Molecular and in vitro comparisons were performed using two isolates of Encephalitozoon hellem, one from an avian host and one from a human host, and one isolate of Encephalitozoon cuniculi from a rabbit. The molecular comparisons were performed by amplifying and sequencing the gene coding for a zinc metallo-aminopeptidase from cDNA and gDNA obtained from each of the isolates. The E. hellem sequences shared >99 % identity between each other and 70% identity with the E. cuniculi sequences. Conserved HEXXH and GXMEN motifs located within the sequences classify the protein as an aminopeptidase of the M1 family, with at least one zinc atom required for catalytic activity. In vitro growth comparisons of the isolates described above were performed under simulated "mammalian and avian conditions". The models utilized mammalian and avian cell lines and sera at incubation temperatures of 37 °C and 40 °C, respectively. Three separate experiments were performed. E. cuniculi grew best under the mammalian model and significantly better than both E. hellem isolates under this model. The E. hellem isolates were able to infect and replicate under both the mammalian and avian models, which reflects the zoonotic potential of these isolates.

Conventional methods such as serology and microscopy are unreliable for diagnosis of encephalitozoonosis in domestic rabbits. Previous studies have reported PCR to be insensitive but it is unclear whether this is because of inherent limitations or the lack of assay optimization and validation. The studies described in this thesis assess DNA quality and quantity for combinations of six DNA extractions kits and four spore disruption methods. The resulting DNA underwent PCR using a published primer set. The optimal method had a detection threshold of 100 spores/ml in saline. However, when repeated in urine, the detection threshold was much higher (10,000 spores/ml) and non-target DNA amplification was present. Various methods were used to improve analytical sensitivity and eliminate non-target amplification. One method involving PEG 8000 treatment produced a detection threshold of 1,000 spores/ml and decreased non-target DNA amplification. Ultimately, new primers were designed and when the optimized method was tested with these primers, a detection threshold of 100 spores/ml with no non-target DNA amplification was achieved. The optimal method and new primers were tested using clinical samples of rabbit urine and 32.4% were found to be positive for E. cuniculi. The final assay was shown to be both analytically sensitive and specific; however further clinical investigation is warranted to determine clinical utility. / OVC Pet Trust

Thesis (Ph. D.)--Cornell University, April, 2008. / Vita. Includes bibliographical references (leaves 151-166).

Microsporidia are obligate intracellular pathogens of medical and ecological importance whose genomes have been studied extensively over the last decade. Their parasitic lifestyle has lead them to lose a great number of genes and, thus, biochemical pathways capacities, but these reductive processes have been often offset by the acquisition of several genes by means of horizontal gene transfer (HGT). First, in this thesis, we will describe the complete genomes of Encephalitozoon hellem and Encephalitozoon romaleae. Both species also were found to harbor a number of protein-coding genes absent in other microsporidia, which products assembled complete metabolic pathways. All these genes are functionally related to DNA and folate metabolism, and all appear to have been acquired from HGT events from different eukaryotic and prokaryotic donors. Interestingly in E. romaleae genes involved in de novo synthesis of folate are all pseudogenes, highlighting the transient nature of transferred genes. Secondly, we took a closer look at the ploidy and sexual status of Encephalitozoon cuniculi, a vertebrate pathogen, by mapping Illumina sequence reads against the genomes of four strains of this species. We identified the presence of low level of heterozygosity in all strains investigated; a feature that revealed the diploid nuclear state of the species. This reductive intra-individual genetic diversity could result from the long-term propagation of these strains under laboratory conditions, but we propose that it could also reflect an intrinsic capacity of these vertebrate pathogens to self-reproduce. Overall, the work presented in this thesis resulted in a much greater understanding of the genome evolution of a medically and economically important group of parasites.

Microsporidia are obligate intracellular pathogens of medical and ecological importance whose genomes have been studied extensively over the last decade. Their parasitic lifestyle has lead them to lose a great number of genes and, thus, biochemical pathways capacities, but these reductive processes have been often offset by the acquisition of several genes by means of horizontal gene transfer (HGT). First, in this thesis, we will describe the complete genomes of Encephalitozoon hellem and Encephalitozoon romaleae. Both species also were found to harbor a number of protein-coding genes absent in other microsporidia, which products assembled complete metabolic pathways. All these genes are functionally related to DNA and folate metabolism, and all appear to have been acquired from HGT events from different eukaryotic and prokaryotic donors. Interestingly in E. romaleae genes involved in de novo synthesis of folate are all pseudogenes, highlighting the transient nature of transferred genes. Secondly, we took a closer look at the ploidy and sexual status of Encephalitozoon cuniculi, a vertebrate pathogen, by mapping Illumina sequence reads against the genomes of four strains of this species. We identified the presence of low level of heterozygosity in all strains investigated; a feature that revealed the diploid nuclear state of the species. This reductive intra-individual genetic diversity could result from the long-term propagation of these strains under laboratory conditions, but we propose that it could also reflect an intrinsic capacity of these vertebrate pathogens to self-reproduce. Overall, the work presented in this thesis resulted in a much greater understanding of the genome evolution of a medically and economically important group of parasites.

Memoria para optar al Título Profesional de Médico Veterinario Departamento de Medicina Preventiva Animal / Los microsporidios son protozoos, parásitos intracelulares obligados de distintas especies animales. Pertenecen al Phylum Microspora, caracterizada por producir esporas muy pequeñas y por carecer de mitocondrias. Entre los microsporidios, está el Encephalitozoon cuniculi, el cual es un parásito oportunista emergente. El reservorio, en estudio, que pone en peligro la salud humana es el conejo (Oryctolagus cuniculus), el cual elimina al parásito principalmente por la orina, contaminando el medio ambiente. En la prevalencia de E. cuniculi en conejos se reportan rangos de 15 al 76 % a nivel mundial (Ansbacher et al, 1988). Con la finalidad de detectar la presencia de este parásito en nuestro país, se realizó la pesquisa de éste, en el laboratorio de parasitología del Instituto de Salud Pública de Chile (ISP). La muestra estuvo constituida por 100 conejos normales sin distinción de sexo, pertenecientes a: Bioterio de ISP, Criaderos familiares de la localidad de Pirque, “Mundo Granja” perteneciente a la Universidad de Chile y a un Criadero comercial. Todos los conejos fueron previamente evaluados mediante un examen clínico. Luego de procesar la muestra, de deposición y de orina, se procedió a teñirlas con colorantes tricrómicos: Cromotropo 2R y Gram Cromotropo. Se obtuvo una positividad en un 55% de los individuos, donde el mayor porcentaje se presenta en conejos inmunosuprimidos y de crianza convencional. El objetivo de este estudio es contribuir a la salud, en especial la del ser humano como la de los animales determinando la presencia de E. cuniculi en conejos de distintos orígenes / Servicio Agrícola y Ganadero SAG) y Proyecto FAVET /04-35.

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.

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

Encephalitozoon cuniculi, Toxoplasma gondii and Sarcocystis neurona are intracellular parasites that infect a wide range of mammalian host species including domestic cats. The prevalence of antibodies to these parasites in cats was examined using an indirect immunofluorescence antibody assay. E. cuniculi targets the kidneys of rabbits but the prevalence of disease in cats is unknown. Chronic kidney disease (CKD) is a common cause of illness in cats. T. gondii is a widespread parasite of cats; however, it is not considered a major causative agent of CKD. The first hypothesis was that E. cuniculi and T. gondii are unrecognized causes of chronic kidney disease in domestic cats. Serum and plasma samples were examined for protozoal antibodies from 232 feline patients at the VMRCVM Teaching Hospital. Thirty-six of the 232 samples met the IRIS criteria for CKD. Antibodies to E. cuniculi were found in 15 samples, 4 of which came from cats with CKD. Antibodies to T. gondii were found in 63 samples; 10 cats of the 63 had CKD. These were not significantly different from cats with no CKD and the null hypothesis was rejected. Domestic cats, armadillos, raccoons and skunks are intermediate hosts (IH) for S. neurona while opossums are the definitive host (DH). The seroprevalence of S. neurona was examined in domestic cats from Virginia and Pennsylvania. The second hypothesis was that domestic cats are important IH for S. neurona transmission. A low seroprevalence was found in 32 of the 441 cats and the null hypothesis was rejected. / Master of Science in Life Sciences

Microsporidia are obligate intracellular pathogens that cause severe disease in immunocompromised humans. While albendazole is the treatment of choice, no therapy exists that effectively treats all forms or causes of human microsporidiosis. Recent studies show that the microsporidian Encephalitozoon intestinalis binds glycosaminoglycans (GAGs) associated with the host cell surface, and that the divalent cations manganese (Mn2+) and magnesium (Mg2+) augment spore adherence to host cells by activating a constituent on the spore surface. These studies also illustrate a direct relationship between spore adherence and host cell infection; inhibition of spore adherence leads to reduced host cell infection while augmentation of spore adherence increases host cell infection. In light of recent studies, microsporidia spore adherence has become a promising target for the development of novel therapeutics to treat or even prevent human microsporidiosis. The goal of this study was to further characterize the molecular mechanisms governing spore adherence by identifying specific constituents on microsporidia spores that participate in spore adherence with host cells. A 40 kDa Encephalitozoon cuniculi host cell binding protein was discovered and identified as ECU01_0820, hereafter known as Encephalitozoon cuniculi microsporidia spore adherence protein (EcMsAP). The gene encoding EcMsAP has multiple heparin-binding motifs and an integrin-binding domain, which are characteristic of proteins that interact with constituents on the cell surface. Immuno-transmission electron microscopy reveals that native EcMsAP is located on the plasma membrane, endospore, exospore, and the anchoring disk of microsporidia spores. Recombinant EcMsAP and antibodies to recombinant EcMsAP both inhibit spore adherence and host cell infection. However, the deletion of heparin-binding motif #1 from the EcMsAP gene results in the loss of ability to inhibit spore adherence and infection. Host cell-binding assays reveal that recombinant EcMsAP binds Vero and CHO cell lines, but exhibits attenuated binding to glycan-deficient CHO cell lines. Finally, biomolecular interactions analysis provides direct evidence that EcMsAP is a glycan binding protein. This study not only identifies a potential microsporidial vaccine candidate, it further supports the assertion that microsporidia spore adherence is a critical step in the host cell infection process.