The infection process of intracellular opportunistic microsporidia involves the forcible eversion of a coiled hollow polar filament that pierces the host cell membrane, allowing the passage of infectious sporoplasm into the host cell cytoplasm. Although the exact mechanism of spore activation leading to polar filament discharge is unknown, we have shown that spore adherence to host cells, which is mediated by sulfated glycosaminoglycans, may play a vital role. When adherence is inhibited, host cell infection decreases, indicating a direct link between adherence and infection. The goal of this study was to evaluate the effects of exogenous divalent cations on microsporidia spore adherence and infection. Data generated using an in vitro spore adherence assay show that spore adherence is augmented by manganese (Mn2+) and magnesium (Mg2+), but not by calcium (Ca2+). However, each of the three divalent cations contributed to increased host cell infection when included in the assay. Finally, we show that Mn2+ and Mg2+ may activate a constituent on the microsporidia spore, not on the host cell, leading to higher infection efficiency. This report further supports recent evidence that spore adherence to the host cell surface is an important aspect of the microsporidial infection process.

Microsporidia are obligate intracellular, spore-forming, fungal-related pathogens that employ a unique organelle, the polar tube, to transfer infectious spore contents into host cells to initiate infection. Spore adherence to host cells may provide the proximity required for polar tube/host cell interaction during in vivo infection. In previous in vitro studies, host sulfated glycosaminoglycans (GAGs) or recombinant microsporidia endospore protein (EnP1) was implicated in the pathogen adherence and infection process; however, complete ablation of spore adherence and infection could not be achieved, suggesting that additional or alternative spore and host cell determinants of adherence and infection may exist. Analysis of the Encephalitozoon intestinalis genome revealed about 100 predicted proteins containing the canonical integrin-binding motif arginine-glycine-aspartic acid (RGD); and, many pathogens have been shown to engage integrin molecules on cell surfaces. We hypothesized that host cell integrins play a role in microsporidia adherence and infection. In this study, we demonstrated that addition of exogenous integrin ligands or recombinant alpha 3 beta 1 integrin or alpha 5 beta 1 integrin to assays of E. intestinalis adherence and infection significantly reduced spore adherence and infection of host cells, supporting our hypothesis and implicating these specific integrins as putative host cell receptors for E. intestinalis spores.

The microsporidian parasite Nadelspora canceri infects Dungeness crabs, Cancer magister, along the United States Pacific Northwest coast. The prevalence and seasonal variation of N. canceri in Dungeness crabs from Alsea Bay, Oregon, are described based on examination of 2991 crabs collected at monthly intervals from October, 1991 to June, 1993. The average prevalence in monthly samples was 21.0% and ranged from 8.2% to 33.0%. No significant differences in monthly or seasonal parasite prevalence were observed. A total of 3061 Dungeness crabs was examined from an additional seven Pacific Northwest estuaries and Puget Sound to document the geographic distribution of N. canceri and the prevalence of the parasite in these locations. The estuaries sampled and the prevalences observed were: Humboldt Bay, California (14.6%), Coos Bay (10.6%), Yaquina Bay (2.0%), Tillamook Bay (41.2%), and Nehalem Bay, Oregon (14.2%), Willapa Bay (6.9%), and Grays Harbor Washington (0.44%). Dungeness crabs were examined from the Dungeness spit, Kala Point, and Mukilteo areas in Puget Sound and no infected crabs were found. A total of 9317 male Dungeness crabs > 15.9 cm carapace width (CW) captured in the commercial ocean crab fishery was examined for N. canceri and 27 (0.3%) were infected with the parasite. No infections were found in crabs smaller than 3.0 cm CW and the prevalence of infection generally increased with crab size reaching a peak of 22.2% in 14 cm CW crabs. The overall infection prevalence in male crabs (19.2%) was more than twice that of female crabs (8.0%), and of the 821 infected crabs found, 629 (76.6%) were males. The mortality of laboratory-held Dungeness crabs naturally infected with N. canceri was compared to that of uninfected crabs in two separate experiments and in both cases a significantly higher mortality was observed for infected crabs. Nadelspora canceri infections were established in both juvenile and adult Dungeness crabs that were fed parasite spores in laboratory experiments indicating that transmission is direct and intermediate hosts or vectors are not required for transmitting the parasite between hosts. / Graduation date: 1995

In this study was described the role of the inflammation in activation of latent microsporidiosis in immunocompetent BALB/c and MMF knock out mice and immunodeficient SCID mice and the correlation between inflammation induction and microsporidia occurrence. The number of spores in selected organs was located by using molecular and histology methods. Moreover, the occurrence of spores in faecal samples was monitored during the whole time of experiment.

The small nemertean <i>Maculaura alaskensis</i> is used as a model for studies of pilidiophoran development. During several microinjection experiments, it became clear several batches of oocytes obtained from wild-caught females contained an intracellular pathogen. Infected oocytes have large vesicles containing dozens to hundreds of refractile oval objects. Examination of oocytes with DIC and confocal microscopy showed the spores within the vesicles were diplokaryotic and contained a coiled tube, traits that are diagnostic of the phylum Microsporidia. The Microsporidia are a group of obligate intracellular parasites that infect cells of protists and animals. No other microsporidian has ever been found infecting cells of nemerteans and the association between <i>M. alaskensis</i> and this microsporidian is, thus far, undocumented. For my thesis, I described morphological characteristics, molecular phylogeny, and geographic range of the microsporidian. Additionally, I observed parasitic influences on development of infected <i>M. alaskensis</i> and explored other potential host species. / 2021-04-30

One hundred cats at different levels of habituation, house kept and free living, from Czech republic were screened for the presence of Cryptosporidium, Encephalitozoon spp. and Enterocytozoon bieneusi to consider the risk of close human - cat contact on cryptosporidial and microsporidial infections. The prevalence of Cryptosporidium, Encephalitozoon, and Enterocytozoon bieneusi in cats was determined from fecal specimens by genus-specific PCR. All PCR-positive specimens were sequenced to determine the genotype present. Out of 100 cats, three (3 %) were positive for Cryptosporidium, namely C. felis. Nine cats (9 %) were infected with E. bieneusi genotype D. None out of 35 house kept cats was infected with any tested parasites. No effect of age of animals on occurrence both Cryptosporidium and microsporidia was proved. To our knowledge, this is the first report on the presence of zoonotic species/genotypes of Cryptosporidium, and E. bieneusi in cats in the Czech republic.

The cryptosporidia and microsporidia are worldwide spread opportunistic parasites. With regard to their wide host spectra and zoonotic potential there is a significant risk for immunocompromised humans, especially AIDS patients. Natural Cryptosporidium infections in pigs are widespread but generally apathogenic. A total of 411 faecal samples, 39 of sows, 127 of pre-weaned, 122 of post-weaned piglets and 123 of pre-growers from three farms were screened for Cryptosporidium spp. infection using the aniline-carbol-methyl violet staining method and positive molecular characterized (direct sequencing of partial SSU rRNA partial genes and PCR-RFLP at the SSU rRNA). Overall, infections were detected in 84 out of 411 animals (20 %), with highest infection rates among post-weaners (27 %). All positive samples were genotyped based on SSU rRNA sequence analysis. C. suis, Cryptosporidium pig genotype II, and C. muris were detected. The microsporidial infections are very frequent in breeds of pigs. Pigs have been frequently reported to be infected with E. bieneusi. Spores were detected in 258 samples of 342 (75 %). The high prevalence was determined in pre-growers (86 %). Genotyping based on the ITS regions of the rRNA gene identified that most pigs were infected with the species-specific genotype F, while two animals had the zoonotic genotype D and two had genotype Peru 9.

Since their first discovery in the 19th century, microsporidian species have been found to be successful obligate intracellular parasites capable of infecting a wide variety of hosts including economically and ecologically important organisms as well as model organisms for biomedical research. Recently, cases of infection of the widely used animal model Danio rerio, commonly known as the zebrafish, by the newly described microsporidium Pseudoloma neurophilia have been reported in an increasing number of research facilities. Current knowledge of the biology of this parasite found in 75% of the Zebrafish Resource Center facilities is limited to microscopic analyses on its lifecycle as well as its physical, behavioral and psychic impact on its hosts. Despite the growing relevance of this parasite in biomedical research no current data is available on its genome. In this dissertation, I provide additional knowledge on the basic biology of P. neurophilia by acquiring and exploring the content and structure of the first genome draft of the zebrafish parasite. My findings reveal that the 5.25 Mb genome of P. neurophilia harbors an unusually high amount of transposable elements as well as numerous inserts found in coding regions typically conserved in microsporidia and other organisms. This peculiar obligate parasite demonstrates strong phylogenetic and genetic relationships with other fish-mosquito microsporidia. Similar to what is observed in closely related species, intra-genomic analyses of P. neurophilia’s genome suggest that it is diploid and possesses a large repertoire of over a thousand putative genes unique to this specie. Overall, my findings provide new insights into the basic biology of this parasite and represent a milestone in the understanding of P. neurophilia and D. rerio host-parasite interaction and ultimately in the development of treatments against this parasite that has been infecting the zebrafish research industry for the past decades.

Microsporidia exploit several targets for binding to host cells. Attachment is known to be an important first step before infection, and by blocking attachment, host cell infection decreases. This project seeks to determine if microsporidia use an ADAM (A Disintegrin And Metalloprotease) like protein to bind to host integrins. To test this, spore adherence assays employed a small, generated peptide that selected the integrin binding domain of the ADAM like protein. Afterwards, spore attachment to cell culture was quantified to determine if the peptide blocked spore attachment to cell monolayers. This project expands previous work by testing attachment of an additional microsporidia species. Finally, cell lysates pretreated with the peptide were screened for phosphorylation of FAK (Focal Adhesion Kinase), a common signaling pathway for activated integrins. Preliminary results suggest that microsporidial ADAM peptides bind to host cell integrins to decrease spore adherence and induce host cell signaling under the FAK pathway.

Encephalitozoon cuniculi is a zoonotic protozoan parasite in the phylum Microspora that has been shown to naturally infect several host species, including humans, rabbits and dogs. Currently, serological diagnosis of infection is made using the immunofluorescense assay (IFA) or enzyme-linked immunosorbent assay (ELISA). Although these methods are sensitive and reliable, there are several drawbacks to both tests. Cross-reactivity between other Encephalitozoon species is common, and specialized equipment is required for IFA and ELISA. Most wildlife species are unable to be tested using these methods, because species-specific antibodies are required. One goal of this work was to develop a new serological test for diagnosing E. cuniculi infection that would be more practical for use in small veterinary and medical clinics. The effectiveness of the agglutination test was examined in CD-1 and C3H/He mice infected with E. cuniculi or one of 2 other Encephalitozoon species. The results indicate that the agglutination test is 86% sensitive and 98% specific for E. cuniculi, with limited cross-reactivity to E. intestinalis. The test is fast and easy to conduct, and requires no specialized equipment or species-specific antibodies. Recent reports of microsporidial DNA in crop irrigation waters suggest that unpasteurized juice products may be contaminated with E. cuniculi. High pressure processing (HPP) is an effective means of eliminating bacteria and extending the shelf life of products while maintaining the sensory features of food and beverages. The effect of HPP on the in vitro infectivity of E. cuniculi spores was examined. Spores were exposed to between 140 and 550 MPa for 1 min, and then spores were loaded onto cell culture flasks or were kept for examination by transmission electron microscopy (TEM). Spores treated with between 200 and 275 MPa showed reduction in infectivity. Following treatment of 345 MPa or more, spores were unable to infect host cells. No morphologic changes were observed in pressure-treated spores using TEM. The effect of disinfectants on in vitro infectivity of E. cuniculi spores was also examined. Spores of E. cuniculi were exposed to several dilutions of commercial bleach, HiTor and Roccal, and 70% ethanol for 10 minutes and then loaded onto Hs68 cells. The results of this study showed that all concentrations of disinfectants tested were lethal to E. cuniculi spores. Encephalitozoon cuniculi spores are more sensitive to disinfectants than are coccidian oocysts and other parasite cysts. / Master of Science