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

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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.

Resting stages of parasites are very well resistant to adverse effects of the external environment. They have the ability to survive for long periods and to accommodate to such effects. One of the accurate methods for detection of C. parvum and E. cuniculi is PCR. As the most appropriate storage medium for the droppings collected in the field is recommended RNA later and 2,5 % potassium dichromate. Samples fixed in this way may be stored at temperatures ranging from -20 to 21 °C, event if there is no possibility of storing them in a refrigerator.

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.

La microsporidie Encephalitozoon cuniculi, parasite intracellulaire obligatoire, pathogène de l'homme, est responsable d'infections opportunistes chez des sujets immunodéprimés. Sa spore est protégée par une épaisse paroi protéo-chitineuse pour laquelle peu de données sur les constituants protéiques sont disponibles. Dans ces travaux, nous avons décrit le protéome exprimé dans les stades tardifs de développement d'E. cuniculi. Grâce à des extractions protéiques séquentielles et une double stratégie d'analyse protéomique, après électrophorèse et "Shotgun", 177 protéines différentes ont pû être identifiées, permettant d'obtenir une vision globale de la physiologie de la spore. L'exploitation de ces données et le développement d'un crible bioinformatique a permis l'identification de 4 protéines de paroi. Le premier modèle dynamique de morphogenèse de la paroi microsporidienne a été proposé grâce au suivi de la localisation de ces protéines durant le cycle de développement

The influence of interferon gamma and specific antibodies on the infection with E. cuniculi in vivo has been studied. Reconstruction of SCID mice with CD4+ T-lymphocytes from BALB/c mice and from mice with defect gene for interferon gamma was used. Effects of the treatment with mouse recombinant interferon gamma and anti-E. cuniculi sera on survival of E. cuniculi infected SCID mice were monitored. The influence of the immunization with E. cuniculi antigen on the survival of E. cuniculi infected mice with defect gene for interferon gamma was examined.

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.

A total of 587 faecal samples were collected from 98 horses of different age from three farms with various breeding system (pasture, stable and combination) in the South Bohemia, Czech Republic during three consecutive years (from 2009 to 2011), and screened for the presence of endoparasites, especially these inhabiting gastrointestinal tract and lung, using standard parasitological methods including flotation, sedimentation and staining methods. Moreover presence of Encephalitozoon spp. was detected using genus specific nested PCR. Small strongyles were the most common nematodes among studied horses. The age of animals was evaluated as a risk factor in relation to Eimeria leuckarti, Strongyloides westeri and Parascaris equorum infection in foals. The resistance of small strongyles (Cyathostominae) to benzimidazol anthelmintics has been revealed only on one of the visited farm. Most of examined animals were mono-infected. The breeding of horses in the stable represent management system with lowest risk in relation to parasitic infection of animals. Molecular analysis revealed the presence of human pathogenic Encephalitozoon cuniculi genotype I in horses.