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Globally, infectious diseases are responsible for major conservation and economic losses in wild and farmed fish populations. Prevention tools, including vaccination and breeding for genetic disease resistance, are used in many systems to prevent mortality by such diseases. Studies are often done to evaluate the efficacy of a preventative method at reducing disease, but the impact on transmission is rarely studied. Protection under diverse field conditions, such as variable pathogen exposure dosages, is also not fully understood. Furthermore, there is little information on how preventative methods alter host-pathogen relationships. For example, it is largely unknown how vaccination impacts non-target pathogens that co-infect the host. These knowledge gaps make it difficult to infer the epidemiological impacts of disease prevention tools. In an attempt to fill these gaps, we investigated two leading pathogens in rainbow trout (Oncorhynchus mykiss) aquaculture: infectious hematopoietic necrosis virus (IHNV) and Flavobacterium psychrophilum. We evaluated the impacts of vaccination and genetic disease resistance on mortality and transmission across a range of challenge dosages of IHNV and F. psychrophilum to accurately reflect field variability. There was evidence of a dosage effect; as dosage increased, shedding increased and vaccine efficacy decreased. We also evaluated how vaccination and genetic disease resistance impact transmission dynamics during simultaneous and sequential co-infection of IHNV and F. psychrophilum. Our results indicate co-infected fish shed more of both pathogens than they do in single infections, and the order that the pathogen infected the host may impact transmission in both pathogens. Furthermore, vaccine efficacy may be diminished by co-infection. These studies are aimed at developing a more robust framework for inferring the efficacy of disease prevention strategies. Our results will also help to inform and improve disease management in one of the top aquaculture species in the United States.

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<p> This dissertation focuses on the use of whole genome sequencing (WGS) for the identification of disease causing variants in canine genomes. A brief review on the historical milestones of genetics, the creation and popularization of the fast throughput DNA sequencing technologies and their advantages and potential problems and biases, the importance of the study of canine genetics and the current state of the canine genome assembly is presented. Our lab sequenced ~100 dogs in the attempt to discover disease-causing variants. So far 20 such variants have been identified. This dissertation contains detailed accounts of the discovery variants likely to be responsible for four canine diseases. Those diseases are: Paroxysmal dyskinesia in Soft Coated Wheaten Terriers that is associated to the missense mutation <i>PIGN:c</i>.398C>T; two different forms of neuronal ceroid lipofuscinosis, one in Australian Cattle dogs caused by CLN5:c.619C>T, and one in the Cane Corso caused by the splice site mutation <i>PPT1c.124+1G>A</i>; and a Shiba Inu GM2 gangliosidosis caused by <i>HEXB.c:948_950delCCT</i>. Furthermore, examples of not so successful attempts, possible reasons for failures and suggestions to successfully conclude other ongoing investigations.</p><p>

The disease caused by the protozoan Perkinsus marinus has been a major source of mortality in the eastern oyster, Crassostrea virginica. Variations in susceptibility to P. marinus infection among eastern oysters collected from the Chesapeake Bay and Gulf of Mexico, as well as between eastern and Pacific (Crassostrea gigas) oysters were determined. Since oyster host defense may play a role in determining susceptibility to pathogen infection, cellular and humoral defense activities of the oyster and their interactions with P. marinus were investigated. Procedures also had to be established to isolate, purify, and propagate in vitro, P. marinus. Eastern oysters from all sites were found to be highly susceptible to the pathogen. Cellular and humoral activities were significantly affected by heavy intensity of P. marinus infection. Prevalence and intensity of P. marinus infection were lower in Pacific oysters than in eastern oysters. Pacific oysters may offer a less favorable environment for the development of P. marinus compared to eastern oysters for at least two possible reasons: the elevated cellular and humoral activities may degrade the parasite more effectively, and lower plasma protein levels may limit parasite growth. Incubation of merozoites with hemocytes of eastern and Pacific oysters in vitro suggested that limited intracellular killing of P. marinus occurred but that killing was not mediated by oxygen metabolites. Perkinsus marinus was successfully propagated in vitro in a culture medium containing most of the known constituents of cell-free oyster hemolymph. Cultures of the parasite were initiated from heart fragments of infected oysters. The cultured protozoan was similar in morphology to P. marinus, enlarged in fluid thioglycollate medium, reacted with polyclonal antibodies raised against hypnospores and was infective. Continuous cultures of P. marinus could also be initiated from hypnospores. Two types of division, progressive cleavage and successive bipartition of the mother cell protoplast, were observed.

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The gypsy moth, Lymantria dispar L., is one of the most damaging pests of the deciduous forests in the United States. It was accidentally introduced from Europe in 1868 by an amateur naturalist in eastern Massachusetts. High density gypsy moth populations are regulated primarily by a nuclear polyhedrosis virus (LdNPV). LdNPV is transmitted by feeding the LdNPV contaminated foliage or the contaminated egg chorion on the way out from the egg by a larva. In 1989, an entomophthoralean fungus, Entomophaga maimaiga Humber, Shimazu et Soper was discovered in the northeastern United States, which caused massive epizootic in both low and high density gypsy moth populations. My study focused on the interactions between E. maimaiga and LdNPV. Laboratory bioassays in which I inoculated gypsy moth larvae with LdNPV and E. maimaiga at the same time indicated that the majority of dually inoculated larvae die from E. maimaiga because of the shorter incubation period of E. maimaiga (5-7 days) compared to LdNPV (14 days) at 20$\sp\circ$C. When the larvae were inoculated with E. maimaiga, 10 days after LdNPV inoculation, there was an apparent synergistic effect of E. maimaiga with LdNPV. Dually inoculated larvae died producing LdNPV propagules, 1-2 days earlier than the larvae inoculated with LdNPV alone. Small-scale field experiments conducted in mesh-bags showed that artificial rainfall increases the E. maimaiga transmission. In a naturally occurring, moderate density gypsy moth population, I found that the LdNPV infection level was little affected by the presence of E. maimaiga. Host heterogeneity is suspected as one of the factors leading non-linear LdNPV transmissions. I showed that the host heterogeneity cannot explain the E. maimaiga epizootic observed in low density populations. I experimentally demonstrated this by comparing the E. maimaiga infection rates in feral (experienced the E. maimaiga/LdNPV epizootic in their parental generations) and laboratory reared (with no epizootic experience) larvae. This is probably due to the short period to which the North American gypsy moths have been exposed to E. maimaiga, so these gypsy moths have not had chance to develop resistance against E. maimaiga.

African trypanosomes are parasitic protozoa that cause fatal disease in humans and domestic livestock. Though domestic animals are susceptible to trypanosomiasis, certain wild animals have developed resistance mechanisms. The African cape buffalo can control the early stages of trypanosome infection by increasing plasm and erythrocyte levels of the reactive oxygen intermediate (ROI), hydrogen peroxide (H2O2). Cape buffalo are able to increase the amount of H2O2 produced by the purine catabolic enzyme xanthine oxidase by supplying more substrate in the blood microenvironment and by decreasing the plasma, and erythrocyte levels of catalase, a H2O2 degrading enzyme. My work has been to attempt to manipulate the ROI generating capabilities of mice to recreate this defense mechanism in a small laboratory animal model. Initial experiments revealed that Balb/c, C57Bl/6 and C3H/HeOu mice have all the purine catabolic enzymes necessary to generate trypanocidal amounts of H2O2. However, unlike the buffalo, all of these mouse strains are susceptible to infection by trypanosomes and their serum is not trypanocidal in vitro. Therefore, the ROI generating capabilities of Balb/c, gamma-interferon knockout Balb/c mice and OH catalase-reduced mutant mice were altered by feeding additives, either in a pellet or liquid diet base, to the mice to either inhibit or enhance ROI generation. Only one combination resulted in a slight but significant decrease in parasitemia: C3H catalase-reduced mice fed the catalase inhibitor 3-amino-triazole. Though parasitemia was not effected in any of the other mouse/diet combinations, the mice were effected in some experiments. Gamma-interferon knockout male mice died significantly earlier than female mice, with or without ROI alterations. Mice maintained on a liquid diet had a significantly reduced acceleration of trypanosome-induced inflammation, but this effect was abrogated when the glutathione precursor N-acetyl-cysteine was included in the diet. This indicates that the diets do have an effect on ROI generation in mice, though parasitemia remained largely unaffected. Thus trypanosomes are able to avoid or neutralize ROI in mice. However, they are susceptible to similar ROI levels in buffalo, suggesting a component in the mice that the trypanosomes are able to utilize to inhibit ROI-induced damage.

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