This study aimed to evaluate and document the excystation process of Cryptosporidium muris oocysts in various incubation media, and to monitor the behaviour of excysting and freshly excysted sporozoites. A test of oocyst viability, using fluorescent double staining with fluorescein diacetate and propidium iodide, was performed prior to each experimental assay. Light microscope observations confirmed that relatively often only three sporozoites were released; the fourth one either left the oocyst later together with a residual body or remained trapped within the oocyst wall. These results suggest that successful oocyst excystation is not limited by the viability of all four sporozoites. Darkening of oocysts to opaque and their specific movement (the so-called "oocyst dancing") preceded the final excystation and liberation of sporozoites, while the dormant oocysts appeared refractive. The process of excystation in C. muris is not gradual as generally described in cryptosporidia but very rapid in an eruptive manner. Experiments were performed using oocysts stored at 4 °C for various time periods, as well as oocysts freshly shed from host rodents (Mastomys coucha) of different ages. The most suitable medium supporting high excystation rate (76 %) and prolonged motility of sporozoites was RPMI 1640, enriched with 5 % bovine serum albumin (BSA). Our results emphasize that to reliably evaluate the success of in vitro excystation of cryptosporidia, not only the number of released sporozoites in a set time period should be taken into consideration but also their subsequent activity (motility), as it is expected to be essential for the invasion of host cells.

• Keywords
• Cryptosporidium muris, Excystation rate, Motility, Oocyst, Sporozoite, Viability test,
• MeSH
• Cryptosporidium drug effects   physiology   MeSH
• Rats MeSH
• Culture Media pharmacology   MeSH
• Microbial Viability drug effects   MeSH
• Oocysts physiology   MeSH
• Propidium MeSH
• Sporozoites drug effects   physiology   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Culture Media MeSH
• Propidium 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

The increasing movement of people to wilderness areas, shrinking of wildlife habitats and the resulting urbanisation of wildlife has led to growing concerns about the transfer of parasitic diseases, particularly from contaminated faeces. Faecal samples from wild carnivores in Ireland were examined for the presence of protozoan and nematode parasites. Red fox (Vulpes vulpes) samples (n = 91) were positive for Uncinaria stenocephala (38%), Eucoleus aerophilus (26%), Toxocara canis (20%), Trichuris vulpis (4%) and Isospora-like oocysts (9%). Badger (Meles meles) samples (n = 50) were positive for Uncinaria criniformis (40%), E. aerophilus (6%) and Isospora-like oocysts (16%). No parasites were observed in pine marten (n = 48; Martes martes) faeces. Approximately 5% of American mink (Mustela vison) samples were positive for Cryptosporidium by polymerase chain reaction (identified as Cryptosporidium andersoni (n = 3) and 'mink' genotype (n = 1)). The results suggest that wild carnivores in Ireland have a range of parasites, although it is unclear from the present study to what extent these infections are associated with morbidity. While it can be expected that, via their faeces, wild carnivores contribute to the spread of these parasites, they are unlikely the primary source of environmental contamination. Therefore, they should not always be the principal target of control measures.

• MeSH
• Carnivora * MeSH
• Feces parasitology   MeSH
• Parasitic Diseases, Animal epidemiology   parasitology   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Ireland epidemiology   MeSH

The infectivity of Cryptosporidium muris and Cryptosporidium andersoni in various species of voles was studied using experimental infections. None of the experimental voles inoculated with 1 × 10(5) oocysts of Cryptosporidium spp. shed any oocysts during 40 DPI, except Brandt's vole (Lasiopodomys brandtii), which was susceptible to C. muris infection. Experiments confirmed the resistance of voles of the genus Microtus sensu stricto to infection with mammalian gastric cryptosporidia, which provides a new study model with prospects to more fully understand the processes involved in the phenomenon of host specificity of this group of protists.

• MeSH
• Arvicolinae parasitology   MeSH
• Cryptosporidium pathogenicity   physiology   MeSH
• Host Specificity MeSH
• Cryptosporidiosis parasitology   pathology   MeSH
• Disease Models, Animal MeSH
• Disease Resistance MeSH
• Animals MeSH
• Check Tag
• 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