60/437) of animals were positive for Cryptosporidium by PCR. Phylogenetic analysis of small-subunit rRNA, Cryptosporidium oocyst wall protein and actin gene sequences showed the presence of Cryptosporidium ditrichi (22/60), Cryptosporidium apodemi (13/60), Cryptosporidium apodemus genotype I (8/60), Cryptosporidium apodemus genotype II (9/60), Cryptosporidium parvum (2/60), Cryptosporidium microti (2/60), Cryptosporidium muris (2/60) and Cryptosporidium tyzzeri (2/60). At the gp60 locus, novel gp60 families XVIIa and XVIIIa were identified in Cryptosporidium apodemus genotype I and II, respectively, subtype IIaA16G1R1b was identified in C. parvum, and subtypes IXaA8 and IXcA6 in C. tyzzeri. Only animals infected with C. ditrichi, C. apodemi, and Cryptosporidium apodemus genotypes shed oocysts that were detectable by microscopy, with the infection intensity ranging from 2000 to 52,000 oocysts per gram of faeces. None of the faecal samples was diarrheic in the time of the sampling.

Experimental Encephalitozoon cuniculi Infection Acquired from Fermented Meat Products.

This study describes the prevalence and concentration of Encephalitozoon cuniculi spores in pork meat and evaluates the effect of sausage fermentation on E. cuniculi infectivity for immunodeficient (severe combined immunodeficient) and immunocompetent (BALB/c and C57BL/6) mice. Using a nested polymerase chain reaction (PCR) approach, E. cuniculi genotype II was detected in the meat from 2 out of 50 pig carcasses at slaughter facilities, with 60-250 spores per gram detected by quantitative PCR. Under experimental conditions, 3000 E. cuniculi genotype II spores per gram of meat remained infective for mice following fermentation at 24°C for 48 h. Based on these findings, fermented meat products should be considered as a potential source of E. cuniculi infection in humans.

Diversity of Cryptosporidium in common voles and description of Cryptosporidium alticolis sp. n. and Cryptosporidium microti sp. n. (Apicomplexa: Cryptosporidiidae).

Fecal samples from wild-caught common voles (n = 328) from 16 locations in the Czech Republic were screened for Cryptosporidium by microscopy and PCR/sequencing at loci coding small-subunit rRNA, Cryptosporidium oocyst wall protein, actin and 70 kDa heat shock protein. Cryptosporidium infections were detected in 74 voles (22.6%). Rates of infection did not differ between males and females nor between juveniles and adults. Phylogenetic analysis revealed the presence of eight Cryptosporidium species/genotypes including two new species, C. alticolis and C. microti. These species from wild-caught common voles were able to infect common and meadow voles under experimental conditions, with a prepatent period of 3-5 days post-infection (DPI), but they were not infectious for various other rodents or chickens. Meadow voles lost infection earlier than common voles (11-14 vs 13-16 DPI) and had significantly lower infection intensity. Cryptosporidium alticolis infects the anterior small intestine and has larger oocysts (5.4 × 4.9 µm), whereas C. microti infects the large intestine and has smaller oocysts (4.3 × 4.1 µm). None of the rodents developed clinical signs of infection. Genetic and biological data support the establishment of C. alticolis and C. microti as separate species of the genus Cryptosporidium.

Evidence of transplacental transmission of Encephalitozoon cuniculi genotype II in murine model.

Microsporidia are obligate intracellurar unicellular parasite of wide range of vertebrates. Although ingestion or inhalation of microsporidian spores is the main route of infection, assumed vertical transmission was described in some mammals. The present study was focused on proof of vertical transmission in mice under experimental conditions. Mice were infected with E. cuniculi genotype II intraperitoneally after mating, or perorally followed by mating in acute or chronic phase of infection. Fetuses were delivered by Caesarean section or mice were kept up to the parturition. Some of cubs were immediately after birth transferred to uninfected surrogate mothers. Group of cubs was immunosuppressed. All cubs were examined using polymerase chain reaction for the presence of Encephalitozoon after birth or in their age of 3 or 6 weeks, respectively. All fetuses delivered by Caesarean section, which were intraperitoneally or perorally infected were negative as well as all neonatal mice and youngsters tested in age of 6 weeks. Only immunosuppressed cubs and cubs of immunodeficient mice in age of 21 days were positive for Encephalitozoon cuniculi genotype II. Present results provided the evidence that transplacental transmission of Encephalitozoon cuniculi in mice occurs, but the mechanism of these transport is still unknown.

Host specificity and age-dependent resistance to Cryptosporidium avium infection in chickens, ducks and pheasants.

Host- and age-specificity of Cryptosporidium avium were studied in 1-, 21- and 365-day-old chickens (Gallus gallus), domestic ducks (Anas platyrhynchos) and ring-necked pheasants (Phasianus colchicus) under experimental conditions. Cryptosporidium avium was not infectious for ring-necked pheasants, but it was infectious for ducks and chickens at all age categories. The course of infection in ducks did not differ among age categories, but 365-day-old chickens had less severe infections than 1- and 21-day-old chickens. The patent period in chickens and ducks was >30 DPI, but ducks started to shed oocysts of C. avium earlier (5-6 DPI) and at a lower intensity (accumulated value of infection intensity of 58,000-65,000 OPG) than chickens (9-11 DPI and accumulated value of infection intensity of 100,000-105,000 OPG). Experimentally infected birds showed no clinical signs of cryptosporidiosis.

Cryptosporidium occultus sp. n. (Apicomplexa: Cryptosporidiidae) in rats.

Cryptosporidium parvum VF383 has been reported in humans, domesticated ruminants, and wild rats worldwide and described under several names including Cryptosporidium suis-like, based on its close phylogenetic relationship to C. suis. Unlike C. suis, however, it has never been detected in pigs. In the present work, C. parvum VF383 originating from wild brown rats was not infectious for piglets or calves but was infectious for laboratory brown rats, BALB/c mice, and Mongolian gerbils. The prepatent period was 4-5 days for all rodents. The patent period was longer for rats (>30 days) than other rodents (<20 days). None of the rodents developed clinical signs of infection. In all rodents, life cycle stages were detected in the colon by histology and electron microscopy. Oocysts were morphometrically similar to those of C. parvum and smaller than those of C. suis, measuring 5.20 × 4.94 µm. Phylogenetic analyses of 18S rRNA, actin, and HSP70 gene sequences revealed C. parvum VF383 to be genetically distinct from, C. suis, and other described species of Cryptosporidium. Morphological, genetic, and biological data support the establishment of C. parvum VF383 as a new species, and we propose the name Cryptosporidium occultus sp. n.

Cryptosporidium apodemi sp. n. and Cryptosporidium ditrichi sp. n. (Apicomplexa: Cryptosporidiidae) in Apodemus spp.

Faecal samples from striped field mice (n = 72) and yellow-necked mice (n = 246) were screened for Cryptosporidium by microscopy and PCR/sequencing. Phylogenetic analysis of small-subunit rRNA, Cryptosporidium oocyst wall protein and actin gene sequences revealed the presence of C. parvum, C. hominis, C. muris and two new species, C. apodemi and C. ditrichi. Oocysts of C. apodemi are smaller than C. ditrichi and both are experimentally infectious for yellow-necked mice but not for common voles. Additionally, infection by C. ditrichi was established in one of three BALB/c mice. The prepatent period was 7-9 and 5-6 days post infection for C. apodemi and C. ditrichi, respectively. The patent period was greater than 30 days for both species. Infection intensity of C. ditrichi ranged from 4000-50,000 oocyst per gram of faeces and developmental stages of C. ditrichi were detected in the jejunum and ileum. In contrast, neither oocysts nor endogenous developmental stages of C. apodemi were detected in faecal or tissue samples, although C. apodemi DNA was detected in contents from the small and large intestine. Morphological, genetic, and biological data support the establishment of C. apodemi and C. ditrichi as a separate species of the genus Cryptosporidium.

Stray cats are more frequently infected with zoonotic protists than pet cats.

Faecal samples were collected from cats kept as pets (n = 120) and stray cats (n = 135) in Central Europe (Czech Republic, Poland and Slovakia) and screened for the presence of Cryptosporidium spp., Giardia intestinalis (Kunstler, 1882), Encephalitozoon spp. and Enterocytozoon bieneusi Desportes, Le Charpentier, Galian, Bernard, Cochand-Priollet, Lavergne, Ravisse et Modigliani, 1985 by PCR analysis of the small-subunit of rRNA (Cryptosporidium spp. and G. intestinalis) and ITS (microsporidia) genes. Sequence analysis of targeted genes revealed the presence of C. felis Iseki, 1979, G. intestinalis assemblage F, E. cuniculi Levaditi, Nicolau et Schoen, 1923 genotype II, and E. bieneusi genotype D. There was no correlation between the occurrence of detected parasites and sex, presence of diarrhoea or drug treatment (drug containing pyrantel and praziquantel). Compared to pet cats (7%), stray cats (30%) were statistically more frequently infected with protist parasites and overall may present a greater risk to human health.

The course of infection caused by Encephalitozoon cuniculi genotype III in immunocompetent and immunodeficient mice.

Encephalitozoon cuniculi is probably the most common microsporidia which infects a wide range of vertebrates, including human. So far, four genotypes of this parasite have been identified based on the rRNA internal transcribed spacer variations. The course of infection caused by E. cuniculi III had very massive onset in immunocompetent host characterized by the presence of this parasite in all organs and tissues within one week after peroral infection. Encephalitozoonosis caused by E. cuniculi III had very progressive spreading into all organs within first week post inoculation in immunocompromised SCID mice and led to the death of the host. The experimental treatment with albendazole of immunocompetent BALB/c mice infected with E. cuniculi III have shown very weak effect. Our findings clearly showed that the different course of infection and response to treatment depends not only on the immunological status of the host, but also on the genotype of microsporidia. It could be very important especially for individuals under chemotherapy and transplant recipients of organs originating from infected donors.

Effects of selected Indonesian plant extracts on E. cuniculi infection in vivo.

The present study was conducted to evaluate the methanolic extracts from several plant leaves widely used in traditional medicine to cure digestive tract disorders and in the self-medication of wild animals such as non-human primates, namely Archidendron fagifolium, Diospyros sumatrana, Shorea sumatrana, and Piper betle leaves, with regard to their antimicrosporidial activity against Encephalitozoon cuniculi in immunocompetent BALB/c mice determined using molecular detection of microsporidial DNA (qPCR) in various tissues and body fluids of infected, treated mice. Of the plant extracts tested, Diospyros sumatrana provided the most promising results, reducing spore shedding by 88% compared to untreated controls. Moreover, total burden per 1 g of tissue in the D. sumatrana extract-treated group reached 87% reduction compared to untreated controls, which was comparable to the effect of the standard drug, Albendazole. This data represents the baseline necessary for further research focused on determining the structure, activity and modes of action of the active compounds, mainly of D. sumatrana, enabling subsequent development of antimicrosporidial remedies.

Cryptosporidium testudinis sp. n., Cryptosporidium ducismarci Traversa, 2010 and Cryptosporidium tortoise genotype III (Apicomplexa: Cryptosporidiidae) in tortoises.

Understanding of the diversity of species of Cryptosporidium Tyzzer, 1910 in tortoises remains incomplete due to the limited number of studies on these hosts. The aim of the present study was to characterise the genetic diversity and biology of cryptosporidia in tortoises of the family Testudinidae Batsch. Faecal samples were individually collected immediately after defecation and were screened for presence of cryptosporidia by microscopy using aniline-carbol-methyl violet staining, and by PCR amplification and sequence analysis targeting the small subunit rRNA (SSU), Cryptosporidium oocyst wall protein (COWP) and actin genes. Out of 387 faecal samples from 16 tortoise species belonging to 11 genera, 10 and 46 were positive for cryptosporidia by microscopy and PCR, respectively. All samples positive by microscopy were also PCR positive. Sequence analysis of amplified genes revealed the presence of the Cryptosporidium tortoise genotype I (n = 22), C. ducismarci Traversa, 2010 (n = 23) and tortoise genotype III (n = 1). Phylogenetic analyses of SSU, COWP and actin gene sequences revealed that Cryptosporidium tortoise genotype I and C. ducismarci are genetically distinct from previously described species of Cryptosporidium. Oocysts of Cryptosporidium tortoise genotype I, measuring 5.8-6.9 µm × 5.3-6.5 µm, are morphologically distinguishable from C. ducismarci, measuring 4.4-5.4 µm × 4.3-5.3 µm. Oocysts of Cryptosporidium tortoise genotype I and C. ducismarci obtained from naturally infected Russian tortoises (Testudo horsfieldii Gray) were infectious for the same tortoise but not for Reeve's turtles (Mauremys reevesii [Gray]), common garter snake (Thamnophis sirtalis [Linnaeus]), zebra finches (Taeniopygia guttata [Vieillot]) and SCID mice (Mus musculus Linnaeus). The prepatent period was 11 and 6 days post infection (DPI) for Cryptosporidium tortoise genotype I and C. ducismarci, respectively; the patent period was longer than 200 days for both cryptosporidia. Naturally or experimentally infected tortoises showed no clinical signs of disease. Our morphological, genetic, and biological data support the establishment of Cryptosporidium tortoise genotype I as a new species, Cryptosporidium testudinis sp. n., and confirm the validity of C. ducismarci as a separate species of the genus Cryptosporidium.