Toxoplasma gondii is a zoonotic parasite infecting all warm-blooded animals, including humans. The contribution of environmental contamination by T. gondii oocysts to infections is understudied. The aim of the current work was to explore T. gondii serology as a means of attributing the source of infection using a robust stepwise approach. We identified in silico thirty-two promising oocyst-specific antigens from T. gondii ´omics data, recombinantly expressed and purified them and validated whether serology based on these proteins could discriminate oocyst- from tissue cyst-driven experimental infections. For this, three well-characterized serum panels, sampled from 0 to 6 weeks post-infection, from pigs and sheep experimentally infected with T. gondii oocysts or tissue cysts, were used. Candidate proteins were initially screened by Western blot with sera from pigs or sheep, infected for different times, either with oocysts or tissue cysts, as well as non-infected animals. Only the recombinant proteins TgCCp5A and TgSR1 provoked seroconversion upon infection and appeared to discriminate between oocyst- and tissue cyst-driven infections with pig sera. They were subsequently used to develop an enzyme-linked immunosorbent assay test for pigs. Based on this assay and Western blot analyses, a lack of stage specificity and low antigenicity was observed with all pig sera. The same was true for proteins TgERP, TgSporoSAG, TgOWP1 and TgOWP8, previously described as source-attributing antigens, when analyzed using the whole panels of sera. We conclude that there is currently no antigen that allows the discrimination of T. gondii infections acquired from either oocysts or tissue cysts by serological tests. This work provides robust new knowledge that can inform further research and development toward source-attributing T. gondii serology.
- Keywords
- Toxoplasma gondii, antigen prediction, diagnosis, oocyst-specific proteins, serology,
- Publication type
- Journal Article MeSH
Within two years and a half, the faeces of 620 cats coming from Brno and the area around the city were subjected to parasitological examination with special regard to the occurrence of the oocysts of Toxoplasma gondii. Sucrose solution at the specific weight of 1,150 was used as flotation medium. Oocysts of Toxoplasma gondii were eliminated by eight cats (1.29%) at the age from 16 days to 1.5 years. Six of the eight cats were younger than seven months. The Toxoplasma gondii oocysts were eliminated by the cats for 1-16 days while exhibiting signs of short-term scours and swelling of lymph nodes. In all cases the oocysts of Toxoplasma gondii were produced in the summer and autumn seasons (June-December). During the patent period, other coccidia (Isospora felis and Isospora rivolta) were also present in the cats.
- MeSH
- Feces parasitology MeSH
- Cats parasitology MeSH
- Parasite Egg Count MeSH
- Toxoplasma isolation & purification MeSH
- Animals MeSH
- Check Tag
- Cats parasitology MeSH
- Animals MeSH
- Publication type
- English Abstract MeSH
- Journal Article MeSH
Two hundred and two samples of excrements were investigated for the presence of Toxoplasma gondii oocysts. Fifty-two samples were taken from dead domestic cats and a hundred and fifty samples were obtained from wild cats living in various localities of the CSSR. The investigation was orientative and a flotation method with the Breza flotation solution of the specific weight of 1.300 was used. If the finding was positive, residues of the excrements were floated with a saccharose, solution of the specific weight of 1.150 and containing 0.8% of phenol. The oocysts were rinsed several times, then they were sporulated in Petri dishes with water with a 2.5% solution of potassium dichromate. The sporulated oocysts, after rinsing, were injected i. p. to mice. The excrements of the fifty-two domestic cats were negative. Out of a hundred and fifty samples of the excrements of wild cats, one sample with the oocysts of isospore type was found; a biological test with mice proved the oocysts of Toxoplasma gondii. It may be inferred from the results obtained that the elimination of oocysts by cats is the same as given in foreign literature, and the occurrence rate will be about 2%.
- MeSH
- Feces parasitology MeSH
- Cats parasitology MeSH
- Methods MeSH
- Parasite Egg Count MeSH
- Toxoplasma isolation & purification MeSH
- Animals MeSH
- Check Tag
- Cats parasitology MeSH
- Animals MeSH
- Publication type
- English Abstract MeSH
- Journal Article MeSH
- MeSH
- Cryptosporidium * MeSH
- Feces parasitology MeSH
- Parasite Egg Count methods veterinary MeSH
- Birds parasitology MeSH
- Mammals parasitology MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
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.
- Keywords
- Animal model, C57BL/6J mice, Cryptosporidiosis, Cryptosporidium, Immune responses, α-defensins,
- MeSH
- alpha-Defensins * MeSH
- Cryptosporidium parvum * MeSH
- Cryptosporidium * physiology MeSH
- Feces MeSH
- Cryptosporidiosis * pathology MeSH
- Humans MeSH
- Disease Models, Animal MeSH
- Mice, Inbred C57BL MeSH
- Mice MeSH
- Oocysts MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Research Support, Non-U.S. Gov't MeSH
- Names of Substances
- alpha-Defensins * MeSH
- MeSH
- Apicomplexa drug effects growth & development MeSH
- Disinfectants pharmacology MeSH
- Publication type
- English Abstract MeSH
- Journal Article MeSH
- Comparative Study MeSH
- Names of Substances
- Disinfectants MeSH
- MeSH
- Cloaca parasitology MeSH
- Chickens parasitology MeSH
- Parasite Egg Count methods MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
The morphology of oocysts of E. media Kessel, 1929, has been studied and two types have been found. Significant differences were observed in length of the oocysts (average values 29.05 microns with type I and 32.88 microns with type II), in width of the micropyle (5.21 and 3.72 microns, respectively) and in width of the sporocysts (6.67 and 8.00 microns). Marked differences were found even in the shape of the residual bodies of the sporocysts and in the structure of the oocyst wall in the region of the micropyle.
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
To study effects of experimental cryptosporidiosis, broiler chickens were infected per os with 5 x 10(5) oocysts of Cryptosporidium baileyi and Cryptosporidium meleagridis. In the first experiment, chickens were infected with oocysts of C. baileyi at the age of 7, 14, and 21 days. In the second experiment, chickens were infected with oocysts of C. baileyi, C. meleagridis, or both cryptosporidial species at the age of 7 days. Although clinical signs of infection were apparent, neither final live weight nor mortality was significanty influenced in chickens infected with a single Cryptosporidium species. In chickens infected with C. meleagridis, the growth retardation was observed in the 2-wk period after infection. The compensatory growth, however, started when the oocyst shedding had ceased. The number of oocysts in excreta specimens of chickens infected with C. meleagridis was two to three times lower than in excreta of chickens infected with C. baileyi. Chickens infected with both C. baileyi and C. meleagridis (5 x 10(5) oocysts of each) had significantly lower final live weight and worse feed efficiency than chickens of other groups. Concurrent infection did not influence individual C. baileyi or C. meleagridis oocyst shedding.
- MeSH
- Cryptosporidium growth & development MeSH
- Feces parasitology MeSH
- Cryptosporidiosis parasitology veterinary MeSH
- Chickens parasitology MeSH
- Poultry Diseases parasitology MeSH
- Parasite Egg Count veterinary MeSH
- Body Weight MeSH
- Age Factors MeSH
- Animals MeSH
- Check Tag
- Male MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
