Perkinsus marinus is an alveolate protozoan parasite of the eastern oyster (Crassostrea virginica) which is responsible for much of the decline in United States oyster populations. Perkinsus marinus can be cultured in vitro, but is rapidly attenuated in the process. Supplementation of a protein-free medium with oyster products altered proliferation, changed protease expression in the parasite extracellular products (ECP), induced morphological forms typically seen in vivo, and partially reversed parasite attenuation. Supplements derived from dissected oyster tissues were used to determine if these changes could be differentially elicited. These supplements, with the exception of adductor muscle, reduced proliferation. Whole oyster and digestive gland/gonad supplements favored palintomic, rather than binary, fission. The total ECP protease activity was generally decreased in supplemented cultures, though gill/mantle supplements may have induced proteases. A low molecular weight subset of proteases was upregulated most effectively by heart- and adductor muscle-derived supplements. Serine proteases and other ECP proteins may be virulence factors. Attempts to create antibodies to study P. marinus cells and ECP have been largely unsuccessful due to poor immune responses and crossreactivity. Ultrafiltration-concentrated P. marinus ECP were poorly immunogenic and toxic to experimental animals. Immunogenicity was not substantially affected by heat denaturation or proteolytic inhibition. Co-administration of ECP with oyster plasma caused a suppression in the anti-plasma antibody response with restriction of epitope recognition. Analysis of medium constituents revealed that a surfactant, Pluronic F-68 (PF68), was immunosuppressive. Although isolated protein antigens from the ECP remained immunosuppressive, separation of the antigens from PF68 enabled antibody production. Five monoclonal antibodies were created against ECP from unsupplemented medium and were used to study ECP function, regulation, and mechanism of storage and release. ECP are secreted by release from the cell wall and from two morphologically distinct intracellular compartments. A sandwich ELISA allowed quantification of an ECP protein with significantly reduced expression in supplemented cultures. Another antibody, which specifically bound to trophozoite and tomont walls, was used to investigate morphological and antigenic changes during thioglycollate-induced formation of prezoosporangia, and confirm supplement-induced formation of prezoosporangia. This antibody labeled P. marinus cells in fixed oyster tissue in a species-specific manner.

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In 1999 a mass mortality of the American lobster (Homarus americanus) occurred in western Long Island Sound (WLIS). Although the etiology remains unknown, warm bottom water temperature, hypoxia, heavy metal poisoning, and pesticides have been suggested as casual factors. Subsequently, lobsters from WLIS have continued to display symptoms of morbidity that include lethargy and cloudy grey eyes, caused by idiopathic lesions. The effects of these lesions on lobster vision are unknown. We therefore used electoretinography (ERG) to document changes in visual function in lobsters obtained from WLIS, while simultaneously using histology to quantify the extent of damage. Of the lobsters collected from WLIS, seventy three percent showed damage to photoreceptors and optic nerve fibers including necrosis of the optic nerve, breakdown of the rhabdom, and hemocyte infiltration through the basement membrane into the ommatidia. Animals with more than 15% of photoreceptors exhibiting histological damage also exhibited markedly reduced responses to 10 ms flashes of a broad-spectrum white light. Specifically, the maximum voltage (Vmax) response was significantly lower and occurred at a lower light intensity as compared to responses from lobsters without idiopathic lesions. Lobsters from outside WLIS did not show such reduced changes to their vision. Lobsters from WLIS still appear to be subjected to an unknown stressor with an idiopathic etiology that is causing significant functional damage to their visual system.

Striped bass (Morone saxatilis) in Chesapeake Bay, USA, are experiencing an epizootic of mycobacteriosis. This disease, caused by bacteria in the genus Mycobacterium, causes granulomatous lesions of the skin and viscera. Diseased fish are often emaciated, and fish with skin lesions may be significantly disfigured. The overall goal of this work was to examine aspects of the pathobiology of mycobacteria in striped bass via laboratory exposure studies and cellular assays. Striped bass were injected intraperitoneally with a sublethal dose of Mycobacterium marinum, M. shottsii, or M. gordonae and sampled for histology and bacteriology at regular intervals to 45 weeks post-injection (p.i.). Fish injected with M. marinum developed granulomas in the mesenteries, spleen and anterior kidney. Acid-fast bacilli (AFB) were rare in initial stages of disease whereas granulomas at 8 weeks p.i. and later frequently contained large numbers of AFB. Secondary disease was observed in some fish between 26 and 45 weeks p.i., with granuloma disintegration, severe inflammation, and elevated splenic bacterial densities. Relative to fish injected with M. marinum, fish injected with M. shottsii or M. gordonae did not develop severe pathology. Granulomas were observed in the mesenteries, but were not observed in the spleen or, with one exception, anterior kidney. M. shottsii and M. gordonae both established persistent splenic infections. The ultrastructure of developing M. marinum granulomas in experimentally infected bass was examined. Formation of large macrophage aggregations containing intracellular bacilli was observed within the peritoneal cavity shortly after injection. M. marinum were always contained within phagosomes, and apparent phagolysosomal fusion was frequently observed. Epithelioid transformation of macrophages was observed. Ultrastructural observation of bacilli within granulomas agreed with histologic findings. The in vitro interaction between macrophages and intracellular M. marinum was examined ultrastructurally and with a quantitative bactericidal assay. Phagosomes containing M. marinum were fused at high rates by pre-labeled lysosomes. No differences in lysosomal fusion rates were observed between phagosomes containing live or heat-killed M. marinum. Intracellular M. marinum remained largely viable for the duration of the assay (72 hours). Heat-killed M marinum were resistant to lysis within phagolysosomes.

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Fertilization initiates a series of intracellular calcium [Ca 2+]i oscillations in the oocyte that promote its exit from and completion of meiosis. In mouse oocytes, the [Ca2+] i oscillations arrest at the pronuclear stage and this is accompanied by a down-regulation in the inositol 1,4,5-trisphosphate receptor type 1 protein (IP3R-1). The goal of this dissertation was first, to investigate the signaling pathway responsible for IP3R-1 degradation during fertilization. Eggs were either incubated in or injected with several compounds that induce egg activation, and the IP3R-1 protein content evaluated using Western blot analysis. Exposure to ethanol or ionomycin, which induce a single [Ca 2+]i rise, failed to induce down-regulation of the IP3R-1. However, injection of porcine sperm factor (pSF), which presumably stimulates IP3 production, or adenophostin A, an IP3R-1 agonist, both induced degradation of the receptor. Exposure to thimerosal, that modulates the IP3R without stimulating IP3 production, also initiated down-regulation. [Ca 2+]i oscillations induced by SrCl2, which does not trigger IP3 production, failed to evoke down-regulation. Degradation of the IP3R-1 appears to be mediated by the proteasome pathway because it was inhibited by lactacystin, a specific proteasome inhibitor. It was therefore propose that persistent stimulation of the phosphoinositide pathway by sperm during fertilization leads to down-regulation of the IP3R-1. The second goal of this research was to investigate the factor(s) involved in the arrest of [Ca2+]i oscillations seen at PN formation. This was achieved by generating parthenotes with similar IP3R-1 numbers and similar kinase activity at three different stages of the cell cycle, metaphase II (MII), pronuclear (PN), and the post pronuclear (PPN) stages. These models allowed us to study the effect of different parameters on PN [Ca2+]i arrest: (1) Cell cycle, (2) IP3R-1 number and sensitivity, and (3) content of intracellular Ca2+ stores. Results from these experiments demonstrate that the cell cycle determines the ability of the egg to exhibit [Ca2+] i oscillations by controlling the decrease in sensitivity of the IP3R-1 at the PN stage. This desensitization appears to be due to the inability of the IP3R-1 to undergo a conformational change and this phenomenon seems to be regulated by presently unknown factors.

Equine laminitis is a devastating condition that results in the failure of the tissue responsible for suspending the skeleton within the hoof capsule. The digital laminae is composed of two interdigitated layers, the dermal lamellae surrounding the distal pedal bone, and the epidermal lamellae, which interfaces with the hoof wall. During laminitis, these layers separate, allowing for rotation and sinking of the pedal bone. While there are multiple diseases and physiological conditions associated with the development of laminitis, including sepsis, metabolic syndrome, and unequal weight bearing, the exact cause remains elusive. Prior work by our research group identified the metalloprotease ADAMTS-4 as a potential early instigator of disease. The data presented herein catalogs the distribution of the substrates of this enzyme, aggrecan and versican, the ramifications of ADAMTS-4-mediated versican loss in the laminae, and further expands into the repression of the canonical wnt signaling pathway and potential additional metalloprotease (MMP) involvement in disease, utilizing a model of acute, carbohydrate-induced laminitis. Additionally, samples from other models of laminitis induction and clinical samples were screened for differential expression of relevant gene markers, including versican, members of the canonical wnt signaling pathway, and MMP-1 and -13. Together, these data provide a characterization of laminar pathology in the carbohydrate-induced model, as well as highlighting key similarities and differences amongst multiple methods of disease development, and lay important groundwork for developing clinical therapeutic interventions.

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