The objectives of this study were first, to investigate the effect of stress and ascorbic acid (AA) supplement during stress on intestinal microflora of broilers and layers, and secondly, to determine nitric oxide (NO) production in intestinal tract of broilers during stress, when supplemented with L-arginine, and NO production in cecal bacteria. The intestinal microflora from broilers and layers were analyzed for bacterial populations during stress and when supplemented with ascorbic acid. In both studies, stress response was induced by adrenocorticotropic hormone (ACTH) via a mini osmotic pump for 7 days, and intestinal samples were collected before and after stress response was induced. During stress, there were no significant effects on intestinal bacterial populations, but changes in intestinal microflora were found in stressed layers and broilers. When AA was supplemented during stress, both short-term and long-term, the microbial population was changed. Cecal NO production during stress, cecal bacterial NO production, and large intestinal NO production when L-arginine was supplemented in broilers were determined as nitrite using Griess reagents. The stress response was induced as in the previous studies. The cecal pouches were collected at day 7 after ACTH insertion. Nitric oxide production by the ceca of broilers during stress was decreased. The cecal bacterial NO production was determined in vitro. The cecal bacteria that produced NO were identified as Lactobacillus fermentum, and Clostridium butyricum. Supplementing with L-arginine, Nù -nitro-L- arginine methyl ester (L-NAME), and sodium nitrate did not affect bacterial NO production on MRS agar incubated anaerobically, but sodium nitrate did affect bacterial NO production on tryptic soy and anaerobic agar incubated aerobically and anaerobically, respectively. L-arginine was supplemented in broiler diet to determine the effect on intestinal NO production and microbial populations. Supplemented with L-arginine affected cecal NO production, but did not affect large intestinal NO production or microbial populations. The positive correlation coefficient between NO contents and bacterial populations was only observed in the large intestine when L-arginine was supplemented in the diet.

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Malaria parasite (Plasmodium spp.) infection in the mosquito Anopheles stephensi induces significant expression of A. stephensi nitric oxide synthase (AsNOS) in the midgut epithelium as early as 6 h post-infection and intermittently thereafter. This induction results in the synthesis of inflammatory levels of nitric oxide (NO) in the blood-filled midgut that limit parasite development. However, the Plasmodium-derived factors that can induce AsNOS expression and the signaling pathways responsible for transduction in A. stephensi have not been identified until completion of the work described herein. In my studies, I have determined that P. falciparum glycosylphosphatidylinositol (PfGPIs) can induce AsNOS expression in A. stephensi cells in vitro and in the midgut epithelium in vivo. Based on related work in mammals, I hypothesized that parasite-derived AsNOS-inducing factors signal through the insulin signaling pathway and the NF-kappaB-dependent Toll and Immune deficiency (Imd) signaling pathways. In support of this hypothesis, I have determined that signaling by P. falciparum merozoites and PfGPIs is mediated through A. stephensi protein kinase B (Akt/PKB) and DSOR1 (mitogen activated protein kinase kinase, MEK)/Extracellular signal-regulated protein kinase (ERK), kinases which are associated with the insulin signaling pathway. However, signaling by P. falciparum and PfGPIs is distinctively different from signaling by insulin and these parasite signals are not insulin-mimetic to A. stephensi cells. In other studies, treatment with pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kappaB, reduced AsNOS expression by P. falciparum merozoites in A. stephensi cells. This result suggested the involvement of Toll and Imd pathways in parasite signaling of mosquito cells. Knockout of Pelle, a proximal signaling protein in the Toll pathway, increased AsNOS expression following parasite stimulation, suggesting that the Toll pathway may negatively regulate signaling by Plasmodium-derived AsNOS-inducing factors. In contrast, knockout of TGF-beta-activated kinase 1 (Tak1), a proximal signaling protein in the Imd pathway, reduced AsNOS expression by 20% relative to the control, suggesting that the Imd pathway is required for signaling by Plasmodium-derived AsNOS-inducing factors. Despite the NO-rich environment of the midgut, Plasmodium development is not completely inhibited. This observation suggests that Plasmodium may have efficient detoxification systems during sexual development in A. stephensi. To identify Plasmodium defense genes that may defend parasites against nitrosative stress caused by AsNOS induction, expression of several antioxidant defense genes known to function in nitrosative stress defense in a variety of organisms were examined during sporogonic development. Notably, increased expression levels of P. falciparum peroxiredoxins containing 1 or 2 cysteines (1-cys or 2-cys PfPrx) were associated with periods of parasite development just prior to and during parasite penetration of midgut epithelium, an event associated with significant AsNOS induction in the midgut. The provision of N omega-L-arginine (L-NAME), a known inhibitor of NOS enzyme activity, to A. stephensi with Plasmodium culture by artificial bloodmeal significantly reduced expression of 1-cys and 2-cys PfPrx indicating that these gene products may function to protect parasites against nitrosative stress induced by AsNOS. / Ph. D.