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.

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.

The very potent antibacterial activity of cecropin B makes it a likely candidate to prevent and/or treat Mannheimia haemolytica 1:A infection in the upper respiratory tract (URT) of cattle. The purpose of this study was to ascertain if the URT could be transfected with a gene coding for the antimicrobial peptide cecropin B. By transfecting cattle with a gene coding for cecropin B, this study attempted to inhibit colonization of a virulent strain of M. haemolytica 1:A in the URT while investigating any possible changes in the indigenous and transient nasal flora. In this study the antibacterial efficacy of cecropin B for a virulent strain of M. haemolytica 1:A was determined. In vitro results showed that cecropin B was very effective in inhibiting this virulent strain of M. haemolytica 1:A within 20 minutes of incubation at 37°C. No inhibition of its activity was observed by incubating cecropin B in pooled bovine nasal secretions. The nasal passages of calves were aerosolized with different amounts of plasmid DNA containing a gene coding for cecropin B. Results of this study show that calves transfected with 50 or 100 μg of plasmid DNA per nostril were able to express cecropin B at the mRNA and peptide level. Detection of the cecropin B gene in control calves may indicate the possibility of native bovine cecropin. After challenge with a virulent strain of M. haemolytica 1:A, all calves were stressed by transportation in a crowded trailer 100 miles for 3 hours. Seven out of the 8 control calves yielded detectible levels of M. haemolytica 1:A in nasal aspirates throughout the weeks following challenge. All 4 calves given 25 μg of plasmid DNA per nostril and two of the 4 calves given 50 μg of plasmid DNA per nostril yielded detectible levels of M. haemolytica 1:A in nasal aspirates following challenge. However, M. haemolytica 1:A was not detected in any calf given 100 μg of plasmid DNA per nostril. There appeared to be no change in the normal bacterial nasal flora.

During the course of their disease, leprosy patients may experience two types of inflammatory reactions- erythema nodosum leprosum (ENL) or reversal reaction (RR). Thalidomide is effective treatment for ENL, but not for RR. Using concentrations of thalidomide similar to that achieved in the treatment of ENL, we investigated thalidomides effect on reactions, viability of M. leprae, and integrity of plasma membranes. Cells from patients with and without RR were stimulated with M. leprae (AFB), a cytosol fraction of M. leprae (MLSA) or DHAR (DHAR) antigen, and the effect of thalidomide on lymphocyte proliferation, expression of TNF-a mRNA and synthesis of TNF-a was investigated. Thalidomide enhanced MLSA and DHAR induced proliferation of cells from RR patients. The expression of TNF-a mRNA was variable, but thalidomide generally suppressed the synthesis of TNF-a. In a sub-set of RR patients, thalidomide enhanced AFB-induced cell proliferation, and the expression of TNF-a mRNA and TNF-a. ENL has been described as a consequence of M. leprae antigens released from macrophages binding antibody and inducing inflammation. Thalidomide did not affect the viability of M. leprae residing in IFN-g/LPS activated mouse macrophages, nor did it suppress TNF-a or nitrite. Drugs may be anti-inflammatory by stabilizing cell membranes. Thalidomide failed to protect the plasma membrane of neutrophils and THP-1 cells from osmotic lysis. Thalidomide stabilized the membrane of erythrocytes from plasma free blood, but not from whole blood. In vivo, the stability of erythrocytes membranes from subjects after ingestion of thalidomide was not affected. In conclusion, thalidomide did not alter the viability of M. leprae, nor the integrity of the plasma membrane of inflammatory cells. It could enhance or suppress M. leprae antigen-induced synthesis of TNF-a. Interestingly, in 15 of 75 RR patients cells stimulated with AFB, thalidomide acted as a co-stimulant enhancing cell proliferation, synthesis of mRNA for TNF-a and TNF-a. Thalidomides enhancing effect on TNF-a in RR appears to be dependent on the stimulant and IL-2 signaling. As the inflammation in RR is associated with the emergence of antigen-reactive T-cells and TNF-a, we speculate that the use of thalidomide in the treatment of RR may exacerbate the reaction

The purpose of this study was to investigate methods to induce earlier onset of cyclicity in transitional mares. Two experiments were conducted evaluating the effect of follicular aspiration to advance the onset of cyclicity, more succinctly define criteria for selection of mares for follicular aspiration and to compare aspiration to deslorelin treatment for initiating cyclicity in transitional mares. In Experiment 1, anestrous mares were assigned to control (n=6) or follicular aspiration (n=11). The control mares were monitored twice weekly, until ovulation was detected. The aspiration mares were similarly monitored until a follicle >35 mm was identified, then transvaginal ultrasound guided follicular aspiration was performed. After aspiration, the mares were monitored for luteal tissue formation. In Experiment 2, anestrous mares were assigned to control (n=14), follicular aspiration (n=10), or deslorelin (n=12). The control mares were treated as in Experiment 1. The aspiration mares were monitored in the same manner as Experiment 1 but were treated only if uterine edema was present. The deslorelin treated mares were monitored similarly to the aspiration mares, but instead of aspiration the mares were administered deslorelin. In both experiments, plasma was obtained at each examination from all mares to verify a rise in progesterone. In Experiment 1, the time from January 1 to the first rise in serum progesterone was 23.8 days earlier for aspiration treated mares than for control mares (80.5±7.3 and 104.3±8.8 days, mean±SE, for aspiration and control groups, respectively; P=0.024). In Experiment 2, there was no significant difference in time from January 1 to the first rise in serum progesterone between groups (100.4±5.8, 113.0±3.0 and 110±6.7 days, for the aspiration, control and deslorelin groups, respectively, P=0.328). However, if mares that did not receive a repeat aspiration treatment due to lack of uterine edema are excluded, there was a significant difference between the aspiration and control groups (93.9±6.7 and 113.0±3.0 days, for the aspiration and control groups, respectively, P=0.045). Results of this study indicate that follicular aspiration of a follicle > 35 mm during late transition may be a means to advance the onset of cyclicity in mares.

Brucellosis and pseudorabies lead to abortion in pregnant sows and are perpetuated by feral swine reservoirs. A multivalent oral vaccine for these diseases would improve vaccination and eradication programs worldwide. Previous studies have shown that the rough attenuated Brucella strains RB51 and VTRS1, when administered subcutaneously to swine, stimulate host immune responses, transiently colonize tissues, and provide partial protection against virulent B. suis infection in pregnant sows. A plasmid encoding for the pseudorabies virus glycoprotein D (PRV gD) has also been added to these strains as part of this project. This study evaluates the use of these strains as oral vaccines for swine brucellosis and investigates the ability of these vaccines to colonize lymph nodes and stimulate the production of antibodies against rough Brucella antigens and PRV gD. Orally administered VTRS1 stimulated a higher immune response than RB51 in a shorter period of time, transiently colonized lymph nodes, and did not lead to the production of anti-O-polysaccharide (OPS) antibodies that interfere with brucellosis diagnostic tests. Both RB51 and VTRS1 provided substantial litter protection in orally vaccinated sows challenged with virulent B. suis; however, VTRS1, unlike RB51, also provided partial protection in the sow. These studies support the use of orally administered VTRS1 as an efficacious vaccine for swine brucellosis. The addition of a plasmid encoding for PRV gD to these strains created the multivalent vaccines RB51+gD and VTRS1+gD. Compared to the above strains, the addition of the plasmid did not alter immune response stimulation to rough Brucella antigens and these vaccines were safe when administered to pregnant sows. Swine vaccinated with RB51+gD or VTRS1+gD exhibited low immune responses to the gD antigen and delayed tissue colonization by the vaccine strains. It was found that plasmid addition slowed the growth of the vaccine strains in the laboratory, which may account for the suppressed tissue colonization and immune responses to the PRV gD antigen in swine. Further studies are necessary to evaluate the use of these strains as multivalent vaccines for brucellosis and pseudorabies in swine.

The Herpes Simplex Virus life cycle contains a number of membrane fusion events that must function properly to ensure a productive infection, including: virus attachment and entry into susceptible cells, de-envelopment at the outer nuclear lamellae, and virus-induced cell-to-cell fusion. A virus-free cell fusion assay was recently developed in order to attempt to understand the underlying mechanisms that are responsible for viral fusion events and was utilized in order to investigate the effect of mutations targeted to the carboxyl terminus of gB. We showed that the predicted alpha helical domain H17b within the carboxyl-terminus of gB is involved in both virus-induced and virus-free fusion systems, and heparin was shown to be a specific inhibitor of gB-mediated fusion in both systems, while resistance to heparin inhibition of gB-mediated cell fusion was associated with the predicted alpha helical structure H17b. An important difference between virus-free and virus-induced membrane fusion is that virus-expressed gB mediates an insignificant amount of cell-to-cell fusion, while transiently expressed gB causes extensive cell-to-cell fusion. gK was recently shown to inhibit cell fusion resulting from transiently expressed gB, gD, gH and gL and hypothesized to function as a negative regulator of membrane fusion. However, we show that gK can not solely act as a negative regulator of gB-mediated membrane fusion, since gK is demonstrated to be absolutely required for virus-induced cell fusion to occur, suggesting a more complicated relationship between gK and gB. A recent publication from our laboratory showed that syncytial mutations in either gB or gK failed to cause fusion in the absence of the UL20 gene, suggesting that the UL20 protein was essential for virus-induced cell fusion. Absence of the UL20 gene also caused accumulation of unenveloped capsids into the cytoplasm, indicating that UL20p functioned in cytoplasmic stages of virion envelopment. We delineated via site-directed mutagenesis the functional domains of UL20p involved in infectious virus production and virus-induced cell fusion, revealing that the role of UL20p in virus-induced cell fusion can be functionally separated from its role in cytoplasmic virion morphogenesis.

Columnaris disease, caused by the bacterium Flavobacterium columnare, is an economically significant problem in many warmwater fish species. Difficulties encountered in the isolation and culture of F. columnare have been an impediment to research on the organism and the disease it causes. The goal of this study was to improve the methods for isolation, culture, identification and maintenance of F. columnare. Following the evaluation of different culture media selective cytophaga agar was determined to be the optimum isolation medium, Flavobacterum columnare growth medium proved to be the optimum culture medium, and tryptone yeast extract agar with increased moisture was best for maintenance of cultures. Biochemical characterization of 49 F. columnare isolates was accomplished utilizing the method of Griffin et al. (1992), and the API ZYM system from BioMérieux (Hazelwood Missouri). Using these methods 48 of the 49 strains were presumptively identified as F. columnare. Ten representative F. columnare isolates were further characterized by the API NE system, and all isolates yielded identical phenotypes. Molecular characterization of various strains of F. columnare was accomplished by random amplified polymorphic DNA analysis (reference). Strains evaluated were confirmed as F. columnare by polymerase chain reaction (PCR) using primers and methodology published by Bader et al (2003). The data from RAPD analysis was used to construct three groups based on similarity comparisons. Methods for antimicrobial susceptibility testing by disk diffusion were evaluated on different formulations of dilute Mueller Hinton agar to address the problem of non distinct zones of inhibition, and to reduce the variability of resulting zone data seen when using the published dilute Mueller Hinton formulation (Hawke and Thune 1992). The improved dilute Mueller Hinton medium formulation reduced variability by 40%, increase overall bacterial growth, and also improved the zones of inhibition by producing distinct margins.