Calcineurin (CaN), a member of ser/thr protein phosphatase, was cloned from bovine retina. The peptide sequence of CaN A subunit is consisted of 511 amino acid residues. A 10 amino acid (A-T-V-E-A-I-E-A-D-E-A) deletion before the autoinhibitory domain was observed in bovine retina CaN A compared to bovine brain CaN A. The study on CaN activity and regulation demonstrated that different metal ions have different effects on its phosphatase activity. Ni2+ was found to be the strongest stimulator while Zn2+ was found to inhibit CaN phosphatase activity. Mn2+ was a relatively less effective stimulator compared to Ni2+. Fe2+ was also able to stimulate CaN phosphatase activity; in contrast, a previous study found Fe2+ slightly inhibited bovine brain CaN activity. The residues at 97-201 were found to be essential for bovine retina CaN A phosphatase activity. The residues at 407-456 also had an inhibitory effect on CaN A phosphatase activity in addition to the previously known auto inhibitory domain at 457-480. These observations suggest that bovine retina CaN A might possess some distinct structural characteristics compared to bovine brain CaN A.

Copper (Cu) is an essential trace element in ruminants such as the cow. Its deficiency leads to a number of debilitating symptoms and can eventually cause death. Secondary Cu deficiency is caused by the presence of chemical antagonists such as the thiomolybdates (TMs) (MoOxS4-x2-; x = 0 3). TMs form in the rumen and then form tightly bound insoluble complexes with Cu. These complexes are then excreted and the Cu is unavailable to the animal. The TMs and their effects on ruminant Cu are the focus of this thesis.<p>This study includes three main bodies of work. First, a method for quantifying the composition of TM mixtures in solution was developed. Second, the rate constants of TM interconversions were determined. Third, reactions between Cu and the TMs were studied. This work is part of an ongoing study in the Reid group to model Cu-ligand speciation in the rumen. <p>Thiomolybdates form by successive replacement of molybdate (MoO42-) O ligands with S. This makes synthesis of the uncontaminated compounds difficult. Mixtures of TMs are best analyzed by UV-visible spectroscopy despite the considerable spectral overlap. The combination of these two issues makes the characterization of the spectra difficult. In this study, a method was developed to establish the spectra of the pure TMs despite cross-contamination. Multivariate curve fitting methods were used to establish the composition of synthesized samples simultaneously with the molar absorptivities of the pure TMs. This was done using a Beers Law model. The composition was determined from a fit using assumed molar absorptivities. The absorptivities were then refined using the composition results. These processes were successively reiterated until both were optimized. The optimized absorptivities could then be used to determine the composition of any TM solution mixture. <p>The kinetics of TM formation under biologically relevant conditions were followed by UV-visible spectroscopy. The spectra were then analyzed, using the method developed above, to determine all TM concentrations. Curve fitting methods were used to simultaneously determine the rate constants of all processes that occurred. Integrated rate equations used in the fitting process were developed using the Laplace Transform method. Rate constants were determined at varying pH, ionic strengths and temperatures. From this information it was shown that TM formation and hydrolysis occurs via an associative mechanism. It was also determined that H2S and not HS- was the sulfide nucleophile in TM formation reactions.<p>This study was then extended to include reactions involving Cu and the TMs. First, the UV-visible spectra of the reaction products of Cu with each TM were characterized. This allowed the kinetics of reactions between Cu and the TMs to be followed. This reaction has been found to proceed via an intermediate. Molar absorptivities for this intermediate were optimized along with the appropriate rate constants. This was done using a combination of mathematical simulations and the curve fitting methods used for the TM kinetics. The resulting rate constants can be compared with those obtained previously in the group using a Cu ion selective electrode. <p>These rate constants were used in conjunction with the TM formation rate constants to perform simulations. The results of these simulations provide a picture of what is expected to occur in the rumen. Reactions were also performed in which TM formation occurred in the presence of Cu. This was then extended to mimic bovine feeding habits. Here, portions of the reactions mixture was removed at various time intervals and replaced with fresh reagents. These experiments were used to gain a qualitative picture of Cu speciation in the presence of the TMs over time. The work presented in this thesis provides a crucial step toward understanding the problem of bovine copper deficiency.

Two studies were designed to characterize the effects of a non-steroidal aromatase inhibitor, letrozole, on ovarian function in cattle. The specific objective was to test the hypothesis that letrozole will arrest dominant follicle growth resulting in emergence of a new follicular wave at a predictable interval post-treatment. In a first experiment, postpubertal beef heifers were assigned randomly to four treatment groups and given phosphate-buffered saline (controls; n=10), or letrozole at a dose of 500 (n=9), 250 (n=10), or 125 (n=10) µg/kg intravenously 4 days after follicular ablation (~2.5 days after wave emergence). In a second study, postpubertal beef heifers were assigned randomly to four treatment groups. One group received no treatment (control; n=17) and the other groups (n=9-10) were given 85 µg/kg of letrozole per day (250 µg/kg total dose), from Days 1 to 3, Days 3 to 5, or Days 5 to 7 (Day 0 = pre-treatment ovulation,) corresponding to the periods before, during and after selection of the dominant follicle, respectively. Follicular dynamics were monitored ultrasonically and blood samples were collected for endocrine assays. Follicle diameter profiles and plasma LH, FSH, and estradiol concentrations were analyzed. Additionally, during the second trial, CL diameter profiles and plasma progesterone concentrations were measured. In both studies, the diameter profile of the dominant follicle was larger in heifers treated with letrozole than in control heifers (P<0.05) and the intervals to new wave emergence and onset of regression of the extant dominant follicle were longer (P<0.05) in heifers treated with letrozole than in controls, regardless of the dose (high, medium, or low; single vs multiple) and the stage of the follicle wave in which treatments were initiated. Furthermore, during the second experiment, the mean CL diameter was larger in letrozole-treated heifers, although there were no differences in plasma progesterone concentrations between treated and control animals. The effects on dominant follicle and CL diameter profiles appeared to be the result of the significantly increased plasma LH concentrations observed in letrozole-treated animals during both treatment approaches (single vs multiple dose). Incomplete and inconsistent inhibition of estradiol production and the lack of a surge on FSH observed in both experiments may be a result of insufficient circulating levels of letrozole during the treatment period. In summary, a single or multiple dose of letrozole did not induce regression of the extant dominant follicle, nor did it directly affect FSH release. Conversely, letrozole extended the lifespan of the dominant follicle, in association with increased endogenous levels of LH, thereby delaying the next FSH surge and subsequent follicular wave emergence. Results suggest that letrozole has potential as a non-steroidal method for controlling ovarian function in cattle, but further studies are warranted to clarify dosage and timing of treatment to predictably affect follicular wave dynamics in cattle.

Equine sarcoids are the most common skin tumors of horses. Despite being such a common entity, relatively little is known about many features of sarcoid epidemiology or growth. In addition, due to the detection of Bovine Papillomavirus (BPV) DNA of 2 different types, BPV type 1 (BPV1) and BPV type 2 (BPV2), in equine sarcoids BPV has been suggested as the causative agent of sarcoid development. Recently, however, BPV DNA has also been detected in other skin conditions of horses; the significance of this is unclear. Multiple studies to learn more about sarcoids were undertaken.<p> To investigate the epidemiology of sarcoids in horses in Western Canada the records of five veterinary diagnostic laboratories were searched to identify submissions of sarcoids from horses. The submission record and diagnostic reports of 802 separate submissions of equine sarcoids were reviewed for age, breed, and gender of the horse and the number, location and clinical type of sarcoid. Based on these submissions, horses of a wide variety of ages and 23 different equine breeds were affected, within these breeds, Donkeys were overrepresented.<p> The presence of BPV was determined by Polymerase Chain Reaction (PCR). BPV was found in 74 of 96 (77.1%) samples, and using Restriction Fragment Length Polymorphism, BPV1 and BPV2 were identified in these samples. BPV2 was present in 59 (79.7%) of these. Unlike other areas in the world, in Western Canada, equine sarcoids are most commonly associated with BPV2.<p> A second study examined different clinical types of sarcoids to determine if there was differential expression of immunohistochemical markers associated with apoptosis, Cleaved Caspase 3(ClC3), and antiapoptotic factors, B-Cell Lymphoma 2 (Bcl-2) and Survivin. No differences in the expression of any of these markers regardless of BPV type were noted. Survivin was expressed in equine sarcoids of all types and increased levels of expression are associated with more aggressive clinical behaviour.<p> Finally, the location of BPV DNA was determined in both sarcoids and a variety of non-sarcoid inflammatory skin conditions of horses, as well as, normal skin. PCR for BPV DNA was performed on 86 skin biopsies from horses with non-sarcoid skin conditions, as well as, normal skin. BPV DNA was present in 41 of 86 biopsies. These positive samples, in addition to BPV positive sarcoid samples from the earlier study, were dissected into tissue compartments using laser microdissection followed by 2 forms of BPV DNA amplification, PCR and isothermal loop mediated amplification. BPV DNA was more often located in the epidermis of non-sarcoid skin conditions than in sarcoids. In addition, areas of inflammation within the dermis and epidermis were more likely to contain BPV DNA than non-inflamed areas. These results suggest that while BPV is commonly found in equine skin, the location where it is found differs between sarcoids and non-sarcoid samples. When BPV DNA was found in non-sarcoid samples, it was commonly associated with inflammation suggesting that microscopic damage to the epidermal barrier of the skin maybe an adequate predisposing factor to the development of sarcoids.

The role of neutrophils in the pathogenesis of Salmonella typhimurium-induced ruminant and human enteritis and diarrhea remains incompletely understood. To address this question, the in vivo bovine ligated ileal loop model of non-typhoidal salmonellosis was used in calves with the naturallyoccurring Bovine Leukocyte Adhesion Deficiency (BLAD) mutation whose neutrophils are unable to extravasate and infiltrate the extravascular matrix. Data obtained from BLAD calves were compared to those from genetically normal calves negative for the BLAD mutation. Morphologic studies showed that the absence of significant tissue influx of neutrophils in intestine infected by S. typhimurium resulted in less tissue damage, reduced luminal fluid accumulation, and increased bacterial invasion compared to regular calves. Study of gene expression profile of cytokines by quantitative Real-Time PCR (qRTPCR) revealed that the massive tissue influx of neutrophils during acute infection is mainly driven by the CXC chemokine GRO- α especially in the last stages of acute infection and to a lesser extent, IL-8. In contrast, the pro-inflammatory cytokines IL-1 β and TNF- α were not significantly correlated with the presence or absence of tissue neutrophils. The precise in situ localization of gene expression of these major cytokines and chemokines was investigated by qRTCPR from specific groups of intestinal cells captured by Laser Capture Microdissection in S. typhimuriuminfected ileal loops from BLAD animals. Our data confirmed that gene expression of IL-8, GRO- α, and IL-1 β was predominantly localized to enterocytes of crypts with less expression in enterocytes of villi tips and cells that form the domed villi were not an important source of TNF- α gene expression. Microarray technology was used to determine the global transcriptional profile of bovine intestinal loops inoculated with S. typhimurium. The host samples were hybridized on a 13K bovine-specific oligoarray and microarray data was analyzed using a suite of gene expression analysis and modeling tools. Analysis of our data revealed that the tissue influx of neutrophils in ileal loops greatly influenced the host gene expression. Major differences in gene expression in relevant fields of Salmonella research including inflammation and immune response, Toll-like receptor signaling, cytokine profiles, apoptosis, and intracellular defense against infection are discussed.

Metaphylactic treatment of incoming feedlot cattle is a common preventative action against bovine respiratory disease (BRD). Cattle are dosed based on estimated or actual lot average weights, rather than on an individual basis, to reduce initial processing time. There has been limited research conducted on the effects of accurate weight- based dosing in feedlot cattle. The objective of this study was to evaluate the economic effects of precision weight- based dosing of cattle as compared to dosing the lot average or lot averages plus 50 lb and minus 50 lb. An economic model was created and stochastic simulations performed to evaluate potential outcomes of different dosing scenarios. Economic analyses of the effects of precision weight-based dosing were conducted using SIMETAR© to determine the stochastic dominance and economic effects of different dosing regimens. Data were obtained from a commercial feedlot for different lots of cattle where individual animal weights were available; for this analysis the minimum lot size was 30 animals, and the maximum lot size was 126 animals. Within lots, individual weight deviations were calculated from the lot mean, the lot mean was rounded up to the nearest 50 lb increment or down to the nearest 50 lb increment to represent mild overestimation and mild underestimation, respectively. Tulathromycin (Draxxin®, Pfizer Animal Health, New York, NY), an antimicrobial commonly prescribed for treatment of bovine respiratory disease, was used to illustrate the impacts of uniform dosing versus exact dosing per body weight. Based on the dilution space method used to evaluate time of drug effectiveness, it was estimated that Draxxin® administered at the recommended dosage to cattle weighing between 500 and 1000 lb should be provided with 191 hours (7.96 days) of protection from pneumonia-causing bacteria. Due to the pharmacokinetic properties of Draxxin®, an animal that is administered half the recommended dose is only protected from pneumonia-causing bacteria for 8 hours, which is 4.2 percent of the coverage time of the proper dose. This limits the effectiveness of the prescribed treatment to fully administer therapeutic treatment. In all cases, the correct weight-based dosing strategy cost less than any other dosing technique. Overall, dosing all cattle at the lot average weight costs $6.04 per animal more than dosing at the exact, correct dose. Dosing all animals at the lot average weight plus 50 lb costs $6.24 per animal more; dosing all animals at lot average minus 50 lb costs $4.01 per animal more. The use of individual animal weights to determine per head dosing of Draxxin® is more cost effective than using lot averages. This concept would appear to extend to all weight-based pharmaceutical products in general, and should be considered a necessary management strategy.

No description available.

A quantitative trait locus (QTL) is the location of a gene that affects a trait that is measured on a quantitative (linear) scale. Many important agricultural traits such as weight gain, milk fat content and intramuscular fat in cattle are quantitative traits. There is a need to integrate genomic sequence data with QTL data and to develop an analytical tool to visualize the data. Without integration, application of this data to agricultural enterprise productivity will be slow and inefficient. My thesis presents a web-accessible tool called the Bovine QTL Viewer developed to solve this problem. It consists of an integrated database of bovine QTL and the QTL viewer to view the QTL and their relative chromosomal position. This tool generates dynamic and interactive images and supports research in the field of genomics. For this tool, the data is modeled and the QTL viewer is developed based on the requirements and feedback of experts in the field of bovine genomics.

The major histocompatibility complex (MHC) is a genomic region containing genes of immunomodulatory importance. MHC class I genes encode cell-surface glycoproteins that present peptides to circulating T cells, playing a key role in recognition of self and non-self. Studies of MHC loci in vertebrates have examined levels of polymorphism and molecular evolutionary processes generating diversity. The bovine MHC (BoLA) has been associated with disease susceptibility, resistance and progression. To delineate mechanisms by which MHC class I genes evolved to function optimally in a species like cattle, it is necessary to study genomic organization of BoLA to define gene content, and investigate characteristics of expressed class I molecules. This study describes development of a physical map of BoLA class I region derived from screening two BAC libraries, isolating positive clones and confirming gene content, order and chromosomal location through PCR, novel BAC end sequencing techniques, and selected BAC shotgun cloning and/or sequencing and FISH analysis. To date, this is the most complete ordered BAC array encompassing the BoLA class I region from the class III boundary to the extended class I region. Characterization of a frameshift allele exhibiting trans-species polymorphism in Bos and Bison by flow cytometry, real-time RT-PCR, 1D and 2D gel analysis is also described. This frameshift allele encodes an early termination signal within the antigen recognition site (ARS) of exon 3 of the BoLA BSA-Adel class I gene predicting a truncated class I protein that is soluble. An ability to assess MHC diversity in populations and provision of animals with defined MHC haplotypes and genetic content for experimental research is necessary in developing a basis upon which to build functional studies to elucidate associations between haplotype and disease in bovids. The BoLA class I region is immunologically important for disease association studies in an economically important species. This study provides knowledge of gene content and organization within the class I MHC region in cattle, providing a template for more detailed analysis and elucidation of complex disease associations through functional genomics and comparative analysis, as well as evolution of the MHC in bovids to optimize a population’s immune response.

Hypercholesterolemia is a major risk factor for cardiovascular disease (CVD). Interestingly, the consumption of dairy products, namely milk, has been shown to lower cholesterol. The mechanism of action surrounding this observation has been attributed to the protein fraction of milk. While there have been many studies evaluating the effects of dietary protein sources on cholesterol concentrations, few studies have evaluated specific animal protein components and no human clinical studies regarding the effects of animal plasma protein fractions on cholesterol metabolism have been conducted. This study examined the effect of an oral serum bovine immunoglobulin protein fraction (bIg) derived from US Department of Agriculture approved beef (aged < 30 months) on lipid indices in hypercholesterolemic humans. Participants included men and women (aged 25 – 70 years) with mild hypercholesterolemia (5.44-6.99 mmol/L) who were not receiving cholesterol-lowering medication. Treatment consisted of the randomized, double blind, parallel group, placebo-controlled administration of 5 grams (g) bIg daily for 6 weeks (W) in 52 participants (n = 26 each in treatment and control groups). Mean (± SD) baseline treatment and placebo total cholesterol (TC) was 6.33 ± 0.1 mmol/L and 6.16 ± 0.1 mmol/L respectively. A repeated-measures multivariate analysis of covariance (MANCOVA) covaried for change in total energy and alcohol intake, and a Tukey posthoc examination of the data showed that the bIg-treated group demonstrated a significant reduction in TC at 3-week (W) (5.98 ± 0.5 mmol/L; P < 0.05) and 6-week (W) (5.97 ± 0.7 mmol/L; P > 0.05) intervals compared to baseline. The 6W concentration was significantly lower than the placebo group (P < 0.05). Additionally, study findings displayed no significant changes in the placebo group or in any other lipid indexes or markers associated with hepatorenal or cardiovascular health. Consumption of bIg appears to lower major lipid indexes associated with CVD.