Graduate Thesis Or Dissertation
 

Pseudoloma neurophilia : progression of infection and transmission characteristics of a microsporidian parasite in a model vertebrate, Danio rerio

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  • The microsporidian parasite, Pseudoloma neurophilia, is the most commonly diagnosed infectious disease in laboratory populations of the zebrafish, Danio rerio. Infections by P. neurophilia are generally subclinical, however, they can become acute either incidentally or due to experimental immune suppression. Non-protocol induced variation can confound results in laboratory experiments using such fish. As a result, there has been growing interest in sensitive diagnostic assays for P. neurophilia and demand for P. neurophilia specific-pathogen free zebrafish lines among the zebrafish research community. The high prevalence of P. neurophilia in zebrafish provides the opportunity to investigate the progression of infection and transmission characteristics of a microsporidian parasite in a well-developed model vertebrate host species. I developed a real-time PCR-based assay combined with the use of sonication to improve spore disruption which has a sensitivity that is 10-100 times more sensitive than a previously published conventional PCR-based assay. The microsporidium infects ovaries and eggs, thus, I developed a sampling method for the testing of water from spawning fish and demonstrated the utility of testing spawn water, eggs, and sperm for the non-lethal detection of P. neurophilia in adult fish. The presence of P. neurophilia in spawn water and eggs from infected adults provided the initial evidence of vertical transmission of P. neurophilia in zebrafish. Intraovum transmission of P. neurophilia was directly observed by histological screening of larval fish spawned from infected adults and by analyzing eggs using a transmitted light dissecting microscope. The prevalence of intraovum transmission was determined by screening surface-decontaminated eggs from 27 paired spawns using the real-time PCR based assay. Intraovum transmission was detected in 4 of the 27 spawns and the prevalence of intraovum P. neurophilia in the eggs from these spawns was determined to be approximately 1%. Parasite DNA was also detected in the spawning water from 11 of the 27 spawns, highlighting the potential for extraovum transmission of P. neurophilia. I investigated the early stages of P. neurophilia infection in experimentally infected larval zebrafish. This was accomplished using a combination of standard hematoxylin and eosin stain, the Luna stain, and an in situ hybridization probe specific to the small-subunit ribosomal DNA gene of P. neurophilia, which I developed. At 12 hours post exposure, P. neurophilia was mainly visualized as intact spores in the intestinal lumen, and proliferative stages developing in the epithelial cells of the anterior intestine and the pharynx, and within the hepatocytes of the liver. Proliferative (presporogonic) stages were visualized in these tissues and in the pancreas and kidney at 36 and 48 hours post exposure and in the spinal cord, eye, and skeletal muscle beginning at 72 hours post exposure. The first spore stages of P. neurophilia were observed at 96 hours post exposure in the pharyngeal epithelium, liver, spinal cord and skeletal muscle. The parasite was observed in the brain of larval fish at 120 hours post-exposure. The distribution of the early stages of P. neurophilia and the lack of mature spores until 96 hours post exposure suggests that the parasite gains access to organs distant from the initial site of entry, likely by penetrating the intestinal wall with the polar tubule, and that autoinfection does not occur at any detectable frequency during the initial stages of infection.
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