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.

Among the gastrointestinal nematode parasites that cause the most problems to small ruminants, Haemonchus contortus is one of major concern. Currently, the control of H. contortus and others is almost entirely based on the use of anthelmintics. Consequently, anthelmintic resistance has developed worldwide and this has become a serious problem in small ruminant nematode parasite control programs. In view of this, there is a need for alternative control methods. The use of Copper-Oxide Wire Particles (COWP) to help reduce parasite burden is one such alternative. Three trials were conducted to determine the effect of COWP on the reduction of H. contortus in ewes (Summer, 2002, and Spring, 2003) and lambs (Summer, 2002). Each trial followed similar protocols where the animals were allocated to treatment and control groups based on fecal egg count (FEC). COWP boluses were administered to the treatment group and infection level was monitored over a period of time by weekly determination of FEC and blood PCV. Serum copper levels were determined before and at the end of each trial. Feces were also collected every other week for coproculture, which was used to determine relative distribution of infective larvae genera. Results of all three trials indicated that COWP were effective in reducing FEC for a period of 4-5 weeks. There was no difference in PCV between groups for any trial. Coproculture indicated that the reduced FEC was primarily due to a reduction in H. contortus. Serum copper levels were either below or within normal range before treatment and remained within normal limits at the end of the trials. The results from these trials demonstrated that the use of COWP reduced H. contortus infection and this may be useful in conjunction with other nematode parasite control methods.

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.

These studies were conducted to elucidate the regional analgesic effect that is observed clinically after treatment of orthopedic disorders with application of extracorporeal shock waves in horses. Regional analgesia after treatment with extracorporeal shock waves presents a concern because it may eliminate protective limiting mechanisms and may place equine athletes with predisposing lesions at risk of sustaining career- or life-ending injuries. Direct percutaneous application of non-focused extracorporeal shock waves to palmar digital nerves in the pastern area of horses resulted in decreased sensory nerve conduction velocities compared with untreated control nerves at 3, 7, and 35 days after treatment. Transmission electron microscopy revealed distinct morphological changes consisting of extensive separation and disruption between the different layers of the myelin sheath in large- to medium-sized myelinated axons of treated palmar digital nerves. Treatment of selected areas of the metacarpus in horses with non-focused extracorporeal shock waves failed to identify a regional analgesic effect when cutaneous sensation was assessed by comparing the nociceptive threshold (limb withdrawal reflex latency, LWRL) between treated and non-treated areas after stimulation with a focused light source. The LWRL responses in all horses were comparable in treated and control areas over time with a significant decrease noted at most sites and time points compared with baseline values.

Cranial cruciate ligament (CCL) rupture is a common injury in the dog and major cause of degenerative joint disease. The pathophysiology of CCL rupture in the dog is well described. Osteoarthritis secondary to CCL rupture causes severe pain and lameness. There are many surgical techniques accepted for dogs with CCL rupture. A commonly performed technique is an extracapsular repair with a lateral fabella-tibial suture (LFS) using large diameter nylon leader line (NLL). Mechanical demands placed upon the LFS are high requiring the material used be able to withstand a high amount of force, undergo minimal elongation, and have a high stiffness. Studies evaluating materials for use for LFS have found NLL to have the most appropriate mechanical profile for use. However, the large diameter, low coefficient of friction, and memory of NLL make knot security a concern, as well, the surgical handling of the material is not ideal. Our hypothesis stated that the Securos Cranial Cruciate Ligament Repair System, a commercially available crimp-clamp system used to secure two ends of NLL together for a LFS, would perform mechanically superior to a clamped square knot using NLL. Furthermore, fluorocarbon (polyvinylidene fluoride; PVDF) a novel biomaterial of reduced diameter for a given tensile strength, would mechanically perform better than NLL using a clamped square knot. The Securos Cranial Cruciate Ligament Repair System is an acceptable method of fixation of NLL loops used for LFS. Loops formed with 27 and 36 kgt NLL using the 36 kg Securos® crimp-clamps performed as well or better than a clamped square knot. However, loops secured with the 18 kg Securos® crimp-clamp system using 18 kgt NLL did not perform as well as a clamped square knot, and their use cannot be recommended based on these results. Fluorocarbon leader line (FCL) performed mechanically similar to NLL and eliminated elongation under low load observed with NLL. Steam sterilization has dramatic effects on FCL and is not recommended. Ethylene oxide sterilization showed no significant mechanical or structural changes to FCL and is recommended. Fluorocarbon leader line appears to be an acceptable alternative to NLL for use as a LFS.

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.

Validation of diagnostic assay performance is hampered where no gold standard exists. Bayesian estimates of the sensitivity and specificity were applied to studies of two persistent bovine infections: bovine immunodeficiency virus (BIV), and bovine viral diarrhea virus (BVDV). BVDV pathogenesis is well described. Losses of $20 million in 1992 alone led the Danish government to mandate eradication. Two enzyme-linked immunosorbent assay (ELISA) tests were developed; one antibody-based to detect exposure, one antigen-based to detect persistent infection. Bayesian estimates of the sensitivity and specificity were 96.6% and 99.2%, and 98.2% and 99.8% for the antibody and antigen ELISA, respectively. Maximum Likelihood estimates of the sensitivity and specificity were 96.3% and 100% and 97.9% and 99.9% for the antibody and antigen ELISA, respectively. Estimates of diagnostic test performance by quarter are reported. A high prevalence of BIV has been reported in the southern U.S., but BIV s effect on production and present assay reliability is still undetermined. Using Bayesian estimation, the performance of an immunofluorescence (IFA) assay (sensitivity = 60%, specificity = 88%) and a polymerase chain reaction (PCR) (sensitivity = 80%, specificity = 86%) for BIV detection in two herds with varying estimated BIV prevalence (20%, 71%) was estimated. Although PCR was more sensitive for diagnosing BIV infection, substantial misclassification of infection is possible regardless of which assay was used. Past research into the pathogenesis of BIV is shrouded in misclassification errors. To evaluate the ability of BIV to shift immune responses to a type 2 cytokine response similar to HIV, we measured the effects of bovine herpes virus 1 (BHV1) vaccination on a cohort of 89 lactating cows, all infected with bovine leukemia virus. BIV negative animals had a more appropriate immune response with decreasing IgG1:IgG2 when compared with BIV positive animals. Using Bayesian estimates of IFA performance, the effect of possible misclassification of BIV serostatus on study results was evaluated. Bayesian estimates are useful where no gold standard exists. More sensitive and specific diagnostic tests for BIV need to be developed. Methods for modeling epidemiological relationships that can adjust for misclassifcation errors are needed.

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.