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.

Hysteroscopic insemination in mares with delayed uterine clearance (DUC mares) is controversial. While some authors proposed that insemination with a reduced volume and number of spermatozoa may reduce post-mating endometritis, others proposed that the hysteroscopic procedure is inflammatory and should not be used in DUC mares. The overall objectives of this study were to evaluate the incidence and severity of post-mating endometritis in reproductively normal and DUC mares after hysteroscopic insemination at the uterotubal junction, and to determine if hysteroscopic insemination could be used in DUC mares to reduce post-mating endometritis. The mares were classified as normal or DUC based on the presence of intrauterine fluid 24 or 48 hours after a semen challenge. In Experiment 1, the acute endometritis was evaluated 24 hours after insemination. Each mare (n=5 normal, n=5 DUC) received three treatments in three consecutive estrous cycles: UB: uterine body insemination (one billion spermatozoa, 20 mL), HYST: hysteroscopic insemination (five million spermatozoa, 0.5 mL) and SHAM: sham hysteroscopic insemination (semen extender, 0.5 mL). Intrauterine fluid accumulation and uterine leukocyte numbers were not influenced by treatment. In the second experiment, residual endometritis was assessed 48 hours after insemination, and the effect of insemination method on fertility was evaluated. Each mare (n=4 normal, n=5 DUC) received four treatments in four consecutive estrous cycles: UB: uterine body insemination (one billion spermatozoa, 20 mL), HYST: hysteroscopic insemination (five million spermatozoa, 0.5 mL), SHAM: sham hysteroscopic insemination (semen extender, 0.5 mL) and SP: hysteroscopic infusion of seminal plasma (0.5 mL). There was no difference in intrauterine fluid accumulation between treatments in normal mares. HYST and SHAM treatments resulted in more fluid accumulation 24 hours after the procedures in DUC mares than UB and SP. However, leukocyte numbers were not different. It was concluded that the hysteroscopic procedure itself is inflammatory, so it should not be used with the intention of reducing post-mating endometritis. However, the inflammation was not greater than that induced by routine uterine body insemination and fertility was not affected. Therefore, there is no contraindication to its use in mares with delayed uterine clearance.

The murine pathogen Helicobacter hepaticus has important confounding effects on research. Neonatal fostering has been studied in our laboratory for elimination of infection in mice. The purpose of our study was to examine fostering of pups from experimentally infected dams in male-absent parturition, and to determine the significance of gender and time on quantity of bacterial colonization in the cecum and feces of C57BL/6 mice. Approximately 20 C57BL/6 mice were fostered per day from one to four days of age. None of the C57BL/6 pups tested positive by PCR in fecal or cecal samples through four days of age. This data showed that removal of the male C57BL/6 mouse prior to parturition is crucial for extending the fostering period to obtain Helicobacter-free mice. In a second experiment, H. hepaticus infected mice were housed under varying arrangements to determine the effects of gender and housing on fecal and cecal colonization. Neither time or housing group affected bacterial fecal shedding. However, there was a significant overall effect of gender and a significant difference between male and female mice in both fecal and cecal bacterial copy number. Male fecal and cecal samples contained more copies of H. hepaticus than did female samples. Additionally, significant correlations between fecal and cecal H. hepaticus values were found both overall and by gender. Novel predictive algorithms were formulated to predict cecal bacterial colonization levels in fecal pellets. These findings should prove useful in Helicobacter elimination efforts, and in future work to further elucidate the role of H. hepaticus in transmission and disease.

Intraoperative reductions in serum fentanyl levels in dogs with induced moderate hypothermia and transdermal fentanyl patches (TDF) in place has been documented. The impact of such reductions has not been evaluated nor has the anesthetic sparing effect of the TDF. Reductions in serum levels may be the result of either a decreased uptake of fentanyl from the dermal depot, or a biophase shift. The objective of this investigation was to determine whether the minimum alveolar concentration (MAC) of isoflurane was altered in the presence of TDF in normothermic and hypothermic dogs. Six mature, healthy, mixed breed dogs were anesthetized on four separate occasions, and received each of the 4 following treatments in random order: 1. Sham patch-normothermia (C-NORM), 2. Sham patch-hypothermia (C-HYPO), 3. Fentanyl patch-normothermia (F-NORM), 4. Fentanyl hypothermia (F-HYPO). The appropriate patch was applied twenty four hours prior to induction of anesthesia. Anesthesia was induced with isoflurane in oxygen; the dogs were intubated and mechanical ventilation was initiated. Target temperatures (34.5ºC- HYPO, or within 1ºC of baseline- NORM) were held constant for one hour prior to beginning the MAC determinations. Supramaximal stimulation was produced by an electrical stimulator which delivered a current to needle electrodes in the buccal mucosa of the lower jaw of the dog. The MAC (± SD) for C-NORM, C-HYPO, F-NORM and F-HYPO was 1.2 ± 0.17, 0.855 ± 0.183, 0.763 ± 0.097, and 0.830 ± 0.172 respectively. MAC for C-NORM was significantly higher than the other groups. There was no significant difference between C-HYPO, F-NORM, and F-HYPO. Transdermal fentanyl reduces the isoflurane requirement in normothermic dogs. The presence of hypothermia appears to negate the effects of TDF.