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.

It is important to be able to predict the potential spread of water borne diseases when building dams or redirecting rivers. This study was designed to test whether the use of a growing degree day (GDD) climate model and remotely sensed data (RS) within a geographic information system (GIS), could be used to predict both the distribution and severity of Schistosoma haematobium. Growing degree days are defined as the number of degrees centigrade over the minimum temperature required for development. The base temperature and the number of GDD required to complete one generation varies for each species. A monthly climate surface grid containing the high and low temperature, rainfall, potential evapotranspiration (PET), and the ratio of rain to PET was used to calculate the total number of GDD provisional on suitable moisture content in the soil. The latitude and longitude for known snail locations were used to create a point file. A 5km buffer was made around each point. Mean values were extracted from buffer areas for Advanced Very High Resolution Radiometer (AVHRR) data on maximum land surface temperature (Tmax) and normalized difference vegetation index (NDVI). The values for Tmax ranged from 15-28 and the NDVI values were 130-157. A map query found all areas that meet both criteria and produced a model surface showing the potential distribution of the vectors for this disease. Results indicate that the GDD and AVHRR models can be used together to define both the distribution range and relative risk of S.haematobium in anticipated water development projects and for control program planning and better allocation of health resources in endemic vs. non-endemic areas.

The leading viral killer of commercially produced channel catfish is Channel Catfish Virus (CCV). Studies conducted to evaluate DNA vaccination against CCV compared encoded gene, dose, multiple DNA vaccines, and immune response to vaccination. Genes were selected (ORFs 1 and 3 [immediate early genes], ORFs 6, 19, and 46 [membrane genes], and ORF59 [putative major envelope glycoprotein gene], cloned into a plasmid, and expressed in mammalian and fish cell culture to detect predicted molecular weight proteins. Plasmid vaccines were injected into fish muscle in doses of 50 μg, 25 μg, 5 μg, or 1 μg and efficacy was evaluated upon challenge. Immune responses were measured by Mx gene expression (α/β interferon indicator), serum neutralization, specific antibody stimulation (ELISA), and DNA vaccine specific expression. Comparisons were made between published live attenuated CCVTK-, DNA vaccines (KN59 and KN6), and DNA vaccines tested in this study. In all experiments, no protection was observed. Multiple groups of vaccines delivered per fish were tested for efficacy and protection was not observed. The live attenuated CCVTK- and published CCV DNA vaccines (KN59 and KN6) were not protective. Mx gene expression in response to DNA vaccination showed positive expression profiles at all time points, but most often at day 3 in all experiments. In the multiple group vaccination, Mx gene expression was detected at a higher level overall and at day 35, indicating that multiple antigens induce a stronger innate response with longer duration. In all experiments, serum neutralizing titers were low in most treatments (< 10), but weakly reactive in the multiple group vaccination (3 groups with titers > 10) and the comparison vaccination (4 groups with titers > 10). ELISA of vaccinated fish sera detected low reactivity, but significant levels of reactivity were observed between sera from pORF46 and pORF3 and the negative control. In the comparison vaccination, DNA vaccine specific gene expression was detected in all groups and at most time points, indicating the DNA vaccines were transcriptionally functional. A protective vaccine against CCV is a goal to be striven for, because currently available vaccines may not be suitable for commercial use.

The overall goals of this research were to develop a reproducible method of detecting stable DNA insertion into Japanese quail and provide a method for gene location on avian chromosomes. This research resulted in the development of a different method of obtaining chromosome spreads in Japanese quail, the establishment of primed in situ hybridization as a method for the chromosomal gene detection in birds, development of Teflon-coated coverslip slides to facilitate laser microdissection of 0.5 Ým samples, and chromosomal identification of proinsulin transgene insertions by laser microdissection and nucleotide sequence from G2 Japanese quail. The 28S rDNA was found on a macrochromosome and a microchromosome pair by primed in situ hybridization, fluorescent in situ hybridization, and silver staining. Teflon-coated coverslip slides were created to facilitate laser microdissection of avian chromosomes for DNA amplification and nucleotide sequencing. Transgenic G2 Japanese quail produced in Dr. Richard Cooper¡¦s laboratory were identified by laser microdissection and found to have 2-5 chromosomal insertions of the proinsulin transgene.

Bacillus anthracis is a bacterium that causes severe disease mainly in ruminants, but can affect any mammal, including humans. A popular method for the detection of this organism is susceptibility of the bacterial isolate to g bacteriophage. However, to date no study on the resistance of a wide variety of B. anthracis isolates has been conducted. The following study examines the rate of resistance of a wide range of B. anthracis isolates to g phage as well as another phage specific for B. anthracis known as Cherry phage. We also compared susceptibility to phage with another detection method, susceptibility to penicillin, to determine any association between the two. The origin of the resistant isolates was examined to determine associations between resistance and isolate origin. Finally, the gross structure and resistant rates of the two phages were compared to determine any relation between the two viruses. We found that B. anthracis showed 20% resistance to g phage, which we propose is too high to continue its use as a reliable diagnostic tool. No association was found between resistance to penicillin and resistance to phage. No association was found between isolate origin and resistance. No conclusions could be drawn as to the relationship between the two phages.

The first event in the infection process of Ostertagia ostertagi in cattle is the process of exsheathment. Before trichostrongylid nematodes can transition from a free-living infective stage larva (L₃) on pasture to a parasitic existence within the ruminant host, it must first undergo exsheathment. Exsheathment is the process whereby the L₂ cuticle retained from the previous molt is cast from the L₃. Exsheathment enables the developmental transition from a free-living stage on pasture to a parasitic existence in the bovine host. For those species with a predilection site in the abomasum, such as O. ostertagi, exsheathment is initiated as the larvae pass through the rumen. Although the stimulus for exsheathment is not known, previously reported biochemical studies on exsheathment suggest the role of a carbonic anhydrase (CA). Partial support for this hypothesis comes from the reported failure of the Haemonchus contortus L₃ to exsheath following pretreatment with ethoxzolamide, a known inhibitor of CA's. Although convincing, a CA has not been previously reported from a trichostrongylid nematode. Therefore, the objective of this work was to isolate a CA gene from O. ostertagi L₃ and subsequently quantitatively measure its expression during in vivo exsheathment of O. ostertagi L₃. This work resulted in the successful isolation, cloning and sequencing of a gene that showed 90.5 % sequence identity with the CA eukaryotic consensus sequence and was 78% and 55% similar to the Caenorhabiditis elegans cah-6 and human CAIII isozyme, respectively. This is the first CA isolated from a gastrointestinal nematode parasite. The enzyme was consequently named OoCAIII. The expression pattern of OoCAIII in O. ostertagi L₃ suggests this particular CA is not responsible for initiating exsheathment, but perhaps has a role in immediate early developmental events following initiation of exsheathment. Analysis of the first 1,758 bases of the proximal and distal promoter regions of the gene suggested OoCAIII is regulated in part by transcription factors associated with hypoxic signaling and development.