Document Type

Thesis - University Access Only

Award Date

2008

Degree Name

Master of Science (MS)

Department / School

Biology and Microbiology

Abstract

Microsporidia are a diverse group of eukaryotic, obligate intracellular parasites, and infect a wide range of vertebrate and invertebrate hosts. Encephalitozoon intestinalis is the second most prevalent microsporidian infecting humans and often causes severe wasting diarrhea and disseminating infections. In humans, the pathogenesis and host parasite interactions of E. intestinalis have not been well established. E. intestinalis first infects the intestinal epithelial cells; however, the role of these cells in pathogenesis and immunity to E. intestinalis infection has not been elucidated. To establish and cause disease, E. intestinalis may subvert defense mechanisms of intestinal epithelium and disrupt its barrier function. These changes may contribute to the diarrhea observed in E.intestinalis infection. The present study was undertaken a) To evaluate gnotobiotic pigs as a suitable and clinically relevant animal model for studying human E. intestinalis pathogenesis, b) To test the infectivity of E. intestinalis to porcine intestinal epithelial cultures and c) To investigate in vitro modulation of epithelial barrier function caused by E. intestinalis infection. Eleven caesarean-derived half-sib piglets were divided into 3 groups (4 in control group, 4 in infected group, and 3 in the infected with immunosuppression (S/1) group), and animals from each group were housed in separate gnotobiotic units. At day 3, each piglet from the infected and S/1 group were orally inoculated with approximately 0.75 X 106 E. intestinalis spores. Piglets from the S/1 group were sacrificed at 15, 18 and 21 days of age due to severe loss of condition characterized by ataxia, anorexia, dyspnoea, and distended belly. All remaining piglets were sacrificed at 27 days of age. PCR analysis of fecal samples at 5 days post infection showed that 2 of the 4 piglets from the infected group were shedding E. intestinalis spores; whereas, all 4 control and S/1 piglets were PCR negative. At 11 days post infection, fecal samples from all the pigs of the S/1 group and one pig of the infected group were PCR positive. At necropsy, piglets from the S/1 group showed significant lung lesions characterized by petechial hemorrhages and swollen lungs. Ileum, lung and brain samples from one S/1 piglet tested PCR positive for E. intestinalis. However, no E. intestinalis organisms were seen in tissue sections. Encephalitozoon intestinalis specific antibodies were detected in one infected immunocompetent animal by ELISA. Animals showed little or no symptoms of diarrhea, but this may have been due to the relatively short observation period. Because E. intestinalis spores were only sporadically detected in feces and tissues, it would appear that the infection was relatively mild. The fact that E. intestinalis successfully infected gnotobiotic piglets suggests that they could be used as large animal model of E. intestinalis infections, but the lack of symptoms in this model suggests that it would not be a good in vivo model for studying pathogenesis. A quantitative cell culture infectivity assay was used to compare the susceptibility of porcine intestinal epithelial cells (IPEC-J2, IPEC-1) and human Caco-2 cells to E. intestinal is from day 2 to day 5 post-infection. Infection of these cells with E. intestinalis was further confirmed by more specific techniques such as immunocytochemistry with E. intestinalis specific FITC linked polyclonal antibody and real time PCR with E. intestinalis specific primers. In all three cell types, the number of intracellular spore clusters increased progressively from day 2 to day 5 post infections. However, Caco-2 cells consistently showed a higher number of spore clusters on each day post-infection, followed by IPEC-1 and IPEC-J2 cells. It was observed by real time PCR that overall as multiplicity of infection (MOI) increased; more spores entered the intracellular compartment. Compared to porcine cell lines, the increase in gene copy number with increase in MOI was more pronounced in Caco-2 cells. The effect of E. intestinalis infection on membrane integrity was evaluated by measuring transepithelial electrical resistance (TEER) in polarized IPEC-J2 cells before and after E. intestinalis infection. TEER in infected cultures decreased to 29% of the control value after 8 days of E. intestinalis infection. Immunofluorescence microscopy showed no pronounced changes in the quantity or distribution of either Z0-1 or occludin proteins in polarized IPEC-J2 cell monolayer as a result of E. intestinalis infection compared to control monolayer at day 8 post-infection. Apoptosis assay indicated that there were about 8.14 ± 4.21 % more apoptotic IPEC-J2 cells in 3 days infected cultures compared to uninfected cultures. The results confirmed that both IPEC-J2 and IPEC-1 were infected with E. intestinalis but to a lesser extent when compared with Caco-2. This study also indicated decreased TEER in association with E. intestinalis induced apoptosis.

Library of Congress Subject Headings

Microsporidiosis

Parasitic diseases

Swine -- Diseases

Format

application/pdf

Number of Pages

108

Publisher

South Dakota State University

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