Thesis--Copenhagen. / Summary in Danish. Bibliography: p. 84-89.

Thesis--Copenhagen. / Summary in Danish. Bibliography: p. 84-89.

En la década pasada, la Microsporidiosis humana era considerada como una enfermedad exclusivamente de personas VIH positivas o de otros pacientes también inmunocomprometidos. En la actualidad, se sabe que no solo afecta a estos pacientes sino que además se vienen reportando nuevos casos en pacientes inmunocompetentes, incrementándose cada día mas el número de casos en este grupo de personas. La especie de microsporidio que comúnmente se diagnostica es el Enterocytozoon bieneusi, que hasta el momento es el único microsporidio que no ha podido ser cultivado ya sea in vivo o in Vitro. El presente trabajo de tesis tiene como objetivo demostrar la infección experimental de cerdos de 1 mes de edad con esporas de Enterocytozoon bieneusi, para lo cual se utilizaron 9 lechones procedentes de una granja tecnificada con muy pocas probabilidades de infección a este parásito. Se realizó la inoculación de las esporas a los lechones por vía oral. Para el diagnóstico del parásito se realizaron 2 tipos de coloraciones: La Coloración Tricrómica, para colorear las muestras de heces y la Coloración de Plata, para colorear las diferentes porciones de tejidos intestinales de cada lechón. Se logró la observación de esporas de Enterocytozoon bieneusi en las heces y en los cortes de intestino de todos los lechones post inoculación, comprobando que una de las vías de infección de este parásito es la oral. Este trabajo demuestra que los cerdos de 1 mes de edad son susceptibles a la infección con esporas de Enterocytozoon bieneusi. y puede ser considerado como un posible animal que sirva de modelo experimental en el estudio de la Microsporidiosis humana causada por Enterocytozoon bieneusi. / In the last decade, the human Microsporidiosis was considered like a disease exclusively of positive people VIH or other patients also immunocompromised. At the present time, one knows that no single it affects these patients but who in addition they come reporting new cases in immunocompetent patients, being increased every day but the number of cases in this group of people. The species of microsporidia that commonly is diagnosed is the Enterocytozoon bieneusi, that until the moment is only microsporidia that could not have been cultivated or in alive or in vitro. The present thesis work must like objective demonstrate to the experimental infection of pigs of 1 moth of age with spores of Enterocytozoon bieneusi, for which 9 pigs coming from a farm technified with very few probabilities of infection to this parasite were used. The inoculation of the spores to the pigs was made orally. For the diagnosis of the parasite 2 types of colorations were made: The Tricrómic Blue Stain, to color the samples of heces and the Coloration of Silver, to color the different portions from intestinal weaves of each pig. The observation of spores of Enterocytozoon bieneusi was obtained in the feces and rhe cuts of intestine of all the pigs post inoculation, verifying that one of the routes of infection of this parasite is the oral one. This work demonstrates that the pigs of 1 month of age are susceptible to the infection with spores of Enterocytozoon bieneusi and it can be considered like a possible animal that serves as experimental model in the study of the human Microsporidiosis caused by Enterocytozoon bieneusi.

Investigations into the phylogeny, genome size, and karyotype of microsporidian Nadeispora canceri were initiated to further characterize the organism. Isolates of N. canceri spores were obtained from both Dungeness (Cancer magisrer) and red rock crabs (Cancer productus). Analysis of the ssu rDNA sequence from spore isolates of the two crab species showed 100% sequence identity among 1,081 nucleotide positions, indicating the same species of microsporidian is infecting both species of crabs. Phylogenetic studies based on the ssu rDNA sequences also showed N. canceri to be most closely related to another crustacean parasite, A meson michaelis. Sequence comparison between the two microsporidian species showed 93% sequence identity (1,001/1081 nucleotide positions). Pulsed field gel electrophoresis was used to estimate the genome size and karyotype of N. canceri isolates obtained from Dungeness and red rock crabs. Resolution of DNA bands on the pulsed field gels revealed both isolates to have a karyotype often chromosome-sized DNA bands. Estimation of the genome size revealed spore isolates from C. magister to have a total genome size of 7.44 Mb and spore isolates from C. productus to have a total genome size of 7.32 Mb. Variations detected in chromosome size culminated in a difference in the genome size between the two isolates. However, the variations in chromosome size were found not to be significant based on the Student's t-test. / Graduation date: 2004

En la década pasada, la Microsporidiosis humana era considerada como una enfermedad exclusivamente de personas VIH positivas o de otros pacientes también inmunocomprometidos. En la actualidad, se sabe que no solo afecta a estos pacientes sino que además se vienen reportando nuevos casos en pacientes inmunocompetentes, incrementándose cada día mas el número de casos en este grupo de personas. La especie de microsporidio que comúnmente se diagnostica es el Enterocytozoon bieneusi, que hasta el momento es el único microsporidio que no ha podido ser cultivado ya sea in vivo o in Vitro. El presente trabajo de tesis tiene como objetivo demostrar la infección experimental de cerdos de 1 mes de edad con esporas de Enterocytozoon bieneusi, para lo cual se utilizaron 9 lechones procedentes de una granja tecnificada con muy pocas probabilidades de infección a este parásito. Se realizó la inoculación de las esporas a los lechones por vía oral. Para el diagnóstico del parásito se realizaron 2 tipos de coloraciones: La Coloración Tricrómica, para colorear las muestras de heces y la Coloración de Plata, para colorear las diferentes porciones de tejidos intestinales de cada lechón. Se logró la observación de esporas de Enterocytozoon bieneusi en las heces y en los cortes de intestino de todos los lechones post inoculación, comprobando que una de las vías de infección de este parásito es la oral. Este trabajo demuestra que los cerdos de 1 mes de edad son susceptibles a la infección con esporas de Enterocytozoon bieneusi. y puede ser considerado como un posible animal que sirva de modelo experimental en el estudio de la Microsporidiosis humana causada por Enterocytozoon bieneusi. / In the last decade, the human Microsporidiosis was considered like a disease exclusively of positive people VIH or other patients also immunocompromised. At the present time, one knows that no single it affects these patients but who in addition they come reporting new cases in immunocompetent patients, being increased every day but the number of cases in this group of people. The species of microsporidia that commonly is diagnosed is the Enterocytozoon bieneusi, that until the moment is only microsporidia that could not have been cultivated or in alive or in vitro. The present thesis work must like objective demonstrate to the experimental infection of pigs of 1 moth of age with spores of Enterocytozoon bieneusi, for which 9 pigs coming from a farm technified with very few probabilities of infection to this parasite were used. The inoculation of the spores to the pigs was made orally. For the diagnosis of the parasite 2 types of colorations were made: The Tricrómic Blue Stain, to color the samples of heces and the Coloration of Silver, to color the different portions from intestinal weaves of each pig. The observation of spores of Enterocytozoon bieneusi was obtained in the feces and rhe cuts of intestine of all the pigs post inoculation, verifying that one of the routes of infection of this parasite is the oral one. This work demonstrates that the pigs of 1 month of age are susceptible to the infection with spores of Enterocytozoon bieneusi and it can be considered like a possible animal that serves as experimental model in the study of the human Microsporidiosis caused by Enterocytozoon bieneusi.

Background: Microsporidia are obligate intracellular opportunistic fungi that cause significant pathology in immunocompromised hosts. However, 11 percent of immunocompetent individuals in the general population are microsporidia-seropositive, indicating that severe immune suppression may not be a prerequisite for infection. Encephalitozoon intestinalis is transmitted in contaminated water and initially infects gastro-intestinal enterocytes, leading to diarrheal disease. This organism can also disseminate to many other organs. A recent report suggests that microsporidia can establish persistent infections, which anti-fungal treatment does not eradicate. Like other intracellular pathogens, microsporidia infection stresses the host cell and infected individuals have elevated hydrogen peroxide and free radical levels. Findings. As oxidative stress can lead to DNA damage, we hypothesized that E. intestinalis-infection would increase host cell nuclear mutation rate. Embryo fibroblasts from Big Blue§ssup§TM§esup§ transgenic mice were E. intestinalis-infected and host nuclear mutation frequency was determined by selection of temperature-sensitive c-II gene mutant λ phage. The host mutation frequency in E. intestinalis-infected cultures was 2.5-fold higher than that observed in either mock-infected cells or cells infected with UV-inactivated E. intestinalis spores. Conclusions: These data provide the first evidence that microsporidia infection can directly increase host cellular mutation frequency. Additionally, some event in the microsporidia developmental cycle between host cell attachment and parasitophorous vacuole formation is required for the observed effect. As there is considerable evidence linking infection with other intracellular pathogens and cancer, future studies to dissect the mechanism by which E. intestinalis infection increases host mutation frequency are warranted.

Background: Microsporidia are obligate intracellular opportunistic fungi that cause significant pathology in immunocompromised hosts. However, 11 percent of immunocompetent individuals in the general population are microsporidia-seropositive, indicating that severe immune suppression may not be a prerequisite for infection. Encephalitozoon intestinalis is transmitted in contaminated water and initially infects gastro-intestinal enterocytes, leading to diarrheal disease. This organism can also disseminate to many other organs. A recent report suggests that microsporidia can establish persistent infections, which anti-fungal treatment does not eradicate. Like other intracellular pathogens, microsporidia infection stresses the host cell and infected individuals have elevated hydrogen peroxide and free radical levels. Findings. As oxidative stress can lead to DNA damage, we hypothesized that E. intestinalis-infection would increase host cell nuclear mutation rate. Embryo fibroblasts from Big Blue§ssup§TM§esup§ transgenic mice were E. intestinalis-infected and host nuclear mutation frequency was determined by selection of temperature-sensitive c-II gene mutant λ phage. The host mutation frequency in E. intestinalis-infected cultures was 2.5-fold higher than that observed in either mock-infected cells or cells infected with UV-inactivated E. intestinalis spores. Conclusions: These data provide the first evidence that microsporidia infection can directly increase host cellular mutation frequency. Additionally, some event in the microsporidia developmental cycle between host cell attachment and parasitophorous vacuole formation is required for the observed effect. As there is considerable evidence linking infection with other intracellular pathogens and cancer, future studies to dissect the mechanism by which E. intestinalis infection increases host mutation frequency are warranted.

The course of microsporidiosis caused by Encephalitozoon cuniculi in immunocompetent BALB/c mice and immunodeficient SCID mice was screened using molecular methods. The site of infection in organs was located using molecular and histology methods. The effectiveness of albendazole treatement and possibility of infection relapse after immunosuppresion (cyclosporine A, tacrolimus, mycofenolate mofetil) was also studied. Moreover, the course of excretion of microsporidial spores in feces was monitored during the whole time of experiment.

Wild populations of fish are faced with a multitude of stressors, which may include human interaction, toxins, and disease. Benzo(a)pyrene (BaP), a known carcinogen and immunotoxin, has been reported in the stomach contents of juvenile chinook salmon, Oncorhynchus tshawytscha, in urban waterways. We investigated the impact of chronic dietary exposure of environmentally relevant levels of BaP on the immune system and cytochrome P4501A1 (CYP1A1) expression in juvenile chinook salmon. Two experiments were carried out in which juvenile fish were fed food treated with ethanol (control diet), low or high concentrations of BaP, or cortisol. In the first experiment we measured mitogen-stimulated proliferation of splenic leukocytes using flow cytometry and a colorimetric assay using Alamar Blue[superscript TM] Susceptibility to a microsporidian parasite, Loma salmonae, was evaluated in the second experiment by quantification of xenomas in the gills. Hepatic CYP1A1 and plasma cortisol were measured in both experiments. No significant trends were found in leukocyte mitogen activation or plasma cortisol between treatments or days. However, western blot analysis of CYP1A1 concentration in liver revealed interesting patterns of induction: in cortisol fed groups CYP1A1 was <20% of control on all days, groups fed low levels of BaP were 250% of control values on days 8 and 21 then dropped below control values on day 29, and groups fed high levels of BaP had less CYP1A1 than controls on all days. Similar patterns of CYP1A1 levels were found in the second experiment, and diseased control groups showed about a 55% decrease in CYP1A1 concentration when compared with non-diseased control groups. Susceptibility to L. salmonae was significantly higher in groups receiving cortisol. Whereas there was no effect of the high BaP dose, the low BaP dose appeared to increase disease susceptibility. This study supports concerns of stress and toxin induced immune dysfunction in wild populations of fish. / Graduation date: 2004