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Field monitoring during Summer 2019 highlighted regular occurrence of bottlenose dolphins inside the survied area, Photo Id proved 4 subjects of 46 identified were sighten mulitple times in June July and September 2019. Behavioural data collected suggest bottlenose dolphins exploit this area primarly for hunting activity, taking advance of fishing boats. This is supported also by the absence of dolphins in August, during the fishing ban. Tourist operators often feed bottlenose dolphins to attract them near the boats. Whale watching companies operating in this area frequently do not follow ACCOBAMS guidelines. Often they approach animals with the engine and high music volume on, cutting them off or encircling them. We will continue our project in order to obtain more data on abundance, structure and distribution of bottlenose dolphins in Cesenatico coastal waters. Moreover, we will organize scientific courses for fishermen and tourist operators to explain them how to interact with wild dolphins and how to approach them in a respectful way. A deeoer knowledge of the structure of bottlenose dolphins’popolulation in North Italian Adriatic sea is fundamental to set up concrete actions for future conservation of the specie. A respectufl and conscious approach to these animals is essential to guarantee a positive intraction between animals and human activities. A better communicatio, cooperation and concrete data sharing between research groups working on the same geographic area should be achieved. The institution of a virtual catalog avaliable for each research group should represent a common goal, as well as the organization of scientific seminaries to educate future generations and maritime operators.

Gastrointestinal stromal tumors; MiRNAs; MicroRNAs Tumores del estroma gastrointestinal; MiARN; MicroARN Tumors de l'estroma gastrointestinal; MiARN; MicroARN Gastrointestinal stromal tumors (GISTs) harboring mutations in the PDGFRA gene occur in only about 5–7% of patients. The most common PDGFRA mutation is exon 18 D842V, which is correlated with specific clinico-pathological features compared to the other PDGFRA mutated GISTs. Herein, we present a miRNA expression profile comparison of PDGFRA D842V mutant GISTs and PDGFRA with mutations other than D842V (non-D842V). miRNA expression profiling was carried out on 10 patients using a TLDA miRNA array. Then, miRNA expression was followed by bioinformatic analysis aimed at evaluating differential expression, pathway enrichment, and miRNA-mRNA networks. We highlighted 24 differentially expressed miRNAs between D842V and non-D842V GIST patients. Pathway enrichment analysis showed that deregulated miRNAs targeted genes that are mainly involved in the immune response pathways. The miRNA-mRNA networks highlighted a signature of miRNAs/mRNA that could explain the indolent behavior of the D842V mutated GIST. The results highlighted a different miRNA fingerprint in PDGFRA D842V GISTs compared to non-D842Vmutated patients, which could explain the different biological behavior of this GIST subset. The study was supported by a financial contribution of the Department of Pharmacy and Biotechnology (RFO) to SA and of the AIG (Associazione Italiana GIST) to MA.

International audience; Reports on the occurrence of Campylobacter spp. in dogs in South Africa are non-existent. This study investigated the prevalence of Campylobacter spp. in 481 dogs visiting four rural community veterinary clinics in South Africa. Dogs were screened for Campylobacter spp. by culture and polymerase chain reaction (PCR), and logistic regression analysis was performed to assess the association between sex, clinic, breed and age and the occurrence of Campylobacter spp. in dogs. The prevalence of Campylobacter spp. was 41.50% (95% confidence interval [CI], 37.39% - 46.04%). Campylobacter jejuni, C. upsaliensis and C. coli were detected in 29.31% (95% CI, 25.42% - 33.54%), 13.10% (95% CI, 10.37% - 16.42%) and 5.41% (95% CI, 3.71% - 7.82%) of dogs, respectively. Dogs carrying more than one species of Campylobacter spp. accounted for 6.23% (95% CI, 4.40% - 8.78%). Campylobacter upsaliensis and C. jejuni were detected in 3.74% (95% CI, 2.37% - 5.86%), whereas C. coli and C. jejuni were found in 2.49% (95% CI, 1.42% - 4.34%) of dogs. Age and clinic were the risk factors significantly associated with Campylobacter spp. occurrence, while age, breed and clinic were predictors of C. jejuni carriage. Furthermore, age was the only risk factor associated with a higher likelihood of carrying C. upsaliensis. The prevalence of Campylobacter spp. C. jejuni and C. upsaliensis increased significantly as dogs grew older. In addition, the odds of carrying Campylobacter spp. were higher in the Staffordshire bull terrier breed compared to crossbreed dogs. In conclusion, this study shows that dogs visiting rural community veterinary clinics in South Africa are reservoirs of Campylobacter spp. and may be potential sources of Campylobacter spp. for humans living in close proximity of the dog populations under study.

International audience; Summer mortality episodes in adult Pacific oysters have been described since the 1950s in various farming areas. Starting in 2012, a recrudescence of mortalities in commercial-sized oysters was first observed in France and then in Italy, with seasonality extension and translation later in the year. Moribund individuals collected during an event in Italy in December 2014 showed yellowish lesions of the mantle and adductor muscle. Histological examination revealed filamentous bacteria associated with necrotic areas. Quantitative PCRs targeting OsHV-1 and Vibrio aestuarianus detected only high loads of the pathogenic bacteria in tissues of symptomatic individuals. A lower diversity of the hemolymph microbiota was also evidenced in moribund individuals, with a predominance of Vibrio and Arcobacter species. A strain of Flavobacteriaceae was isolated from all the symptomatic individuals. Sequence analysis of the 16S rRNA gene identified the strain as Tenacibaculum soleae. When strain pathogenicity was tested by injection in adult individuals, it induced mortality rates of up to 45%, even in the absence of V. aestuarianus. As mortality occurred only 11 days post-infection, further investigation is needed to determine its effective virulence in natural conditions. This is the first description of a Tenacibaculum strain associated with bivalve mortalities.

Avian metapneumovirus (aMPV) infects respiratory and reproductive tracts of domestic poultry, often involving secondary infections, and leads to serious economic losses in most parts of the world. While in general disease is effectively controlled by live vaccines, reversion to virulence of those vaccines has been demonstrated on several occasions. Consensus sequence mutations involved in the process have been identified in more than one instance. In one previous subtype A aMPV candidate vaccine study, small subpopulations were implicated. In the current study, the presence of subpopulations in a subtype B vaccine was investigated by deep sequencing. Of the 19 positions where vaccine (strain VCO3/50) and progenitor (strain VCO3/60616) consensus sequences differed, subpopulations were found to have sequence matching progenitor sequence in 4 positions. However none of these mutations occurred in a virulent revertant of that vaccine, thereby demonstrating that the majority progenitor virus population had not survived the attenuation process, hence was not obviously involved in any return to virulence. However within the vaccine, a single nucleotide variation was found which agreed with consensus sequence of a derived virulent revertant virus, hence this and other undetected, potentially virulent subpopulations, can be involved in reversion. Much deeper sequencing of progenitor, vaccine and revertant may clarify whether problematic virulent subpopulations are present and therefore whether these need to be routinely removed during aMPV vaccine preparation prior to registration and release. Avian metapneumovirus (aMPV) infects respiratory and reproductive tracts of domestic poultry, often involving secondary infections, and leads to serious economic losses in most parts of the world. While in general disease is effectively controlled by live vaccines, reversion to virulence of those vaccines has been demonstrated on several occasions. Consensus sequence mutations involved in the process have been identified in more than one instance. In one previous subtype A aMPV candidate vaccine study, small subpopulations were implicated. In the current study, the presence of subpopulations in a subtype B vaccine was investigated by deep sequencing. Of the 19 positions where vaccine (strain VCO3/50) and progenitor (strain VCO3/60616) consensus sequences differed, subpopulations were found to have sequence matching progenitor sequence in 4 positions. However none of these mutations occurred in a virulent revertant of that vaccine, thereby demonstrating that the majority progenitor virus population had not survived the attenuation process, hence was not obviously involved in any return to virulence. However within the vaccine, a single nucleotide variation was found which agreed with consensus sequence of a derived virulent revertant virus, hence this and other undetected, potentially virulent subpopulations, can be involved in reversion. Much deeper sequencing of progenitor, vaccine and revertant may clarify whether problematic virulent subpopulations are present and therefore whether these need to be routinely removed during aMPV vaccine preparation prior to registration and release.

International audience; Infections with Ostreid herpesvirus 1 (OsHV-1) microvariants in young Pacific oysters are associated with massive mortality events and significant economic losses. Previous studies, focusing on few regions of the genome, have revealed the genomic diversity of these genotypes with respect to the reference type. We used a NGS process to sequence the whole genome of the OsHV-1 µVar in infected individuals, collected during mortality events in France and Ireland. The final genome length of OsHV-1 µVar was approximately 205kbp, shorter than the reference genotype and the overall genome organisation resembled herpes simplex viruses. 94.4% similarity was observed with the OsHV-1 reference genotype. Large indels, including five deletions and three insertions were found to induce the loss and the addition of several ORFs, summed with codon substitutions in 64% of genes shared with the reference type. This diversity raises the question of the exact origin and evolution of OsHV-1 µVar.

International audience; Xenocell therapy from neonate or adult pig pancreatic islets is one of the most promising alternatives to allograft in type 1 diabetes for addressing organ shortage. In humans, however, natural and elicited anti-bodies specific for pig xenoantigens, α-(1,3)-galactose (GAL) and N-glycolylneuraminic acid (Neu5Gc), are likely to significantly contribute to xenoislet rejection. We obtained double-knockout (DKO) pigs lacking GAL and Neu5Gc. Because Neu5Gc 2/2 mice exhibit glycemic dys-regulations and pancreatic b-cell dysfunctions, we evaluated islet function and glucose metabolism regulation in DKO pigs. Isolation of islets from neonate piglets yielded identical islet equivalent quantities to quantities obtained from control wild-type pigs. In contrast to wild-type islets, DKO islets did not induce anti-Neu5Gc antibody when grafted in cytidine monophosphate-N-acetylneuraminic acid hydroxylase KO mice and exhibited in vitro normal insulin secretion stimulated by glucose and theophylline. Adult DKO pancreata showed no histological abnormalities , and immunostaining of insulin and glucagon was similar to that from wild-type pancreata. Blood glucose, insulin, C-peptide, the insulin-to-glucagon ratio, and HOMA-insulin resistance in fasted adult DKO pigs and blood glucose and C-peptide changes after intravenous glucose or insulin administration were similar to wild-type pigs. This first evaluation of glucose homeostasis in DKO pigs for two major xenoantigens paves the way to their use in (pre)clinical studies.

Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.anifeedsci.2017.06.015.; The use of organic acids in diets for livestock animals is interest due to their being potentially beneficial in maintaining gut homeostasis and controlling pathogenic bacterial growth. The objective of this study was to evaluate the influence of two doses of formic acid supplementation for six weeks to weaner pigs. The long-term effects of formic acid were tested for growth performance, microbiota composition in the jejunum using 163 rRNA, expression of marker genes of inflammation in the jejunum, and quantification of acid-secreting cells in the gastric oxyntic mucosa using immunohistochemistry. Three diets containing no (control; CO), 1.4 g/kg (low formic acid; LFA) or 6.4 g/kg (high formic acid; HFA) free formic acid were fed to 36 pigs for 42 d from 7 d post-weaning (7.1 +/- 1.0 kg live weight) until the end of the trial (27.7 +/- 5.5 kg live weight); they were housed in individual cages for six weeks. During the first three weeks, formic acid supplementation, at any of the dosage above, increased average daily gain (ADG) (P = 0.004), tended to increase average daily feed intake (ADFI) (P = 0.08) and tended to decrease g of feed to g of gain (F:G) (P = 0.08) while no effect was observed during the last three weeks. At the end of the trial, formic acid supplementation had only a minor effect on the microbiota composition in the jejunum. A higher microbiota diversity (Chaol index P < 0.05) was found in the HFA group than in the CO group. The Control group showed a higher abundance (P < 0.05) of Gemella, Lactobacillus and Parvimimoas than the HFA and LFA groups, higher levels of Acinetobacter, Fusobactrium, Leuconostoc respect to the HFA group and of Turcibacter as compared to the LFA group. The abundance of Streptococcus was lower in the CO group than in the HFA group and higher than in the LFA group. The jejunal expression of C-C Motif Chemokine Ligand 2 (CCL20) was higher in the HFA group respect to the LFA group. Formic acid intake did not affect the thickness of the gastric mucosa, the number of parietal cells and stomach weight. This study showed that the addition of formic acid to piglet diets improved growth performance during the first period after weaning and that the long-term supplementation of formic slightly affected the microbiota composition according to the dose.

One of the most widely European farmed mollusc, the mussel Mytilus sp., has been subjected to massive mortalities located in Charente-Maritime (France) in spring 2014. The national surveillance network for mollusc health has reported a systematic detection of V. splendidus in all dying batches. V. splendidus is the type species of a clade composed of almost 20 known strains with variable pathogenicity on bivalves. In our study, we first developed a Multi Locus Sequence Analysis (MLSA), specific to V. splendidus group, based on fragments of 5 housekeeping genes: atpA , ftsZ , mreB , rpoD and topA . This tool was validated on reference strains and compared with individual gene analyses. It allowed a useful and reliable classification of V. splendidus closely-related strains. Thanks to MLSA, we then tried to classify genetically 23 strains isolated from healthy or dying mussels. 21 were classified within the Splendidus clade: in splendidus cluster (38%), in tasmaniensis cluster (24%), in artabrorum cluster (24%) and on distinct branches (14%). All of them were tested by injection to healthy adult mussels to identify possible pathogenic strains. Experimental trials revealed the presence of a strain called M3H, allied with the splendidus cluster and able to induce mortality in mussels with rates up to 80%. The M3H virulence was demonstrated by the recovery of the injected strain in dying animals, resulting from repeated experimental infections. Further work should be now conducted to explore the pathogenicity of the M3H strain towards different mussel batches and under various conditions. Statement of relevance Our MLSA is useful to identify related V. splendidus strains

Feral swine are a nuisance species across the United States that costs around $1.5 billion each year in agricultural, environmental, and personal property damages. In the last ten years the population of feral swine is estimated to have quadrupled and novel population control methods are needed. Furthermore, feral swine are known carriers of zoonotic diseases such as brucellosis, which threatens both livestock biosecurity and public health. Recombinant multimeric gonadotropin-releasing hormone (mGnRH) has been previously used as a subunit vaccine to induce immunocontraception in feral pigs. However, potent adjuvants and large amounts of purified antigen are needed to elicit a robust anti-GnRH immune response and current delivery methods are limited. Brucella suis strain VTRS2 can be used as a novel platform to deliver mGnRH without the use of antibiotic resistant markers. Strain VTRS2 was created by deletion of the LPS biosynthesis gene wboA as well as the leuB gene required for leucine biosynthesis inside the nutrient-depleted intracellular environment occupied by Brucella. Mutations in wboA are known to attenuate Brucella strains such as the vaccine strain B. abortus RB51, however strain RB51 is rifampin resistant and has poor efficacy in swine. Strain VTRS2 confers significant protection against B. suis challenge in mice and additionally shows evidence of protection in feral swine. Furthermore, the mGnRH antigen can be delivered using the pNS4 plasmid (which expresses leuB under its native promoter) thus maintaining the plasmid in strain VTRS2 under leucine-deficient conditions while expressing recombinant antigen in the host. The murine model was used to determine the clearance kinetics of strain VTRS2-mGnRH and to measure vaccine efficacy against challenge by virulent B. suis 1330. Subsequently the effects of the VTRS2-mGnRH vaccine on fertility were assessed in breeding trials in mice. Strains VTRS2 and VTRS2-mGnRH were found to be protective against virulent Brucella suis challenge. Strain VTRS2-mGnRH elicited an anti-mGnRH antibody response in vaccinated mice, though an effect on fertility was not observed. An improved vaccine against brucellosis in swine, which also confers immunocontraception without the introduction of antibiotic resistance, could become an important tool in the management of this nuisance invasive species. Ph. D.

International audience; Ostreid herpesvirus 1 (OsHV-1) is a significant pathogen affecting the young Pacific oyster Crassostrea gigas, worldwide. A new variant, OsHV-1 μVar, has been associated with recurrent mortality events in Europe since 2008. Epidemiological data collection is key for global risk assessment; however little is known about health status and genotypes present in European wild oyster beds. Most studies to date have involved only cultivated individuals during mortality events, and reported low genotype diversity. With this study, conducted along the Italian coasts, we investigated for the first time the presence of OsHV-1 in European natural oyster beds. Analysis of three genomic regions revealed the presence of at least nine different genotypes, including two variants close to the OsHV-1 reference, known since the early 1990s but with no European record reported since 2010, and highlights relevant genotype diversity in natural environment. Phylogenetic analysis distinguished two distinct clusters and geographical distribution of genotypes, with the exception of a variant very closely related to the μVar, which appeared the single genotype present in all the Adriatic sites. Interestingly, these wild symptom free populations could represent, in Europe, an accessible alternative to the import of OsHV-1-resistant oyster strains from the East Pacific, the native area of C. gigas, avoiding the high-risk of non-native marine species and new pathogen introductions.

International audience

Ornithobacterium rhinotracheale (ORT) is a Gram-negative bacterium that causes respiratory disease in poultry characterized by rhinitis, tracheitis, and pneumonia with mortality averaging 2-3%. In the Shenandoah Valley of Virginia, the seroprevalence for ORT among turkey flocks as determined by enzyme-linked immunosorbent assay (ELISA) was found to be 70.9% (n=175). Additionally, the seroprevalence for hemorrhagic enteritis virus (vaccine induced), Bordetella avium, and paramyxovirus-1 was 100%, 74.8%, and 6.3% respectively. No significant interactions were detected. The type strain of ORT is characteristically non-hemolytic at least for 96 hours at 37°C on Columbia Blood Agar. In recent years, atypical isolates that rapidly produce hemolysis have been isolated with increasing frequency. A variety of in vitro tests were used to determine differences between representative isolates of the hemolytic (H) and non-hemolytic (NH) phenotypes. Findings suggest that the H isolate contains a 4 kb plasmid similar to that found in Reimerella anatipestifer. No plasmid was found in the NH isolate. Differences in growth characteristics and resistance to tetracyclines were also noted. No differences in proteins, biochemical characteristics or 16S rRNA sequences were found, the latter serving as confirmation that the isolates were both ORT. Embryo inoculation was used to assess virulence. No significant differences were observed and most embryos survived through to the day of hatch (pip) despite the fact that ORT could be re-isolated. In turkey poults however, the H phenotype did appear less virulent. A significant depression in weight gain was noted for birds inoculated intratracheally with the NH isolate at 7 days post-inoculation (dpi). NH inoculates also had significantly higher antibody levels on ELISA at 14 and 21 dpi and histopathological lesion scores for lung at 7, 14, and 21 dpi. The NH isolate could be re-isolated from NH-inoculated poults through 21 dpi; whereas the H isolate could only be re-isolated through 14 dpi. In conclusion, there are numerous differences between the NH and H isolates found in the field with the H isolate appearing less virulent and as such, making it a potential vaccine candidate. The phenotypic difference appears to correlate with this, but may not suffice to explain it. Ph. D.

DNA encodes the genetic instructions for the development and function of organisms and hence maintaining genomic integrity is essential for the propagation of life. However, DNA molecules are under constant threat of metabolic and environmental insults resulting in DNA damages including DNA double strand breaks (DSB), which are considered as a serious threat to cell survival. The majority of these DSB are repaired by Non-homologous end joining (NHEJ). Unrepaired DSB can lead to genomic instability resulting in cell cycle arrest, apoptosis, and mutations. Thus, delineating this DNA repair process is important in understanding the molecular mechanisms of aging and malignant progression. B lymphocytes undergo physiological DNA breaks and NHEJ-mediated DNA repair during their bone marrow differentiation and peripheral class switch recombination (CSR), thus lending them as a good model system in which to delineate the DNA repair mechanisms. To determine the effect of aging on NHEJ, B lymphocytes from old mice were analyzed. The results showed compromised DNA repair in cells from old mice compared to cells from adult mice. These results suggest that NHEJ is compromised during aging and might play critical roles in the aging process and age-associated conditions. To delineate the role of a CT in regulating the immune system, transgenic mice expressing NUP98-HOXD13 (NHD13) were analyzed for B lymphocyte differentiation, peripheral development, CSR, and antibody production. The results showed impaired B cell development and antibody production, which worsened with antigenic stimulation, suggesting the role of NHD13 in immune regulation. These studies explored the possibility of altered NHEJ-mediated DNA repair as a contributing reason for aging process and age-associated conditions. Also, the results from NHD13 study suggested that a primary CT can result in impaired NHEJ and regulate immune cell development and function. Furthermore, the results pointed to the possibility that a primary CT may lead to secondary mutations through altered NHEJ. Thus, these studies shed insight into the molecular mechanisms of altered NHEJ and may help in developing preventive or therapeutic strategies against accumulation of DNA damage, aging process and secondary mutations. Ph. D.

The purpose of this study was to determine the safety and pharmacokinetic profile of firocoxib in healthy neonatal foals. Foals are more sensitive to the side effects of nonsteroidal anti-inflammatory drugs, (NSAIDs), particularly due to immature renal clearance mechanisms and ulcerogenic effects on gastric mucosa. Firocoxib, a novel second generation NSAID, is reported to have reduced side effects due to its COX-2 selectivity. The pharmacokinetic profile of firocoxib in neonates has not been established, making reliable dosing difficult. We hypothesized that firocoxib given per os at the labeled dose to neonatal foals would be absorbed and not be associated with clinically significant adverse events. Seven healthy American Quarter Horse foals of mixed gender were administered 0.1mg/kg firocoxib orally q24h for nine consecutive days, commencing at 36h of age. Blood samples were collected for firocoxib analysis using high pressure liquid chromatography with fluorescence detection at 0 (dose #1 only), 0.25, 0.5, 1, 2, 4, 8, 16 and 24 hours after doses #1, 5 and 9. For all other doses (2, 3, 4, 6, 7 and 8) blood was collected immediately prior to the next dose (24 hour trough). Elimination samples (36, 48, 72, 96, 120 and 144 hours) were collected after dose #9. Safety was assessed via physical examinations, changes in body weight, gastroscopy, complete blood count, serum biochemistry and urinalysis. Firocoxib was rapidly absorbed following oral administration with minimal accumulation after repeat dosing. After the initial dose, an average peak serum concentration (Cmax) of 89.50 ° 53.36 ng/mL (mean ° SD) was achieved (Tmax) in 0.54 ° 0.65 hours. Steady state was obtained after approximately 4 doses and the average maximum concentration (Cavg) in serum was 39.1 ° 8.4 ng/mL. After the final dose, the mean terminal half-life (T½?») was 10.46 ° 4.97 hours. Firocoxib was not detected in plasma 72 hours after the final dose (

Hypothyroidism is the most common endocrine disease in dogs and has been shown to have a hereditary nature in many breeds. Previous studies have documented decreased fertility in bitches with experimentally-induced hypothyroidism, decreased viability at birth, increased periparturient mortality, and reduced birth weight in pups born to hypothyroid dogs. Hypothyroid women have an increased demand for exogenous thyroxine throughout gestation in order to maintain normal plasma concentrations of thyroid hormones and produce neuropsychologically normal children. This study was performed to determine if pregnancy causes a similar need for increased levothyroxine dosages in dogs to maintain a euthyroid state. Serum was harvested from blood collected from six bitches with experimentally-induced hypothyroidism that were receiving standard thyroid hormone replacement therapy and from four euthyroid control bitches. Thyroid function tests performed on these samples included total thyroxine (T4), free T4 (FT4), thyroid stimulating hormone (TSH), and 3,5,3'-triiodinine (T3). Thyroid concentrations were measured from ovluation through the end of pregnancy. All bitches whelped normal litters. Euthyroid bitches had no significant alterations in their hormone concentrations throughout pregnancy. None of the supplemented hypothyroid bitches had clinical signs of hypothyroidism throughout the study. Serum concentrations of T4 and FT4 were elevated at multiple sample points during gestation. The results from this study indicate that standard levothyroxine supplementation is adequate to maintain a euthyroid state during pregnancy in experimentally-induced hypothyroid dogs. In addition, there is no evidence that canine thyroid profiles in euthyroid dogs are altered during gestation. Master of Science

Recurrent Airway Obstruction (RAO)-affected horses experience bronchoconstriction and airway inflammation in response to inhalation of irritants including hay molds. Steaming hay reduces fungal content, but the effect on the antigenic potential has not been investigated. We tested the hypothesis that RAO-affected horses develop less severe clinical disease when fed steamed versus non-steamed hay and this reduction coincides with decreased hay fungal content. Six RAO-affected horses in clinical remission were divided in two groups and fed steamed or non-steamed hay for 10 days using a two-way cross-over design. Hay was steamed using a commercial hay-steamer. Clinical assessment was performed daily. Full assessment, including airway endoscopy, tracheal mucous scores and maximal change in pleural pressure, was performed on days 1, 5, and 10. Bronchial fluid sampling and cytology were performed on days 1 and 10. Hay core samples were collected pre- and post-steaming and cultured to determine fungal and bacterial concentrations. Statistical analysis was based on data distribution and quantity and performed using SAS®. P-value

Interleukin-17 (IL-17) plays a major role in inflammation by regulating the induction of various proinflammatory genes, which aid in the recruitment and activation of neutrophils. Although IL-17 is considered to be protective in infection, overproduction of IL-17 in conditions like autoimmune diseases has been shown to aggravate these diseases and contribute to tissue injury. One of the principal focus of our laboratory is to decipher molecular mechanisms involved in inflammatory cytokine regulation and response in inflammatory disorders. To study this aspect, we employ a murine model of pro-inflammation induced by exposure to a natural immunomodulator, estrogen. In this novel study, we have comprehensively investigated the effect of estrogen on IL-17 induction, an aspect not studied thus far. We are the first to demonstrate that estrogen increases the ability of lymphocytes to secrete IL-17A, and its isoforms IL-17F, IL-17A/F. In addition to the cytokine levels, the percentages of IL-17⁺ cells are also increased by estrogen. Impressively, we found that estrogen fine tunes the balance of multiple transcription factors/signaling pathways. Estrogen upregulates IL-17 by promoting the activity and expression of positive regulators (RORγt, RORα, NF-κB, JAK-2) and decreases the activity and/or expression of negative regulators (IRF8, ETS-1). In addition, we found that estrogen epigenetically regulates IL-17 induction by miRNAs (miR-326 and miR-223). We also found that majority of IL-17 positive cells are CD8⁺ suggesting that estrogen-mediated IL-17 induction is predominantly from Tc17 cells. This is possibly due to increased proliferation of CD8⁺ cells from estrogen-treated mice, as demonstrated by CFSE cell proliferation assay. Furthermore, estrogen also enhances the ability of IL-17-target cells to release proinflammatory molecules when exposed to IL-17. Together, this is the first study to comprehensively show that estrogen calibrates transcription factors and miRNAs to enhance IL-17 induction and promote IL-17 response. This dissertation work will provide a platform to continue further research in estrogen modulation of IL-17 in inflammation and disease conditions. Ph. D.

Objectives - To evaluate the effects of equine sCD14 and monoclonal antibodies (mAbs) to equine CD14 on LPS-induced TNF° expression of equine peripheral blood mononuclear cells (PBMCs). To determine serum concentrations of soluble (sCD14) in a population of horses with gastrointestinal diseases or other illnesses likely to result in endotoxemia; and identify relationships with clinical data. Animals - Part 1; 10 healthy horses. Part 2; 55 clinical cases and 23 healthy control horses. Procedure - Part 1; PBMCs were incubated with Escherichia coli LPS, CD14 mAb, sCD14, CD14 mAb plus E coli LPS or sCD14 plus E coli LPS. Supernatants were collected at 6 hours and assayed for tumor necrosis factor ° (TNF°) activity. Part 2; Serum sCD14 was measured at admission and then at 24 and 48 hours after admission using a bead-based multiplex assay. Results - Part 1; Pre-incubation with CD14 mAb did not inhibit LPS-induced TNF° protein production in isolated equine monocytes. Use of sCD14 inhibited LPS-induced TNF° protein production in isolated monocytes in a concentration-dependent manner. Part 2; Serum concentration of sCD14 was positively related to duration of clinical signs (P = 0.007), respiratory rate (P=0.04) and band neutrophil count (P = 0.0002). There was no correlation between serum concentration of sCD14 and heart rate, temperature, hematocrit, lactate, white blood cell count, fibrinogen, creatinine, urea nitrogen, glucose and anion gap values. Serum sCD14 did not correlate with outcome at any time point for clinical cases. Master of Science

Influenza epidemics and pandemics remain a significant burden to world health and economy. Low efficacy of current inactivated influenza vaccines in the elderly and immunocompromized and the inability to protect against antigenically drifted or shifted strains of influenza virus are the two major problems in influenza vaccine research. To overcome these hurdles, we have utilized an in vitro cell culture vaccine platform, which results in whole inactivated influenza vaccine (WIV) bearing bioactive membrane-anchored immunomodulatory proteins such as cytokines on the virion surface, collectively known as CYT-IVACs (Cytokine bearing-Inactivated Vaccine). In addition, we tested whether a multimeric M2e peptide presented on WIV can serve to enhance immunogenicity and augment protective efficacy of whole virus vaccines. Our panel of cytokines includes IL-2, IL-4, IL-12, IL-23, and Flt3L as well as the multimeric M2e peptide, all fused to the membrane anchoring regions of influenza virus hemagglutinin protein and constitutively expressed in virus permissive MDCK cell line. Subsequent infection with influenza virus results in incorporation of fusion constructs directly into budding progeny virions that are harvested, purified and inactivated to generate distinct CYT-IVAC formulations. Following validation of immunomodulator incorporation, vaccines were tested for in vivo efficacy in either "young adult" or "aged" female Balb/c mice. Our results demonstrate that our CYT-IVAC~IL-12/HA and CYT-IVAC~IL-23/HA serve as potent mucosal adjuvants in young adult mice elicited significantly high levels of mucosal IgA antibodies and afford superior protection against lethal virus challenge. Our Flt3L/HA formulation was the most effective stimulator of systemic anti-viral antibody levels. In "aged" mice a single dose formulation of IL-12 bearing CYTIVAC was superior at affording protection against lethal homotypic virus challenge. Finally, administration of multimeric M2e molecule co-presented on WIV elicited prolonged antibody responses in "young adult mice" and provided cross-protection from challenge with the heterologous influenza A pandemic strain 2009 H1N1. In conclusion, the CYT-IVAC approach represents a novel tailored advancement to current WIV approaches that has the potential to elicit both potent mucosal and systemic immune responses in young and old. Ph. D.

Animal classification needs vary by use and application. The Linnaean taxonomy is an important animal classification scheme but does not portray key animal identifying information like sex, age group, physiologic stage, living environment and role in production systems such as farms. Ontologies are created and used for defining, organizing and classifying information in a domain to enable learning and sharing of information. This work develops an ontology of animal classes that form the basis for communication of animal identifying information among animal managers, medical professionals caring for animals and biomedical researchers involved in disciplines as diverse as wildlife ecology and dairy science. The Animals in Context Ontology (ACO) was created from an extension and subset of the Systematized Nomenclature of Medicine — Clinical Terms (SNOMED-CT). The principles of the Open Biological and Biomedical Ontologies (OBO) Foundry were followed and freely available tools were used. ACO includes normal development and physiologic animal classes as well animal classes where humans have assigned the animal's role. ACO is interoperable with and includes classes from other OBO Foundry ontologies such as the Gene Ontology (GO). Meeting many of the OBO Foundry principles was straightforward but difficulties were encountered with missing and problematic content in some of the OBO ontologies. Additions and corrections were submitted to four ontologies. Some information in ACO could not be represented formally because of inconsistency in husbandry practices. ACO classes are of interest to science, medicine and agriculture, and can connect information between animal and human systems to enable knowledge discovery. Master of Science

Platelet-rich plasma (PRP) is a popular treatment for equine tendon and ligament injuries; however, commercial PRP systems are expensive. Development of a safe, inexpensive alternative would make PRP therapy more widely available to horse owners. The purpose of this study was to evaluate the quality and bacteriologic safety of PRP produced by three simple, inexpensive tube centrifugation methods and compare the results to a commercial system. Citrated blood collected from 26 normal horses was processed by four methods: blood collection tubes centrifuged at 1200 and 2000 x g, a 50ml conical tube, and a commercial system. Platelet and cell counts and mean platelet volume (MPV) in whole blood and PRP were determined using an automated hematology analyzer. Results were analyzed using mixed model ANOVA with post-hoc comparisons (MPV and fold change for RBC, WBC, and platelets) and binary logistic generalized estimating equations with horse as a blocking factor (absolute numbers of WBC, and platelets). Aerobic and anaerobic cultures were performed. Significance was set at p250,000 platelets/?l. WBC counts were lowest using the commercial system and unacceptably high using the red top methods. The incidence of bacterial contamination was low (2.1%). Based on these results, the conical tube method may be a suitable alternative to commercial PRP systems in cases with budgetary constraints. Master of Science

Paratuberculosis is an intestinal condition in ruminants infected with Mycobacterium avium subspecies paratuberculosis (MAP) and precedes Johne's disease, a chronic enteric disorder in ruminants caused by MAP infection. Necropsy with histopathology provides definitive diagnosis of Johne's disease and positive culture of MAP from tissues provides definitive diagnosis of paratuberculosis. To determine assay sensitivity, 85 formalin-fixed paraffin-embedded (FFPE) tissues from ruminants diagnosed with Johne's disease were tested with a commercial paratuberculosis quantitative polymerase chain reaction (qPCR) assay and had a sensitivity of 92%. To determine assay specificity, 21 FFPE tissues from animals without gastrointestinal disease combined with 13 FFPE tissues from non-ruminant animals (frog, dove, turtle, dog, and 2 cats) with non-paratuberculosis mycobacterial diseases were tested with the commercial qPCR assay and had a specificity of 100%. Slides prepared from the FFPE tissue blocks were stained with hematoxylin & eosin (H&E) and Ziehl-Neelsen's (acid fast stain), then examined for granulomatous inflammation and scored on a scale from 0-4 based on the quantity of acid fast bacteria (AFB). Digital microscopy and morphometric software were used to compute an acid fast bacteria area index (AFBAI) to evaluate a more precise correlation with the qPCR results. The quantity of AFB in tissue slides showed medium to strong correlation with the appropriate qPCR results. The results indicate that the commercial qPCR assay can be used on FFPE tissues with good results and the qPCR results have medium-strong correlation with quantitative acid fast histopathology. Master of Science

Objectives: To determine the effects of prednisone and prednisone plus ultralow-dose aspirin on coagulation in healthy dogs, and to determine intra-individual variation in thromboelastography (TEG). Animals: 14 healthy experimental dogs and 10 healthy client-owned dogs Procedures: Prospective, randomized, blinded study. TEG was performed twice three days apart on each experimental dog prior to treatment and intra-individual variation was calculated. Dogs were given prednisone (2 mg/kg/day) plus aspirin (0.5 mg/kg/day) or prednisone (2 mg/kg/day) plus placebo for 14 days, after which TEG and other baseline tests were repeated. Changes from baseline between and within each group were compared using t-tests or Wilcoxon 2 sample tests. Client owned dogs had TEG performed twice three days apart to determine intra-individual variation. Results: Intra-individual variation in TEG parameters were

Objectives: Phase 1) Establish echocardiographic reference intervals for measurements of the normal canine right heart. Phase 2) Describe the repeatability of normal right heart echocardiographic measurements. Phase 3) Describe the repeatability of right heart echocardiographic measurements which predict pulmonary artery pressure. Materials and Methods: Phase 1) 45 healthy adult dogs. Dogs underwent one echocardiographic examination by the same operator. Phase 2) 6 randomly selected dogs from the pool of Phase 1 dogs. Dogs underwent repeated echocardiograms by two operators. Phase 3) 4 client-owned dogs. Dogs underwent repeated echocardiographic examination by two operators. Results: Phase 1) The linear relationship between dimension and transformed body weight was highly variable. For linear dimensions, most of the scaling exponents were close to the theoretical value of 1/3. For area measurements, most of the scaling exponents were close to 2/3. Phase 2) Of the 168 within-day, between-day and between-operator coefficients of variation (CV) generated, 154 (91.7%) were below 15% and 135 (80.4%) were less than 10%. Phase 3) Of the 100 within-day, between-day and between-operator CVs generated, 72 (72%) were below 20% and 46 (46%) were below 10%. Conclusions: The right heart can be measured with relatively low repeatability. Measurement of the tricuspid regurgitation velocity should be the first priority when attempting to predict pulmonary artery pressure. If tricuspid regurgitation is not present, the use of transpulmonic acceleration time (AT) and the ratio of transpulmonic acceleration-to-ejection time (AT:ET) to indirectly assess pulmonary artery pressure is recommended. Master of Science

Brucellosis is the most common zoonotic disease worldwide. The intracellular localization of Brucella hinders the action of drugs that poorly cross cell membrane barriers. Additionally, when the immune response fails to clear the infection, chronic brucellosis ensues that becomes more challenging to treat with antibiotics. Therefore, two approaches, intracellular drug delivery and immunostimulation, have been explored in this dissertation, with an aim to develop a better therapeutic against Brucella infection in mice. First, to overcome the cell membrane barriers, drug loaded nanoparticles were tested to treat B. melitensis infection in mice. Gentamicin loaded block-ionomer complexes (BICs) and magnetite block-ionomer complexes (MBICs) were tested in vitro and along with clusters of MBICs (MBIClusters) were tested in vivo as tools to deliver gentamicin intracellularly. While these complexes showed very high efficacy compared to free gentamicin against Brucella in macrophage cell culture, they failed to show similar efficacies in mice. Histopathological examination of kidneys from mice treated with MBICs or MBIClusters showed deposition of brown pigment-laden macrophages in peri-renal adipose tissue and the pigment was confirmed as MBICs or MBIClusters based on special staining for iron. Additionally, it was found that doxycycline-gentamicin (DG) treatment results in better clearance of Brucella from infected mice compared to doxycycline alone. Secondly, two vaccine candidates, irradiated B. neotomae (IBN) and outer membrane vesicles (OMVs), were tested as immunostimulants to treat chronic B. melitensis infection in mice in combination with antibiotics. The non-ionic block co-polymer Pluronic P85, when mixed with OMVs as an adjuvant showed significantly higher protection against B. melitensis challenge in vaccinated mice compared to those vaccinated with OMVs alone. When tested as immunostimulants, there was no additive effect of vaccines and antibiotics on Brucella clearance from mice. However, IBN enhanced the production of IFN-γ while OMVs were associated with enhanced antibody production. This enhancement in the immune system resulted in the control of Brucella growth after the end of treatment. When given without antibiotics, vaccine alone failed to clear any Brucella from infected mice. The use of these vaccine candidates in combination with antibiotics shows a potential to prevent relapses in cases of brucellosis. Ph. D.

My dissertation research focused on studying mechanisms of immunomodulation by probiotic lactobacilli on innate and T cell immune responses induced by rotavirus infection and vaccines in a gnotobiotic pig model of human rotavirus (HRV) infection and vaccination. We first studied the effects of probiotics on antigen-presenting cells (APCs) through TLR activation. We found that a mixture of Lactobacilli acidophilus strain NCFM (LA) and L. reuteri (ATCC# 23272) induced strong TLR2-expressing APC responses and virulent HRV induced a TLR3 response. Probiotics and HRV had an additive effect on TLR2- and TLR9-expressing APC responses, consistent with the adjuvant effect of lactobacilli. Dose effects of LA on T cell immune responses were investigated. We found that low dose LA significantly enhanced frequencies of HRV-specific IFN-γ producing CD4⁺ and CD8+ T cells whereas high dose LA reduced frequencies of HRV-specific IFN-γ producing CD4+ T cells. Low dose LA reduced frequencies of induced regulatory (iTreg) cells and TGF-β expression in the iTreg cells whereas high dose LA increased frequencies of iTreg cells and IL-10 expression in the iTreg cells. The dose effects of LA were independent of HRV infection/vaccination. In addition, we demonstrated that TCR-γδ T cells play an important role in modulating immune responses to rotavirus infections. All three γδ T cell subsets showed evidence of activation after HRV infection by increasing TLR2, TLR3, TLR9 expression and IFN-γ production during the acute phase of infection. There was an additive effect between lactobacilli and HRV in inducing total γδ T cell expansion in ileum and in recruiting the cells from blood. HRV infection induced a significant expansion of the CD2+CD8+ γδ T cell subset in the ileum. This subset mainly exerts regulatory functions as evident by expressing FoxP3, secreting TGF-β and IL-10 or increasing production of the anti-inflammatory cytokines by CD4+ and/or CD8+ αβ T cells in the co-cultures. CD2+CD8- and CD2-CD8- γδ T cell subsets have mainly pro-inflammatory and anti-viral functions as evident by secreting IFN-γ or promoting CD4+ αβ T cell proliferation and IFN-γ production. This knowledge will facilitate the development of more effective vaccination and therapeutic strategies to protect children and young animals against rotavirus gastroenteritis. Ph. D.

Eimerian parasites infect cattle worldwide. Information on the infection dynamics of these parasites is lacking in the central Appalachian region of the United States. Studies aimed at characterizing the seasonal dynamics of eimerian parasites in this region were carried out in order to assess the impact of these organisms in grazing systems. In these studies the prevalence of Eimeria spp. infection was highest in calves less than one year of age and subsequently decreased to stable levels in older animals. Although E. bovis was the most common species identified in calves, heifers and cows, mixed species infections dominated. Additional studies were carried out to investigate the effect of stress on Eimeria spp. infection in beef calves. Lower stress, two-stage, weaning methods had no effect on Eimeria spp. infection dynamics in beef calves. These findings must be interpreted in light of the fact that calves used in this study were not managed in a way typical of many calves in the U.S.A. The fact that they were only transported short distances, never commingled, or exposed to a livestock market may explain why a rise in post weaning FOC was not observed. A model of stress- induced coccidiosis was developed using dexamethasone and E. bovis challenge. In this model, an oral challenge of at least 500,000 sporulated E. bovis oocysts in addition to dexamethasone injection at 7 days post challenge increased subsequent FOC. Further investigation of the immune response to E. bovis challenge during times of stress indicates that stress-induced suppression of cell mediated immunity and E. bovis challenge are required to increase subsequent oocyst shedding. These findings may represent the mechanism associated with stress-induced outbreaks of coccidiosis reported to occur in beef cattle in the United States. Ph. D.

Lipopolysaccharide (LPS), a Gram-negative endotoxin, has been well-established as the trigger for the effects of sepsis and septic shock through its binding with the innate immune receptor, Toll-like receptor 4 (TLR4). High doses of LPS signal through TLR4 to produce a massive release of pro-inflammatory cytokines including IL-6, TNFα, and other. Additionally, several recent publications have demonstrated severe metabolic alterations after LPS challenge, suppressing lipid oxidation and concurrently up-regulating glucose oxidation. Unfortunately, this switch in metabolism is inefficient for the great energy demands of the host during a systemic microbial infection which can result in vital organ failure. Meanwhile, a novel concept in several chronic disease pathologies also implicates LPS, although at very low doses. The presence of subclinically elevated circulating endotoxin levels has been termed metabolic endotoxemia and is beginning to be investigated in disease pathologies including insulin resistance and type II diabetes, atherosclerosis, cancer metastasis and Parkinson's disease. These disease phenotypes all possess a component of chronic inflammation whose source has not largely been understood, but examining the effects of very low doses of LPS may provide vital information in understanding their etiologies. However, most information on LPS signaling has been obtained using high doses of LPS (10-200ng/ml) while little to no studies have been published regarding the effects of very low doses of LPS (1pg-100pg/ml) on inflammatory and metabolic alterations. Thus, we use in vivo and in vitro models to determine that both IRAK1 and JNK are critical points of crosstalk downstream of TLR4 for the metabolic and inflammatory alterations associated with metabolic endotoxemia. Additionally, we observed significant down-regulation of nuclear receptors responsible for fatty acid metabolism, including PGC1α, PPARα, and PPARγ after very low dose LPS challenge. Further, we observe phenotypic changes in fatty acid oxidation and glucose oxidation, as well as subsequent changes in cytosolic acetyl-CoA levels and acetylation of pro-inflammatory transcription factor ATF2. Overall our studies point to several mechanisms of cross-talk between metabolism and inflammation and offer significant support to the concept of metabolic endotoxemia in the development of chronic disease. Ph. D.

Evidence suggests that the neurotropic parasite Toxoplasma gondii may play a role in the development of cognitive impairments. My hypothesis was that congenital exposure to T. gondii would lead to detectable age and sex related differences in behavior and neurotransmitter levels in mice. The neurotransmitter dopamine and commonly used anti-schizophrenic agents were evaluated against T. gondii in human fibroblast cells. Dopamine caused a significant increase in tachyzoite numbers at 250 nM but not 100 nM and the drugs valproic acid, fluphenazine, thioridazine and trifluoperazine inhibited T. gondii development. The effects T. gondii infection had on behavior were examined using a congenital mouse model. Previous work demonstrated maternal immune stimulation (MIS) with interferon gamma (INF-g) resulted in decreased fetal mortality from congenital T. gondii infections; therefore I examined the effects of INF- g treatment of mothers to determine if protection from the behavioral effects of T. gondii occurred in their offspring. No differences in concentrations of neurotransmitters in the brains of congenitally infected mice were observed. I found that mice infected with T. gondii developed adult onset behavior impairments with decreased rate of learning, increased activity and decreased memory, indicating cognitive impairment for male mice and not female mice. My findings support the evidence T. gondii is a factor in the development of cognitive impairments. My results for T. gondii exposed male mice are consistent with the convention that males have more cognitive impairments in the prodromal stage of schizophrenia. MIS with IFN-g had a minimal effect on behavior post sexual maturity but had a greater effect on pre sexual maturity female mice which exhibited difficulties with spatial memory, coordination and the ability to process stimuli. The results indicate the behavior alterations from IFN- g are transient. When MIS is given prior to congenital infection with T. gondii, we detected no behavior deficits in any group of mice, including male mice post sexual maturity. Based on the results of my study, I must reject the hypothesis that neurotransmitter levels are influenced by congenital toxoplasmosis and accept the hypothesis that congenital T. gondii infection caused cognitive impairments in male mice post sexual maturity. Ph. D.

Flexor tendon lacerations in horses are traumatic injuries that can be career ending and life threatening. In the horse, a tendon repair must withstand the strains placed on the tenorrhaphy by immediate weight bearing and locomotion post-operatively. Despite the use of external coaptation, such strains can lead to significant gap formation, construct failure, longer healing time and poor quality of the healed tendon. Similar to equine surgery, gap formation and construct failure are common concerns in human medicine, with early return to post-operative physiotherapy challenging the primary repair. Early return to exercise and decreased gap formation has been shown to reduce adhesion formation. Based on these concerns, the ideal tenorrhaphy suture pattern for equines would provide: 1) high ultimate failure load, 2) resistance to gap formation, 3) minimal alteration in blood supply, and 4) minimal adhesion formation. Historically, various suture patterns and materials have been evaluated for human and equine flexor tendon repair. Results of equine studies suggest the three-loop pulley pattern (3LP) compares favorably to other patterns and is recommended for primary tenorrhaphy. However, this pattern still experiences significant gap formation and can result in failure. As a result, a technique which decreases the problems inherent in the 3LP is warranted for tenorrhaphy of equine flexor tendons. A review of the human literature highlights certain characteristics of the tenorrhaphy that may improve results including core purchase length and suture loop characteristics. Optimization of these tenorrhaphy characteristics can increase tenorrhaphy performance and patient outcome. The six-strand Savage technique (SSS) is a pattern routinely used in human hand surgery for tendon repair, and possesses high ultimate failure load and resistance to gap formation that may be beneficial for application in equine tendon repair. This study compared a novel tenorrhaphy pattern for horses, the SSS, with the currently recommended pattern, the 3LP, in an in vitro model. We hypothesize the SSS will fail at a higher ultimate load, resist pull through, and resist gap formation better than the 3LP. All testing used cadaveric equine superficial digital flexor tendons from horses euthanized for reasons other than musculoskeletal injury. All testing was approved by the IACUC. The two techniques were applied to cadaveric equine superficial digital flexor tendons. The same investigator performed all repairs (EE). Biomechanical properties were determined in a blinded, randomized pair design. Ultimate failure load, mode of failure and load required to form a 3mm gap were recorded on an Instron Electropuls materials testing system. Gap formation was determined using synchronized high-speed video analysis. Results are reported as mean + standard deviation. Statistical comparisons were made using Student's T test, with significance set at p

A variety of neurologic illnesses including peripheral and cranial neuropathies, central vestibular disease, seizures and coma have been associated with hypothyroidism in dogs. Repeated studies have shown that there is loss of blood brain barrier (BBB) integrity in these animals. Current research has also shown the development cerebrospinal fluid abnormalities in neurologically normal hypothyroid dogs; a finding that is related to BBB degradation. This derangement may be secondary to atherosclerosis and vascular accidents. One possible mediator of vasospasm and ischemic brain injury is endothelin-1 (ET-1). Another group of mediators of vascular dysfunction that has been found in CSF of dogs with various other CNS diseases is matrix metalloproteinases (MMP). The purpose of this study was to assay molecular markers that may contribute to disruption in the blood brain barrier in chronically hypothyroid canines. We hypothesized that BBB disruption in hypothyroidism is mediated by ET-1 and MMPs, as evidenced by increased concentrations of these proteins in CSF compared to controls. Cerebrospinal fluid (CSF) previously collected from 9 control and 9 experimentally induced hypothyroid dogs was used. Administration of I-131 was used to create the experimental model. CSF from time points 0, 6, 12, and 18 months post-induction were evaluated using an ELISA kit for endothelin-1. CSF from each time point was also evaluated using gelatinase zymography to detect MMP-2,9, and 14. The endothelin assay was able to detect ET-1 in CSF as determined by a spike and recovery method. However, ET-1 was undetectable in CSF of control and hypothyroid dogs at all time points. Constitutively expressed MMP-2 was detectable in all dogs at all time points. No other MMPs were detectable in CSF. ET-1 and gelatinase MMP,-9, and -14 are not primary mediators of BBB damage in chronically hypothyroid dogs. They could be involved secondarily and may be better evaluated with different assays or in temporal association with the development of clinical signs of neurologic dysfunction. Additional research is needed to confirm this finding and to evaluate biomarkers of non-vascular components of the BBB. Master of Science

Dysregulation of apoptosis during development is a possible mechanism for teratogen-induced birth defects. Neural tube defects (NTDs) are the second most common fetal malformations. Non-specific stimulation of maternal immune system prevents birth defects. This study investigated the role of dysregulated apoptosis in formation of NTDs from two teratogens: valproic acid (VA) and an unknown teratogen found in tap water. Interferon- γ (IFN γ) was used to stimulate maternal immunity to evaluate the role of altered apoptosis in this protective mechanism. Apoptosis was evaluated using flow cytometry, Terminal Transferase dUTP Nick End Labeling (TUNEL) assay and gene expression changes by RT2 Profiler PCR arrays. Additionally, changes in the expression of key signal transduction pathway genes that play a role in development were determined. Increased apoptosis, suggesting involvement in VA teratogenicity, was observed along the neural tube in both normal and abnormal embryos from VA-exposed dams. Increased apoptosis in normal VA-exposed embryos suggests that VA may alter other cellular processes such as cell proliferation and differentiation in addition to apoptosis. Apoptotic percentages in embryos with NTDs from IFNγ+VA dams were similar to controls, which indicated resistance to teratogen-induced apoptosis. In IFNγ+VA-exposed embryos with NTDs, immune stimulation failed to prevent apoptosis. VA initiated both death and survival signaling in the embryos; however, upregulation of the apoptotic genes and down regulation of anti-apoptotic genes of p53 and Bcl2 family tended to shift the balance towards death signaling. This change in gene expression patterns could result in increased apoptosis and NTDs in VA-exposed embryos. Immune stimulation normalized changes in the expression of pro-apoptotic signaling molecules. These results suggest immune stimulation protects embryos from teratogenicity of VA by preventing VA-induced apoptosis. VA altered the hedgehog, Wnt, retinoic acid and fibronectin signaling pathways in embryos with NTDs. These results suggest that VA also disrupted signaling pathways required for various morphogenic events during organogenesis. Immune stimulation normalized the expression of Fn1 and Hspb1 and thus may mediate protection through these signaling pathways. In tap water exposed embryos, no change in apoptotic pattern was observed by flow cytometry, TUNEL assay and RT-PCR. Also, none of the signal transduction pathway genes tested were significantly altered in tap water-exposed embryos. This suggests that apoptosis is not a mechanism for teratogenicity resulting from exposure to the contaminant in tap water. Ph. D.

Hepatitis E virus (HEV) is a major cause of enterically transmitted acute viral hepatitis in developing countries that lack proper hygienic infrastructure. Hepatitis E is globally distributed and has emerged as an important public health disease in both developing and industrialized countries. HEV is a non-enveloped virus carrying a single-stranded positive-sense RNA genome of approximately 7.200 bp in length. The life cycle of HEV is poorly understood due to the lack of an efficient cell culture system. Animal model systems, including non-human primates, swine, and chickens are being used to study some fundamental aspects of the HEV biology. Recently, novel animal strains of rat and rabbit HEV have been discovered, and whose usage as animal model systems needs to be established. HEV infections in pigs and chickens provide excellent model systems to study the replication and pathogenesis aspects of HEV. Recently, we identified a hypervariable region (HVR) in the open reading frame 1 (ORF1) of HEV. The objectives of this dissertation were to utilize chicken and swine model systems to study the role of HVR in HEV infection in vivo, to determine the effects of HVR on replication of HEV in vitro, and to analyze the effect of exchange of HVR among different genotypes on the replication-competency and virion production in vitro. Extensive sequence variability in the HVR among HEV strains of different genotypes prompted us to study the dispensability of this region. Initially we constructed two partial deletion mutants of genotype 1 human HEV, hHVRd1 and hHVRd2, with in-frame deletion of amino acids (aa) 711 to 777 and 747 to 761 in the HVR of a sub-genomic GFP HEV replicon. Expression of enhanced green fluorescent protein by the mutant hHVRd2 confirmed the dispensability of amino acid residues 747-761 of the HVR. To confirm our in vitro results, specific-pathogen-free (SPF) chickens were intra-hepatically inoculated with capped RNA transcripts from three avian HEV HVR-deletion mutants: mutants aHVRd1 (Δ557-585), aHVRd2 (Δ612-641), and aHVRd3 (Δ557-641). Chickens intra-hepatically inoculated with the mutants, aHVRd1 and aHVRd2, developed active viral infection as evidenced by seroconversion, viremia, and fecal virus shedding. Mutant aHVRd3, with a larger HVR deletion, was apparently attenuated in chickens. Additionally, we used the swine model system to further verify our results from the chicken study. The infectivity of four genotype 3 swine HEV HVR-deletion mutants, sHVRd1 (Δ712-790), sHVRd2 (Δ722-781), sHVRd3 (Δ735-765), and sHVRd4 (Δ712-765) constructed using the genotype 3 swine HEV as the backbone was determined in SPF pigs. Pigs intra-hepatically inoculated with capped RNA transcripts from the mutants sHVRd2, sHVRd3, and sHVRd4 developed active viral infection, whereas mutant sHVRd1 (Δ712-790), with a nearly complete HVR deletion, exhibited an attenuation phenotype. The data from these studies indicate that deletions in HVR do not abolish HEV infectivity in vitro or in vivo, although evidence for attenuation was observed for HEV mutants with a larger or nearly complete HVR deletion. To further elucidate the role of HVR in HEV replication, we investigated the effects of serial amino acid deletions in HVR on the replication of HEV. We first constructed a genotype 1 human HEV luciferase replicon by replacing the ORF2 gene that encodes for the capsid protein with the fire fly luciferase reporter gene. Using the backbone of human HEV genotype 1 luciferase replicon, we constructed a series of HVR-deletion mutants with deletions of variable lengths in the HVR. Amino acid deletions Δ711-725, 711-740 and Δ711-750 were engineered at the N-terminus, deletions Δ729-754, Δ721-766, and Δ716-771 were engineered in the central region, and deletions Δ761-775, Δ746-775, and Δ736-775 were engineered at C-terminus of the HVR. The effects of these serial deletions on HEV RNA replication in the human liver carcinoma cell line, Huh7, were examined. Replication levels of mutants carrying these deletions were compared with that of the wild-type HEV in Huh7 cells. We observed that deletions in the HVR did not abolish viral RNA synthesis but substantially reduced the replication levels of viral RNA, as measured by the reporter luciferase activity. To further verify the effects of HVR deletions on viral RNA replication as observed with the genotype 1 human HEV replicon, we subsequently used a genetically-distinct strain of HEV, avian HEV, and constructed an avian HEV sub-genomic luciferase replicon by substituting the ORF2 gene of avian HEV with the fire fly luciferase gene. Avian HEV HVR-deletion mutants Δ557-603, Δ566-595, and Δ573-587 were then engineered using the backbone of avian HEV luciferase replicon. The replication efficiency of the three deletion mutants of avian HEV in chicken liver hepatoma cell line, LMH, was evaluated. Compared with the wild-type avian HEV, the viral RNA synthesis of the avian HEV HVR-deletion mutants was considerably reduced by the HVR deletions. To analyze the impact of the complete HVR deletion on avian HEV infectivity, we constructed an avian HEV mutant with a deletion of the entire HVR region (aaΔ557-603) using the avian HEV infectious cDNA clone as the backbone. After confirming the viability of the complete HVR-deletion mutant in LMH cells, SPF chickens were intrahepatically inoculated with capped RNA transcripts generated from the mutant. None of the chickens inoculated with the complete HVR-deletion mutant showed evidence of HEV infection, indicating that drastic reduction in replication levels due to complete HVR deletion has resulted in the loss of virus infectivity. The results indicated that HVR may have critical residues that may interact with viral/and or host factors and modulate the replication efficiency of HEV. In the final part of the dissertation research, we sought to determine if the variable sequences of HVR are genotype-specific for in vitro virus replication. By using the genotype 1 human HEV as the backbone, we swapped the HVR of genotype 1 human HEV with the HVRs of the genotype 3 swine HEV and the distantly-related avian HEV to construct two inter-genotypic chimeras, pSKHEV2-Sw and pSKHEV2-Av. Similarly, by using the genotype 3 swine HEV as the backbone, the HVR of genotype 3 swine HEV was swapped with the HVR of genotype 1 human HEV to construct the chimera, pSHEV3-Hu. The viability of these chimeras was tested in Huh7 cells that are permissive for HEV replication. Immunofluorescence assay (IFA) with anti-HEV antibodies revealed that all the three chimeras were replication-competent in Huh7 cells. The infectivity of these chimeras was subsequently evaluated in HepG2 cells. The results showed that exchange of the HVR between different genotypes of mammalian HEVs does not abolish the replication competency and infectivity of HEV. This finding suggests that HVR is not genotype-specific with respect to viral replication and infectivity. The absence of detectable viral antigen in HepG2 cells infected with chimera pSKHEV2-Av suggested a functional incompatibility of the HVR of avian HEV in the mammalian HEV genome. In summary, we identified a highly variable sequence, HVR, in the ORF1 of the HEV genome, and the sequences of the HVR vary significantly among HEV strains of different genotypes. We found that the HVR contain sequences that are dispensable for virus infectivity both in vitro and in vivo. Deletion analysis of HVR revealed that the region may play a role in modulating the replication efficiency of HEV RNA by interacting with viral and/or host factors. Finally, we demonstrated that HVR is not genotype-specific for virus replication and the region can be functionally replaced between mammalian HEV genotypes for virus replication and virion production in vitro. The results from this dissertation research have important implications for better understanding the biology and mechanism of HEV replication and may aid in our efforts to eventually develop a modified live-attenuated vaccine against HEV. Ph. D.

Many oncolytic virotherapies have shown great advantages for rapid, rational design through recombinant DNA technology to facilitate the targeting of a broad spectrum of malignancies. Newcastle disease virus (NDV), an avian paramyxovirus, is naturally tumor-selective and inherently oncolytic. Our approach is to develop NDV-based nanoparticles (VBNP) for oncolytic virotherapy. VBNPs are non-infectious and non-replicating and are relatively safe. We obtained VBNPs by co-expressing matrix (M), hemagglutinin (HN), and fusion (F) proteins of NDV in avian/ mammalian cells. The budding characteristics, size and morphology of VBNPs were similar to authentic virions. As a proof of concept, we engineered the apoptin (VP3) gene of chicken anemia virus in VBNPs and specifically targeted them to folate-receptor bearing tumor cells by surface conjugation to folate. The VBNPs killed tumor cells by apoptosis and induced proinflammatory and chemotactic cytokines. The VBNPs, although not curative, were able to limit the progression of xenotransplanted fibrosarcoma and malignant glioma tumors and provided a survival advantage in nude mice. We also engineered NDV M based particles with nipah virus surface glycorporteins to target ephrin B receptors. NDV based nipah Virus BNPs (NiV-ndBNP) were morphologically similar to authentic NiV virions. NiV glycoproteins were incorporated into the NDV M based particles, despite poor sequence homology in the transmembrane domain and cytoplasmic tails of glycoproteins. Our results suggest that VBNPs could be used to deliver small molecules, tumor antigens, anti-tumor/ reporter genes and also aid in generating tumor specific immunity by rational design. Master of Science

Oxidative stress plays a vital role in the pathogenesis of many chronic and acute inflammatory diseases. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are two key mediators that are known to induce cellular and tissue oxidative stress. The generation of ROS and RNS is mediated by innate immune signaling processes. Lipopolysaccharide (LPS), a major inflammatory signal, is known to be a potent inducer of ROS/RNS. Thus, strategies that may block LPS-mediated generation of free radicals may hold promise in treating various inflammatory disease processes. However, the molecular mechanisms underlying LPS-mediated ROS/RNS production are not fully defined. Interleukin-1 Receptor associated kinase (IRAK-1), an intracellular kinase downstream of Toll-like Receptor 4 (TLR4) has been shown to contribute to the inflammatory cascade associated with LPS-TLR4 signaling pathway. However, its role in ROS production has not been defined. Therefore, we tested the hypothesis that IRAK-1 plays an important role in regulating ROS/RNS production. Both in vitro and in vivo studies were conducted to investigate the role of IRAK-1 in modulating free radicals as well as oxidative stress. In vitro studies demonstrate that IRAK-1 is a critical molecule involved in the induction of ROS/RNS. IRAK-1 deletion ablated free radical production following LPS challenge in a variety of cell types including macrophages, fibroblasts and microglia. Mechanistically, we observed that IRAK-1 is required for optimal expression and activity of NADPH oxidase subunits and iNOS. IRAK-1 deletion reduced LPS-triggered p47phox membrane translocation, suppressed NOX-1 expression and protein levels as well as hampered Rac1 activation. On the other hand, IRAK-1 deletion sustained antioxidative enzyme activity and levels in IRAK-1-/- macrophages and fibroblasts. In terms of the in vivo physiological consequences, IRAK-1-/- mice exhibited attenuated lipid peroxidation in vital organs, attenuated histopathological lesions in liver and kidney, and reduced endotoxemia-associated mortality. Taken together, IRAK-1 may, at least in part, serve as an important therapeutic target in the treatment of various inflammatory disease processes. Ph. D.

Utilization of DNA vaccines in aquaculture has been gaining interest and recent efforts have been focused on methods of delivering DNA vaccines to fish. In the present study, a methodology was sought that could protect DNA vaccines such that they could be orally administered. The main objective of the study was to determine if a DNA vaccine could be effectively compounded into an orally administrable formulation with chitosan or polylactide-co-glycolide (PLG). The immune response of hybrid striped bass (Morone saxatilis x Morone chrysops) following oral delivery of a DNA vaccine containing Mycobacterium marinum Ag85A plasmid in either chitosan or PLG nanoparticle encapsulation was evaluated. Hybrid striped bass were divided into four experimental groups: IM immunization of the DNA vaccine as a positive control, oral delivery of uncomplexed DNA vaccine, oral delivery of chitosan or PLG alone as a negative control, and oral delivery of complexed chitosan or complexed PLG DNA vaccine. Fish were bled at regular intervals and an ELISA was used to evaluate antibody levels in individual fish. While the chitosan /plasmid DNA complex containing the Mycobacterium marinum Ag85A gene failed to produce a significant antibody response, the PLG/plasmid DNA matrix stimulated humoral immune response in the fish. Master of Science

Weaning is defined as the physical separation of the cow-calf pair and the end of milk feeding. Natural weaning occurs between 7 and 14 months and is a gradual process. However, domestic weaning occurs between 6 and 8 months and occurs rapidly. Calves that are abruptly separated from their dam respond with increased vocalization and walking, and decreased eating and resting. The psychological stress the calf undergoes during weaning causes elevated glucocorticoid and catecholamine hormone concentrations that may predispose to increased morbidity and/or mortality from infectious diseases such as Bovine Respiratory Disease Complex. As an attempt to counter these changes, alternative weaning methods have been implemented and normally occur in two stages. Two-stage weaning begins with the cessation of milk feeding for approximately one week with the calf maintaining some contact with their dam and then permanent separation occurs. One of these methods uses a single fence to separate the cow-calf pair; this process allows the calf to see, hear and smell their dam, but does not allow the calf to suckle from its dam. Increases in cortisol, a glucocorticoid, have been linked to immunological alterations. Most notably, elevated cortisol concentrations decrease neutrophil function by down regulating the gene expression of CD62L and Fas. Cortisol also alters lymphocyte phenotype by decreasing ?δ T cells and increasing°? T cells in the circulation. Lastly, increases in cortisol can modify T cell cytokine production. The cytokines IL-12 and IFN? are secreted from T helper 1 cells while T helper 2 cells secrete IL-4 and IL-10; these T cells subsets also inhibit one another. During higher cortisol concentrations, these T cells are biased toward T helper 2 cytokine production. All these changes in immune function can lead to increased susceptibility to disease around the time of weaning. Therefore, two trials were conducted to test the hypotheses that abrupt weaning results in elevated concentrations of cortisol and subsequently alters immunological functions, and that fenceline weaning alleviates the increase in cortisol and alterations to immune function associated with weaning. In the fall of 2008, 12 Angus and Angus-X heifers (186°21 kgs; 174°16 days of age) were blocked by age and weight and randomly allotted into two groups, fenceline and abrupt. Blood samples were taken on day -7, 0, 7, 14, 21, and 42; fecal samples were taken on day -7, 0, and 3. All calves were weighed on day -7, 0, 7, 14, and 42. On day -1 all calves were separated from their dam and transported for 2 hours to another facility. On day 0 all calves were vaccinated with Brucella abortus (strain RB51). Serum was analyzed for IFN? and IL-4 as well as IgG1 and IgG2 specific antibodies to RB51. Fecal samples were analyzed for cortisol metabolites. Both IgG1 and IgG2 antibodies to RB51 increased from day 0 to day 14 (P

Brucella are Gram-negative intracellular bacteria that cause abortion and infertility in livestock and chronic disease in humans. The Centers for Disease Control and Prevention (CDC) categorizes them as class B pathogens due to their zoonotic potential. Currently, there are no efficacious Brucella vaccines for humans available. Very few studies have focused on identifying protective vaccines against respiratory exposure. Protection by B. abortus rough vaccine strains RB51 and RB51SOD is through strong CD4⁺ Th₁ and CD8⁺ Tc₁ adaptive immunity. However, limited information is available on how they stimulate innate immunity. This knowledge is critical for improving these vaccines for their potential use in humans. Dendritic cells (DCs) play a crucial role bridging innate and adaptive immunity. Therefore, enhancing the ability of rough vaccine strains to induce DC maturation and function could be critical for upregulating protective T-cell responses. Herein, we demonstrated that live vaccine strain RB51 induced significantly better (p≤0.05) DC maturation and function in vitro and upon intranasal inoculation in vivo compared to strain RB51SOD or strain 2308. Due to safety concerns of live vaccines, irradiated and heat killed vaccines were also tested; only live strain RB51 infected DCs induced significant (p≤0.05) DC function based on TNF-α and IL-12 secretion. DC activation occurs through Toll-like receptors (TLRs) 2, 4 and 9. Our study reported that strain RB51 induced significant (p≤0.05) DC activation compared to strain 2308, which was not dependent on a specific TLR. However, strain RB51 induced TNF – α production was TLR2 and TLR9 dependent and IL-12 production was TLR2 and TLR4 dependent. TLR4 KO mice had significantly (p≤0.05) higher number of strain RB51 colonies present at day 14 post infection. By unraveling the innate immune responses to Brucella, the ultimate goal of these studies is to develop a protective vaccine for animals and people against respiratory challenge. As such, we tested several vaccination strategies. Despite enhanced DC activation and function achieved by vaccine strains, they failed to protect mice against intranasal challenge with strain 2308. Future experiments will address host-pathogen interaction at the lung microenvironment and elucidate immune mechanisms that will enhance protection against aerosol exposure. Ph. D.

Tendon injuries are a significant cause of morbidity in performance horses with superficial digital flexor tendon injury reported to represent up to 43% of overall Thoroughbred racehorse injuries. Natural repair is slow and results in inferior structural organization and biomechanical properties and, therefore, reinjury is common. The inability of tendon to regenerate after injury, or to heal with mechanical properties comparable to the original tissue, is likely attributable to low vascularity and cellularity of the tissue, low number of resident progenitor cells, and healing under weight-bearing conditions. Strategies to improve tendon healing have focused on enhancing the metabolic response of tenocytes, modulating the organization of the newly synthesized extracellular matrix, or administering progenitor cells to enhance repair. Significant research effort has been directed at the use of adult mesenchymal stem cells as a source of progenitor cells for equine tendon repair and recent clinical applications have utilized adult autologous stem cells derived either from adipose tissue or bone marrow aspirates. Isolation of a homogenous population of stem cells from bone marrow is time-consuming, and there is much variation in cell numbers, cell viability and growth rates among samples. Recently, a population of progenitor cells has been isolated from equine flexor tendons, thus providing an alternative source of progenitor cells from the target tissue for therapeutic intervention. The interaction between cells and the extracellular matrix (ECM) is an important factor in regulation of cell function. Proliferation, migration, differentiation and gene expression of many cell types are altered by adhesion to and interaction with matrix proteins and the extracellular environment. Tendon progenitor cells reside within a niche that comprises primarily parallel collagen fibers, and this niche plays an important role in regulating their function and differentiation. Culture conditions replicating this environment could be beneficial for both cell growth and matrix gene expression. The objectives of the study were to compare cell growth kinetics and biosynthetic capabilities of bone marrow mesenchymal stem cells (BMMSCs) and tendon derived progenitor cells (TPCs) cultured on commercially available bovine, highly purified bovine, porcine, and rattus collagen sources and standard tissue culture surfaces. We hypothesized that collagen type I matrix would preferentially support TPC proliferation and up regulate gene expression for collagens and organizational components of tendon and therefore provide a culture system and progenitor cell type with advantages over the current practice of BMMSC expansion on standard cell culture plastic surfaces. Cells were isolated from 6 young adult horses, expanded, and cultured on collagen-coated tissue culture plates, and no collagen control for 7 days. Samples were analyzed for cell number on days 4 and 7, and for mRNA expression of collagen type I, collagen type III, cartilage oligomeric matrix protein (COMP), and decorin on day 7. Glycosaminoglycan (GAG) synthesis was analyzed on day 7. Differences of cell number between collagen groups and cell type, and in gene expression and GAG synthesis between collagen groups and cell types, were evaluated by use of mixed-model repeated measures ANOVA. Pair-wise comparisons were made on significant differences identified with ANOVA using Tukey's post hoc test. Statistical significance was set at P

Extracorporeal circulation damages blood and causes harmful side effects such as stroke and/or systemic inflammatory response in patients. Reactions of blood components to extracorporeal circulation include complement and inflammatory reactions, coagulation and thrombogenesis, frank hemolysis, and platelet activation and adhesion to the extracorporeal circuit. Non-physiologic pressure and flow produced by blood pumps contribute to blood injury. Two pump types, roller and centrifugal, are used for maintaining flow, with various models available from different manufacturers. This study compared the effects of these two pumps in identical, isolated, artificial circuits to a non-pumped control for a period of 24 hours on heparinized porcine blood. Hematology parameters were used to evaluate blood damage. Mean corpuscular volume, mean corpuscular hemoglobin, white blood cell count, platelet count, and mean platelet volume were affected by time of circulation. Mean corpuscular hemoglobin, platelet count, and red cell distribution width were different between circulated and non-circulated blood, however no differences were found between the pumping systems in any parameter. Red blood cell count, total hemoglobin, and hematocrit were not affected by time or treatment. The changes observed in this study have implications for the use of extracorporeal circulation in the clinical setting and in future use of blood as a potential organ perfusion medium. Master of Science

Objective: To determine the ability of the Tibial Plateau Leveling Osteotomy (TPLO) to restore normal joint kinematics in a cranial cruciate ligament (CrCL)-deficient stifle through a loaded range of motion. Methods: Paired pelvic limbs from 12 dogs were compared in an in vitro biomechanical study. Each limb was placed in a custom designed jig at 120° of stifle extension under an axial load of 20% body weight. Electromagnetic motion tracking sensors were placed on the distal femur and proximal tibia. A force was applied at approximately 10 N/sec to mimic the action of the quadriceps muscle. Force application allowed the limb to move from 120° to maximal extension. Positional data was acquired at 60 points/second. Each limb was tested under normal, CrCL-deficient, and TPLO-treated conditions. Results: The TPLO failed to normalize CTT within the CrCL-deficient stifle; however, values trended towards intact values throughout the range of motion. No significant differences were noted in internal rotation in any of the three conditions from 120° – 137°. Hyperextension values did not differ significantly between conditions. Conclusion: Data from this biomechanical model suggests that the TPLO fails to neutralize CTT throughout a loaded range of motion. Internal rotation and hyperextension values were not found to differ significantly between intact, CrCL-deficient and TPLO repaired stifles. The effectiveness of the TPLO in restoring normal biomechanics is more significant at greater angles of flexion. Master of Science

Brucella abortus is a zoonotic agent that primarily infects cattle and causes brucellosis. B. abortus strain RB51 is a live, attenuated vaccine licensed for cattle. However, there is no available vaccine to prevent human brucellosis. Outer membrane vesicles have been tested as potential vaccines to prevent diseases caused by bacterial species. OMV are constantly released from Gram-negative bacteria. They are comprised principally of the outer membrane components and periplasmic proteins from the bacterial cell envelope. The research in this thesis examined the adjuvant property of non-replicative, metabolically active irradiated strain RB51 and the protective ability of OMV derived from strain RB51. Irradiated B. abortus strain RB51 was assessed for its ability to act as an adjuvant to induce protection against malaria. It was found that irradiated B. abortus strain RB51 administered along with fasciclin related adhesive protein (FRAP) to mice induced a protective immune response and a significant decrease in parasitemia after challenge with Plasmodium berghei. Strain RB51 and strain RB51 over-producing Cu/Zn superoxide dismutase (Cu/Zn SOD) were used to produce OMV. Western blotting and SDS-PAGE gel staining confirmed the presence of OMV and the over-production of Cu/Zn SOD. OMV were delivered to mice using an intraperitoneal route and, in some cases, with aluminum hydroxide adjuvant. The immune response was assessed by antibody isotyping with respect to OMV and measuring splenic clearance (i.e. protection) from a B. abortus strain 2308 challenge. The results demonstrate that OMV from B. abortus strain RB51 or strain RB51 over producing Cu/Zn SOD produced a Th1 polarized immune response as measured by specific OMV antibodies and cytokines but no statistically significant protection was observed. Master of Science

Intracellular pathogens like Salmonella have developed various mechanisms to evade host defenses, and they can establish infections. Treatment and eradication are difficult due to our inability in achieving the optimum concentrations of cell-impermeable aminoglycosides like gentamicin within these cells. In this dissertation, we hypothesize that developing a novel core-shell methodology for incorporating high amounts of gentamicin into the cores with either hydrophilic or amphiphilic shell will be more effective than the free gentamicin in clearing intracellular Salmonella infection. Hydrophilic core-shell nanostructures (N1) were made with block co-polymers of poly (ethylene oxide-b-sodium acrylate) blended with sodium polyacrylate (PAA-+Na) and complexed with the polycationic antibiotic gentamicin. N1 showed 20-25 fold higher gentamicin loading than the currently existing materials and reduced numbers of viable Salmonella in the liver and spleen compared to free gentamicin. To further improve the rate and route of uptake, the shell of the nanostructures were made amphiphilic by incorporating pluronics F68 (PPO)₆₈ in the block copolymer. We showed that core-shell nanostructures encapsulating gentamicin having (PPO)₆₈ in the shell (N2) enhances the rate and modulates the route of uptake into macrophages, thus promoting significant reduction in the intracellular Salmonella in-vitro and in-vivo. The main drawback of N2 was its poor stability at physiological pH of 7.4, 0.1 M NaCl. Therefore, core-shell nanostructures encapsulating gentamicin containing pluronic P85 (PPO)₈₅ in the shell (N3) with improved colloidal and ionic stability were designed. N3 achieved significant intracellular reduction of vacuolar Salmonella (0.53 log₁₀) and cytoplasm resident Listeria (3.11 log₁₀) compared to free gentamicin in-vitro. However, greater reduction of Listeria suggested that sub-cellular localization of bacterium influences targeting by N3. Even though oral administration of N3 was not effective compared to free gentamicin, parenteral (I.P.) administration significantly reduced the intracellular Salmonella from liver and spleen compared to free gentamicin and appeared to have no abnormal in-vivo toxicity. In summary, core-shell nanostructures encapsulating gentamicin (N) with improved encapsulation efficiency and different shell chemistry (N1, N2 and N3) were developed with enhanced efficacy against intracellular Salmonella. The novel gentamicin delivery approach developed in this study may be applicable for therapy of many intracellular infections. Ph. D.

Vascular endothelial growth factor (VEGF) is a potent angiogenic agent. VEGF activates its biologic responses through two cell-surface receptors, Flt1 and Flk1. In addition to the transmembrane form of Flt1, the Flt1 gene also encodes a secreted, truncated form of the receptor (sFlt1) translated from an mRNA in which a portion of intron 13 is preserved. sFlt1 retains high affinity for VEGF and thereby inhibits its angiogenic activity. Intron 13 contains important cis elements involved in sFlt1 mRNA formation. Here, we test the hypothesis that polymorphisms in the human Flt1 gene, particularly SNPs at sites suspected to contain splicing or cleavage-polyadenylation signals, influence Flt1 pre-mRNA processing and rates of Flt1 and sFlt1 expression. The NCBI SNP database contained 23 SNPs in the region of interest, one each in exons 13 and 14. An independent human SNP screen confirmed several of the reported SNPs. The web-based ESEfinder software predicted that the exon 13/14 SNPs had reduced potential for recruitment of splicing components. To test effects of exonic SNPs on Flt1 pre-mRNA processing, wild type and mutant Flt1 minigene plasmids were transfected into NIH/3T3 cells. Both exonic SNPs were associated with ~40% decreases in Flt1:sFlt1 mRNA ratios determined by real-time PCR. To facilitate exploration of ESEs in regulated RNA splicing, a PERL computer program, "EXONerator" was written to silence predicted ESEs without altering polypeptide sequence. These results support the notion that small changes in exon composition can have significant effects on splicing activity and underscore the utility of software tools for hypothesis generation. Ph. D.

Flexor tendonitis is a common cause of lameness and wastage in the equine athlete. Current techniques for tendonitis therapy provide limited success, and horses that do recover tend to return at a decreased level of performance. Current treatment techniques have begun to focus on regenerative medicine to improve tissue healing. Investigations of new treatments are made difficult by the lack of reliable in vitro models that allow for accurate comparison of treatment protocols. New techniques are often implemented into the clinical setting prior to thorough investigation for safety and efficacy. In vitro testing is an important step in the development of new therapeutic agents. However, results of in vitro tests should only be deemed as useful if the model used is one that is reliable and mimics the clinical situation that the reseachers are attempting to investigate. Equine flexor tendonitis is believed to be the result of microdamage caused by cyclic loading of tendons. Cyclic loading of fibroblasts results in increased production of the inflammatory cytokine prostaglandin E2 (PGE2). Thus the exposure of tendon fibroblasts to exogenous PGE2 may induce metabolic changes in the cells similar to what is seen in clinically affected animals making this a useful model for the investigation of therapeutic techniques. Currently a variety of techniques exist for treatment of flexor tendonitis; however, no single treatment has separated itself as superior. A new technique using autogenous conditioned serum (ACS) in humans for treatment of muscle injury has been shown to speed tissue regeneration. ACS produced from human blood has been shown to contain significantly increased levels of III growth factors that may improve tendon fibril formation and strength. We propose to investigate the effect of ACS on cellular metabolism in equine tendon fibroblast monolayers. This will involve cell culture, PGE2-induced cellular injury, and analysis of the cellular response to injury when treated with ACS. Controls will include fetal bovine serum, normal equine serum, and ACS without PGE2-induced cellular injury. The cellular response will be investigated biochemically by quantification of DNA, glycosaminoglycan, and soluble collagen levels and by real time PCR to assess gene expression for matrix metalloproteinases (MMP)-1, MMP-3, and MMP-13, collagen types I and III, and the non-collagenous proteins cartilage oligomeric matrix protein (COMP) and decorin. Data will be analyzed by analysis of variance and post-hoc comparisons. Significance will be set at p

Lidocaine continuous-rate infusions (CRI) are the most commonly used prokinetic in equine practice for the treatment of post-operative ileus and are also increasingly being used in pain management, such as in cases of severe laminitis, and are often used for prolonged durations. To date only limited time/concentration relationships of lidocaine administered as a short term (24hours) CRI to horses are reported. This study examined the time/concentration profile of lidocaine and its active metabolites (GX, MEGX) during a 96 hour lidocaine infusion in eight mature healthy horses. Serum lidocaine concentrations reached steady state by three hours and did not accumulate thereafter. The serum concentration of lidocaine was above the target therapeutic concentration (980ng/ml) only at 6 and 48 hours. The serum lidocaine concentration did not reach the range described as potentially causing toxicity (>1850ng/ml). The MEGX metabolite did not accumulate over time, while the GX metabolite accumulated significantly up to 48 hours and then remained constant. The serum concentrations of lidocaine, MEGX and GX were below the limit of detection within 24 hours of discontinuation of the infusion. None of the horses developed any signs of lidocaine toxicity during the study. It was concluded that the metabolism of lidocaine was not significantly impaired by prolonged infusion, contrasting with studies in dogs and humans. No adverse effects were observed in this study, which with the lack of lidocaine accumulation suggests that prolonged infusions are safe. However the accumulation of GX, a potentially toxic active metabolite, is cause for concern. Master of Science