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.

• Keywords
• Cryptosporidium sp. ferret genotype, biology, course of infection, infectivity, occurrence, oocyst size, phylogeny,
• Publication type
• Journal Article MeSH

BACKGROUND: Avian cryptosporidiosis is a common parasitic disease that is caused by five species, which are well characterised at the molecular and biological level, and more than 18 genotypes for which we have limited information. In this study, we determined the occurrence and molecular characteristics of Cryptosporidium spp. in farmed ostriches in the Czech Republic. METHODS: The occurrence and genetic identity of Cryptosporidium spp. were analysed by microscopy and PCR/sequencing of the small subunit rRNA, actin, HSP70 and gp60 genes. Cryptosporidium avian genotype II was examined from naturally and experimentally infected hosts and measured using differential interference contrast. The localisation of the life-cycle stages was studied by electron microscopy and histologically. Infectivity of Cryptosporidium avian genotype II for cockatiels (Nymphicus hollandicus (Kerr)), chickens (Gallus gallus f. domestica (L.)), geese (Anser anser f. domestica (L.)), SCID and BALB/c mice (Mus musculus L.) was verified. RESULTS: A total of 204 individual faecal samples were examined for Cryptosporidium spp. using differential staining and PCR/sequencing. Phylogenetic analysis of small subunit rRNA, actin, HSP70 and gp60 gene sequences showed the presence of Cryptosporidium avian genotype II (n = 7) and C. ubiquitum Fayer, Santín & Macarisin, 2010 IXa (n = 5). Only ostriches infected with Cryptosporidium avian genotype II shed oocysts that were detectable by microscopy. Oocysts were purified from a pooled sample of four birds, characterised morphometrically and used in experimental infections to determine biological characteristics. Oocysts of Cryptosporidium avian genotype II measure on average 6.13 × 5.15 μm, and are indistinguishable by size from C. baileyi Current, Upton & Haynes, 1986 and C. avium Holubová, Sak, Horčičková, Hlásková, Květoňová, Menchaca, McEvoy & Kváč, 2016. Cryptosporidium avian genotype II was experimentally infectious for geese, chickens and cockatiels, with a prepatent period of four, seven and eight days post-infection, respectively. The infection intensity ranged from 1000 to 16,000 oocysts per gram. None of the naturally or experimentally infected birds developed clinical signs in the present study. CONCLUSIONS: The molecular and biological characteristics of Cryptosporidium avian genotype II, described here, support the establishment of a new species, Cryptosporidium ornithophilus n. sp.

• Keywords
• C. ubiquitum, Cryptosporidium avian genotype II, Cryptosporidium ornithophilus n. sp., Experimental infections, Occurrence, Oocyst size, PCR,
• MeSH
• Cryptosporidium classification   genetics   ultrastructure   MeSH
• Phylogeny MeSH
• Animals, Domestic parasitology   MeSH
• Host Specificity MeSH
• Classification MeSH
• Cryptosporidiosis parasitology   MeSH
• Bird Diseases parasitology   MeSH
• Genes, Protozoan genetics   MeSH
• Birds parasitology   MeSH
• Life Cycle Stages MeSH
• Struthioniformes parasitology   MeSH
• DNA Barcoding, Taxonomic veterinary   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH

The morphological, biological, and molecular characteristics of Cryptosporidium muris strain TS03 are described, and the species name Cryptosporidium proliferans n. sp. is proposed. Cryptosporidium proliferans obtained from a naturally infected East African mole rat (Tachyoryctes splendens) in Kenya was propagated under laboratory conditions in rodents (SCID mice and southern multimammate mice, Mastomys coucha) and used in experiments to examine oocyst morphology and transmission. DNA from the propagated C. proliferans isolate, and C. proliferans DNA isolated from the feces of an African buffalo (Syncerus caffer) in Central African Republic, a donkey (Equus africanus) in Algeria, and a domestic horse (Equus caballus) in the Czech Republic were used for phylogenetic analyses. Oocysts of C. proliferans are morphologically distinguishable from C. parvum and C. muris HZ206, measuring 6.8-8.8 (mean = 7.7 μm) × 4.8-6.2 μm (mean = 5.3) with a length to width ratio of 1.48 (n = 100). Experimental studies using an isolate originated from T. splendens have shown that the course of C. proliferans infection in rodent hosts differs from that of C. muris and C. andersoni. The prepatent period of 18-21 days post infection (DPI) for C. proliferans in southern multimammate mice (Mastomys coucha) was similar to that of C. andersoni and longer than the 6-8 DPI prepatent period for C. muris RN66 and HZ206 in the same host. Histopatologicaly, stomach glands of southern multimammate mice infected with C. proliferans were markedly dilated and filled with necrotic material, mucus, and numerous Cryptosporidium developmental stages. Epithelial cells of infected glands were atrophic, exhibited cuboidal or squamous metaplasia, and significantly proliferated into the lumen of the stomach, forming papillary structures. The epithelial height and stomach weight were six-fold greater than in non-infected controls. Phylogenetic analyses based on small subunit rRNA, Cryptosporidium oocyst wall protein, thrombospondin-related adhesive protein of Cryptosporidium-1, heat shock protein 70, actin, heat shock protein 90 (MS2), MS1, MS3, and M16 gene sequences revealed that C. proliferans is genetically distinct from C. muris and other previously described Cryptosporidium species.

• MeSH
• Cryptosporidium classification   genetics   MeSH
• Phylogeny MeSH
• Cryptosporidiosis parasitology   MeSH
• Mole Rats MeSH
• Mice, SCID MeSH
• Mice MeSH
• Oocysts metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Gastric cryptosporidia only inhabit the glandular part of the stomach of all age categories of their hosts and can cause chronic life-long infections independent of a host's immune status. The immune response in the stomach mucosa during the primary infection and re-infection with Cryptosporidium muris (TS03 and CB03) in immunocompetent BALB/c mice was characterized using flow cytometry analysis and measurement of IFN-gamma and IL10 by enzyme-linked immunosorbent assays (ELISA). Significantly, elevated migration of T lymphocytes (more than 1,000-fold), especially CD8+ T lymphocytes, to the stomach mucosa occurred during primary infection and persisted for more than 2 months after its resolution. The ex vivo cultures of splenocytes revealed very low levels of IFN-gamma production during the course of the primary infection (0.5 ng/ml), whereas in the following re-exposure to the parasites, the concentration of IFN-gamma rapidly increased 22-fold. Although the two parasite strains that were tested were genetically distinct, they yielded similar results in the induction of cellular immune responses, suggesting that these patterns are not unique to a single parasite strain. These results imply that the CD8+ T lymphocytes are involved in the immune response to gastric cryptosporidiosis and could play an important role in the elimination of C. muris infection in mice.

• MeSH
• Immunity, Cellular * MeSH
• Cryptosporidium immunology   MeSH
• Enzyme-Linked Immunosorbent Assay MeSH
• Interferon-gamma metabolism   MeSH
• Interleukin-10 metabolism   MeSH
• Cryptosporidiosis immunology   MeSH
• Humans MeSH
• Mice, Inbred BALB C MeSH
• Mice MeSH
• Flow Cytometry MeSH
• Spleen immunology   MeSH
• Immunity, Mucosal * MeSH
• T-Lymphocyte Subsets immunology   MeSH
• Gastric Mucosa immunology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Interferon-gamma MeSH
• Interleukin-10 MeSH