Thesis (Ph. D.)--Texas A&M University, 2005. / "Major Subject: Veterinary Microbiology" Title from author supplied metadata (automated record created on Apr. 27, 2007.) Vita. Abstract. Includes bibliographical references.

Thesis (Ph. D.)--Texas A&M University, 2007. / "Major Subject: Veterinary Microbiology" Title from author supplied metadata (automated record created on Oct. 13, 2008.) Vita. Abstract. Includes bibliographical references.

Thesis (Ph. D.)--Texas A&M University, 2008. / "Major Subject: Veterinary Microbiology" Title from author supplied metadata (automated record created on Oct. 13, 2008.) Vita. Abstract. Includes bibliographical references.

Sentinel herds were used to study the epidemiology of arboviruses in Oman and North Yemen. The results indicate that bluetongue virus (BTV) is fairly widely distributed and is enzootic in Northern Oman, all year round. Virus type specific antibodies to five BTV serotypes (3, 4, 17, 20 and 22) were detected. Antibodies to epizootichaemorrhagic disease virus (EHDV) type 2 and EHDV318 were also present although to a lesser extent. No EHDV1 antibodies could be detected among the sentinel animals during the entire period. Prevalence of neutralizing antibodies against Akabane virus was found in a wide range of domestic animals in all countries of the Arabian Peninsula but the virus does not seem to be enzootic there. The possibility of windborne, infected vectors, from virus enzootic areas initiating these incursions into the Arabian Peninsula is discussed. A total of four arboviruses were isolated and identified (two BTV4 and two Akabane virus). Three of them from vertebrate hosts and one was from a species of Culicoides. This isolation of Akabane virus from C. imicola is the first record of this virus from this species of midge. Entomological investigations were undertaken, into the population dynamics of potential Culicoides vectors and the results correlated with the climatic conditions. Sixteen species of Culicoides were identified among which four were new to science, these included C. arabiensis, C. ibriensis, C. bueltikeri and C. neoschultzei. Vector competence studies have shown that the Omani virus isolates multiply in C. variipennis after oral ingestion and that both viruses are maintained for at least 10 days post-infection. Biochemical analysis and comparison of the proteins induced in infected cells and genomic profiles of the Omani virus isolates were analysed by polyacrylamide gel electrophoresis and formaldehyde-agarose gel electrophoresis and compared with several other prototype reference strains. Minor differences in BTV specific proteins and dsRNA profiles between various strains were observed. Akabane virus specific proteins were indistinguishable between the different Akabane strains, but minor differences were observed in their genomic profiles and that their RNA was polyadenylated.

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Photobacterium damselae subspecies piscicida, previously known as Pasteurella piscicida, is an important pathogen of hybrid striped bass and many fish species cultured in brackish water in the United States, Japan, Europe, and the Mediterranean. The purpose of this study is to investigate virulence mechanisms that contribute to the pathogenesis of this organism. The ability of P. damselae to survive/replicate within hybrid striped bass macrophages was evaluated with an in vitro killing assay. Results indicated that the numbers of bacteria recovered from macrophages at 3, 6, 12, and 18 hours of incubation increased significantly over time. In contrast, the numbers of Escherichia coli control strain recovered from macrophages declined at the same designated incubation times. Light and electron microscopy confirmed internalization, uptake, and multiplication of bacteria within spacious, clear vacuoles in the macrophages. Using acid phosphatase as a lysosomal marker, it was shown that P. damselae inhibits phagolysosomal fusion. Invasion and replication of P. damselae within epithelioma papillosum carpio (EPC), channel catfish ovary (CCO), and fathead minnow (FHM) cells lines was also evaluated using an in vitro invasion assay. All three cell lines were susceptible to invasion and supported replication of P. damselae. Fathead minnow cells were more susceptible to invasion than the other two cell lines as indicated by greater numbers of infected cells and recovered bacteria at time 0. Using light and electron microscopy, invasion of cells by bacteria was observed as early as 30 minutes after infection, and intracellular bacteria were observed in large, clear, membrane-bound vacuoles. The intracellular location of P. damselae was confirmed using ruthenium red staining to discriminate between the extra- and intra-cellular spaces. Using flow cytometry, results indicated that P. damselae induces apoptosis in phagocytes obtained from hybrid striped bass head kidney and after 12, 18, and 24 hours of incubation, the relative numbers of cells infected with P. damselae showing signs of apoptosis increased over time and were significantly greater than the controls. The relative numbers of apoptotic cells that were infected with the formalin-killed strain increased, but not significantly, above the control after the same designated times of incubation.

Vaccination programs are designed to protect an animal from infection, however, depending upon the age and health of the animal vaccination may not stimulate a protective humoral response. It is possible that, as in the human and mouse models, geriatric equines may be less responsive than their younger counterparts to current vaccination protocols. The purpose of this study was to identify an age related diminution in the primary and secondary immune responses of geriatric horses in response to vaccination. Two groups of horses were sampled. The first group consisted of an open herd of 39 privately owned horses, varying in age from 2-27 years of age. The second group consisted of a closed herd of 24 horses ranging in age from 7-over 30 years of age. Each group was vaccinated twice intramuscularly with a commercial equine influenza virus vaccine (Ft Dodge, A/Eq/KY/97). Additionally, one group was vaccinated with keyhole limpet hemocyanin (KLH) in order to study a primary immune response to a novel antigen. The other group was vaccinated with ovalbumin (OVA) for the same purpose. Blood samples were obtained via jugular venipuncture prior to vaccination and monthly thereafter for 5 months. All sera samples were analyzed for antigen-specific antibodies using an ELISA assay. Our results show that all horses responded to primary vaccination with either KLH or OVA, irrespective of age. In contrast, when vaccinated with influenza the middle aged and older horses showed significant differences in the their response between both the herds and the age groups. Thus we were able to demonstrate that age was not a factor in the generation of a primary immune response but was a factor in the generation of a memory immune response. Future research should focus on whether increased frequency of vaccination does in fact increase or maintain vaccine responses and whether antibody responses measured in vitro actually correlate with protection in vivo.

To study the protective responses of cyathostome-infected ponies, two challenges were performed employing animals with different histories of exposure to these parasites. The hypothesis developed and to be tested in these experiments was that ponies that had longer exposure to cyathostome contaminated pastures would express acquired resistance to infection. The assumption behind this hypothesis was that helminth-naïve ponies infected with cyathostomes would eliminate the infection using only innate immune responses. Whereas previously exposed ponies would eliminate the infection with acquired immune responses, and these would be more effective in ponies with longer exposure to cyathostomes. Thus, helminth-naïve animals would acquire the largest number of worms, followed in decreasing order by young and then adult ponies. Two types of challenges were used: an experimental infection with 150,000 cyathostome infective third stage (L₃) given over a period of 5 days, and the natural acquisition of infection by grazing a cyathostome contaminated pasture for 7 weeks. The natural challenge was performed to confirm the data obtained with the experimental challenge, therefore to validate its use. The parasitological data recovered showed that ponies with acquired resistance to cyathostome infections had reduced total number of worms, of developing larvae, luminal fourth stage larvae, adult parasites and of cyathostome species. The acquisition of resistance was also observed as elevations in the hypobiotic larvae numbers and of intestinal mast cells, intestinal and peripheral eosinophils, and antibody responses. These responses were consistent with increases in Th2 type cytokines, principally IL-4. The data obtained suggest that the immune mechanisms of resistance developed in ponies with acquired protection to cyathostome are slow to develop and are targeted against each parasite stage present in the host. These results warrant further research in the area, especially in the difference between immune mechanisms of helminth naïve ponies and animals with short exposures to cyathostome contaminated pastures.

Breast cancer is one of the most common malignancies in women. It continues to be a major burden and cause of death among women worldwide. Molecular oncology is now one of the most promising fields that may contribute considerably to diagnosis of breast cancer and its metastases addressing major problems with early detection, accurate staging, and monitoring of breast cancer patients. The overall objective of these feasibility studies was to contribute to improved diagnosis, prognosis, and prediction of breast cancer disease through the development of reagents and protocols for the use of molecular biological advances and the assessment of the relative potential of these diagnostic procedures for the detection and quantification of multiple specific mRNA tumor markers. Newest molecular technologies such as real-time quantitative TaqMan RT-PCR assays, microarray analyses, and production of in-house arrays were included in the study. Tissue, blood, and bone marrow samples were obtained from surgeries of confirmed and suspected breast cancer patients. TaqMan assays were performed for six mRNA markers: MAGE 3, HER2/NEU, MGB 1, CK 20, PSA, and HPR. Low-density nylon arrays with 265 immobilized genes included in cell to cell interactions were used for microarray analyses. Three highly overexpressed genes from microarray analyses and negative controls were selected for custom spotting on nylon membranes to produce in-house arrays. It was concluded that TaqMan assays can be easily designed and implemented for the screening of a large number of clinical specimens when including carefully selected controls, high purity RNA from samples, and a set of mRNA markers. Custom arrays can be produced incorporating multiple selected mRNA markers. It is suggested that the initial screening of biological samples could be done by microarray analyses and individual positive samples could be confirmed by additional tests using real-time quantitative TaqMan assays.

Complete sequencing and annotation of the M. leprae genome has provided new information related to proteins constituting its hypothetical proteome. Since M. leprae can not be grown in vitro, novel approaches are needed to determine which proteins are expressed during infection and whether these proteins are related to pathogenesis. Secreted proteins represent a distinct group of protein with respect to their structure and function, contribution to virulence and are of particular importance for vaccine development because they are often immunogenic and have the potential to be recognized early in infection. The objectives of this study were: 1) to identify putatively secreted proteins of M. leprae based on protein sequences homologies with known MT secreted proteins; 2) to apply bioinformatic tools designed to assess proteins for secretion, to proteins selected in objective 1 with the goal of improving the likelihood that selected proteins are secreted by M. leprae, 3) to validate secretion of selected ML proteins through genetic cloning of predicted secreted ML protein genes using surrogate host bacteria, E. coli and M. smegmatis. Bioinformatics identified 24 proteins with high probability for secretion in M. leprae. Fifteen of 24 ML genes showed more than 50% amino acid homology with their M. tuberculosis counterparts and were studied for gene expression and secretion. mRNA analysis identified transcripts for all Sec-dependent pathway proteins of 15 genes predicted to be secreted in M. leprae. PhoA fusion studies in E. coli showed that 5 of 6 (83%) ML proteins (ML0091, ML0097, ML0620, ML1811 and ML1812) were secreted in E. coli and 2 of 7 (29%) proteins (ML0715 and ML2569) were secreted in M. smegmatis. Only lipoproteins were secreted in M. smegmatis suggesting the importance of mycobacterial-related characteristics for secretion of ML lipoproteins. These results suggest that bioinformatic tools are reliable predictors for identifying secreted proteins in M. leprae and support the hypothesis that Sec-dependent secretion exists in M. leprae.