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.
- MeSH
- alfa-defensiny * MeSH
- Cryptosporidium parvum * MeSH
- Cryptosporidium * fyziologie MeSH
- feces MeSH
- kryptosporidióza * patologie MeSH
- lidé MeSH
- modely nemocí na zvířatech MeSH
- myši inbrední C57BL MeSH
- myši MeSH
- oocysty MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- MeSH
- Cryptosporidium fyziologie MeSH
- druhová specificita MeSH
- feces parazitologie MeSH
- genotyp MeSH
- hostitelská specificita MeSH
- Psittaciformes parazitologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
The need for an effective treatment against cryptosporidiosis has triggered studies in the search for a working in vitro model. The peculiar niche of cryptosporidia at the brush border of host epithelial cells has been the subject of extensive debates. Despite extensive research on the invasion process, it remains enigmatic whether cryptosporidian host-parasite interactions result from an active invasion process or through encapsulation. We used HCT-8 and HT-29 cell lines for in vitro cultivation of the gastric parasite Cryptosporidium proliferans strain TS03. Using electron and confocal laser scanning microscopy, observations were carried out 24, 48 and 72 h after inoculation with a mixture of C. proliferans oocysts and sporozoites. Free sporozoites and putative merozoites were observed apparently searching for an appropriate infection site. Advanced stages, corresponding to trophozoites and meronts/gamonts enveloped by parasitophorous sac, and emptied sacs were detected. As our observations showed that even unexcysted oocysts became enveloped by cultured cell projections, using polystyrene microspheres, we evaluated the response of cell lines to simulated inoculation with cryptosporidian oocysts to verify innate and parasite-induced behaviour. We found that cultured cell encapsulation of oocysts is induced by parasite antigens, independent of any active invasion/motility.
- MeSH
- antigeny protozoální metabolismus MeSH
- buněčné linie MeSH
- buňky HT-29 MeSH
- Cryptosporidium fyziologie MeSH
- interakce hostitele a parazita fyziologie MeSH
- konfokální mikroskopie MeSH
- kryptosporidióza metabolismus parazitologie MeSH
- lidé MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
We undertook a study on Cryptosporidium spp. in wild cricetid rodents. Fecal samples were collected from meadow voles (Microtus pennsylvanicus), southern red-backed voles (Myodes gapperi), woodland voles (Microtus pinetorum), muskrats (Ondatra zibethicus) and Peromyscus spp. mice in North America, and from bank voles (Myodes glareolus) and common voles (Microtus arvalis) in Europe. Isolates were characterized by sequence and phylogenetic analyses of the small subunit ribosomal RNA (SSU) and actin genes. Overall, 33·2% (362/1089) of cricetids tested positive for Cryptosporidium, with a greater prevalence in cricetids from North America (50·7%; 302/596) than Europe (12·1%; 60/493). Principal Coordinate analysis separated SSU sequences into three major groups (G1-G3), each represented by sequences from North American and European cricetids. A maximum likelihood tree of SSU sequences had low bootstrap support and showed G1 to be more heterogeneous than G2 or G3. Actin and concatenated actin-SSU trees, which were better resolved and had higher bootstrap support than the SSU phylogeny, showed that closely related cricetid hosts in Europe and North America are infected with closely related Cryptosporidium genotypes. Cricetids were not major reservoirs of human pathogenic Cryptosporidium spp.
- MeSH
- Arvicolinae parazitologie MeSH
- Cryptosporidium klasifikace izolace a purifikace patogenita fyziologie MeSH
- divoká zvířata parazitologie MeSH
- feces parazitologie MeSH
- fylogeneze MeSH
- fylogeografie MeSH
- genotyp MeSH
- hlodavci parazitologie MeSH
- kryptosporidióza epidemiologie parazitologie MeSH
- myši parazitologie MeSH
- RNA ribozomální genetika MeSH
- sekvenční analýza DNA MeSH
- zdroje nemoci parazitologie MeSH
- zvířata MeSH
- Check Tag
- myši parazitologie MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Evropa MeSH
- Severní Amerika MeSH
The present study was undertaken to describe Cryptosporidium spp. infection in tree squirrels from 17 locations in Northern Italy. A total of 357 squirrels were examined, including species native to Europe (Sciurus vulgaris; n=123), and species introduced from North America (Sciurus carolinensis; n=162) and Southeast Asia (Callosciurus erythraeus; n=72). Faecal samples of all squirrels were examined for the presence of Cryptosporidium infection by microscopy (flotation method) and PCR/sequence analysis of the Cryptosporidium 18S rRNA, actin, and gp60 genes. Despite the overlapping ranges of native and introduced tree squirrel species in the study area, they host different Cryptosporidium spp. Sciurus vulgaris were exclusively infected with Cryptosporidium ferret genotype (n=13) belonging to three novel gp60 subtypes, VIIIb-VIIId. Sciurus carolinensis hosted C. ubiquitum subtype XIIb (n=2), Cryptosporidium skunk genotype subtype XVIa (n=3), and chipmunk genotype I subtype XIVa (n=1). Cryptosporidium chipmunk genotype I subtype XIVa was also found in two C. erythraeus. Comparing data from this and previous studies, we propose that Cryptosporidium skunk genotype and possibly C. ubiquitum subtype XIIb were introduced to Europe with eastern grey squirrels. Cryptosporidium chipmunk genotype I and ferret genotype were associated with high intensity infections, but there was no association with diarrhoea.
- MeSH
- Cryptosporidium klasifikace genetika fyziologie MeSH
- feces parazitologie MeSH
- hostitelská specificita MeSH
- interakce hostitele a parazita MeSH
- kryptosporidióza parazitologie MeSH
- protozoální geny genetika MeSH
- rozšíření zvířat MeSH
- Sciuridae parazitologie MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Itálie MeSH
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.
- MeSH
- Cryptosporidium účinky léků fyziologie MeSH
- krysa rodu rattus MeSH
- kultivační média farmakologie MeSH
- mikrobiální viabilita účinky léků MeSH
- oocysty fyziologie MeSH
- propidium MeSH
- sporozoiti účinky léků fyziologie MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
This study describes cryptosporidiosis in an overwintering group of 15 European hedgehogs (Erinaceus europaeus), comprising 3 adults and 12 juveniles. Four juvenile hedgehogs were hospitalised with anorexia, malodorous diarrhoea and dehydration. Immediate parasitological examinations revealed the presence of Cryptosporidium sp. in these animals and also in 5 other juveniles. All hedgehogs were coproscopically monitored for 4 months over the winter season. Shedding of Cryptosporidium oocysts persisted from 6 to 70 days. Repeated shedding of Cryptosporidium oocysts occurred in 3 animals after 4 months subsequent to the first outbreak. Clinical signs were observed only at the beginning of the outbreak (apathy, anorexia, general weakness, mild dehydration, and malodorous faeces with changed consistence - soft/diarrhoea) in the 4 hospitalised juveniles. Overall 11 hedgehogs were Cryptosporidium-positive, both microscopically and by PCR methods. Sequence analyses of SSU rRNA and gp60 genes revealed the presence of C. parvum IIdA18G1 subtype in all positive hedgehogs. Moreover, 3 hedgehogs had a mixed infection of the zoonotic C. parvum and C. erinacei XIIIaA19R13 subtype. Cryptosporidium infections can be rapidly spread among debilitated animals and the positive hedgehogs released back into the wild can be a source of the infection for individuals weakened after hibernation.
- MeSH
- Cryptosporidium parvum klasifikace genetika fyziologie MeSH
- Cryptosporidium klasifikace genetika fyziologie MeSH
- feces parazitologie MeSH
- fylogeneze MeSH
- geny rRNA genetika MeSH
- ježkovití parazitologie MeSH
- kryptosporidióza parazitologie patologie MeSH
- nemocnice veterinární MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- kazuistiky MeSH
This study focuses on mapping the life cycle of Cryptosporidium muris in two laboratory rodents; BALB/c mice and the southern multimammate rat Mastomys coucha, differing in their prepatent and patent periods. Both rodents were simultaneously experimentally inoculated with viable oocysts of C. muris (strain TS03). Animals were dissected and screened for the presence of the parasite using a combined morphological approach and nested PCR (SSU rRNA) at different times after inoculation. The occurrence of first developmental stages of C. muris in stomach was detected at 2.5 days post-infection (dpi). The presence of Type II merogony, appearing 36 h later than Type I merogony, was confirmed in both rodents. Oocysts exhibiting different size and thickness of their wall were observed from 5 dpi onwards in stomachs of both host models. The early phase of parasitization in BALB/c mice progressed rapidly, with a prepatent period of 7.5-10 days; whereas in M. coucha, the developmental stages of C. muris were first observed 12 h later in comparison with BALB/c mice and prepatent period was longer (18-21 days). Similarly, the patent periods of BALB/c mice and M. coucha differed considerably, i.e. 10-15 days vs chronic infection throughout the life of the host, respectively.
- MeSH
- Cryptosporidium růst a vývoj fyziologie MeSH
- druhová specificita MeSH
- feces parazitologie MeSH
- kryptosporidióza parazitologie patologie MeSH
- modely nemocí na zvířatech MeSH
- Murinae MeSH
- myši inbrední BALB C MeSH
- myši MeSH
- oocysty MeSH
- stadia vývoje * MeSH
- trofozoiti MeSH
- žaludeční sliznice patologie MeSH
- zvířata MeSH
- Check Tag
- mužské pohlaví MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Three and 8 week old pigs were inoculated with Cryptosporidium muris HZ206 (Mus musculus musculus isolate), Cryptosporidium tyzerri CR2090 (M. m. musculus isolate) or C. tyzzeri CR4293 (isolate from a hybrid between Mus musculus domesticus and M. m. musculus) at a dose of 1 × 10(7) oocysts per animal. Inoculated pigs showed no detectable infection and no clinical symptoms of cryptosporidiosis during 30 days post infection (DPI), and no macroscopic changes were detected in the digestive tract following necropsy. Developmental stages were not detected in gastrointestinal tract tissue by histology or PCR throughout the duration of the experiment. The infectivity of isolates was verified on SCID mice, in which oocysts shedding started from 4 to 8 DPI. Based on our findings, it can be concluded that pigs are not susceptible to C. muris or C. tyzzeri infection.
- MeSH
- Cryptosporidium klasifikace genetika fyziologie MeSH
- feces parazitologie MeSH
- genotyp MeSH
- hostitelská specificita MeSH
- kryptosporidióza parazitologie veterinární MeSH
- myši SCID MeSH
- myši parazitologie MeSH
- nemoci hlodavců parazitologie MeSH
- nemoci prasat parazitologie MeSH
- prasata MeSH
- protozoální DNA chemie izolace a purifikace MeSH
- RNA ribozomální genetika MeSH
- sekvenční seřazení veterinární MeSH
- zvířata MeSH
- Check Tag
- myši parazitologie MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Geografické názvy
- Česká republika 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 parazitologie MeSH
- Cryptosporidium patogenita fyziologie MeSH
- hostitelská specificita MeSH
- kryptosporidióza parazitologie patologie MeSH
- modely nemocí na zvířatech MeSH
- odolnost vůči nemocem MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
