<p>In this Ph.D. dissertation, the capacity of cultured bovine polymorphonuclear neutrophils (PMNs) to passively acquire functional membrane proteins from apoptotic or necrotic cells was examined. The rapid transfer of membrane proteins from a variety of syngeneic, allogeneic and xenogeneic donor cells to PMNs was observed. In contrast to PMNs from other species, bovine PMNs did not express endogenous major histocompatability class II (MHC II) protein, either constitutively or inducibly. The entire bovine PMN population was, however, able to acquire detectable levels of surface MHC II or cluster of differentiation (CD) 3 protein following PMN co-culture with cells in conditions which permitted close contact with dieing cells. Therefore, it was hypothesized that membrane lipids and proteins were acquired by bovine PMN following fusion with microparticles (MPs) shed from either apoptotic or necrotic cells. </p> <p>It was then determined whether the lifespan of bovine PMNs could be sufficient to provide an opportunity for PMNs to interact with T cells. Lymphocyte recruitment to sites of inflammation often occurs 3-5 days after the initial PMN recruitment. PMN survival would need to span this interval to provide an opportunity for an interaction between PMNs and lymphocytes. Pro-inflammatory cytokines, such as interferon (IFN)-ã and granulocyte macrophage colony stimulating factor (GM-CSF), and bacterial lipopolysaccharide (LPS) were observed to prolong the lifespan of cultured PMNs beyond 96 hours. These observations supported the conclusion that it was biologically possible for PMNs and T cells to interact at sites of inflammation.</p> <p>Using confocal microscopy, direct evidence was provided for the formation and release of MPs from peripheral blood mononuclear cells (PBMCs) and the attachment of these MPs to bovine PMNs. A time-dependent integration of both MP membranes and integral membrane proteins into the PMN plasma membrane was also observed. The passively acquired membrane lipids and proteins then diffused throughout the PMN plasma membrane. Another observation was the formation of MPs which contained donor cell cytoplasmic proteins and subsequent transfer this cytoplasmic protein to recipient PMNs. These observations raised the possibility that MPs could also transfer genetic material. Thus, confocal microscopy provided direct evidence that MPs were one mechanism by which bovine PMNs could passively acquire membrane lipids and integral membrane proteins.</p> <p>Finally, the functional consequences of passive acquisition of membrane proteins were examined using two different approaches. A significant increase in green fluorescent protein (GFP) transgene expression was observed following PMN infection using the GFP expressing bovine adenovirus vector (BAV304). These PMNs had passively acquired membranes from an adenovirus permissive cell line. This observation provided indirect evidence for the passive acquisition of a functional viral receptor protein. Direct evidence that PMNs passively acquired functional membrane proteins was provided by the observation that the passive transfer of ovine MHC II molecules to bovine PMNs enabled these cells to induce antigen-specific proliferation and cytokine expression by xenoreactive T cell lines. Despite a reduction in amplitude and duration, T cell responses induced by PMNs were qualitatively similar to those observed following activation by the stimulator B cell line. These observations supported the conclusion that PMNs could function as antigen presenting cells (APCs) following the passive acquisition of MHC II protein.</p> <p>In conclusion, this research project provided evidence that bovine PMNs have an impressive ability to acquire membranes and functional integral membrane proteins from dead or dying cells. The implications of this transfer of immunological information are discussed within the context of the role which PMNs might play in both innate and adaptive immune responses. </p>

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.

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 main objective of this study was to investigate if natural honey can be used as a cryoprotecting agent (CP) in vitrification medium to improve the viability of vitrified-warmed bovine oocytes. The first study was conducted to investigate the dehydration capability of natural honey compared with sucrose, and to determine the proper concentration of honey-based medium and the optimum time for sufficiently safe dehydration of bovine oocytes. Matured cumulus-oocyte complexs (COCs) were denuded and introduced individually into different concentrations (0.25, 0.5, 1.0, 1.5 or 2.0 M) of honey and sucrose-based medium followed by rehydration in control media (TCM). Video images were recorded during dehydration and rehydration, and oocyte images were captured at 12 time intervals to calculate oocyte-volume changes during dehydration and rehydration. Results demonstrated that, in honey-based media, the maximum oocyte shrinkage was achieved after 60 sec exposure in 0.25M, 0.5M and 1.0M concentrations; while at higher concentrations 1.5M and 2.0M, the maximum dehydration occurred at 30 and 20 seconds respectively. In sucrose-based medium, the maximum oocyte shrinkage was achieved after 60 sec exposure in 0.25 or 0.5M concentrations. However, at higher concentrations (1M, 1.5M or 2M), the maximum dehydration occurred at 30, 20 and 10 sec. For rehydration, oocytes dehydrated in honey or sucrose-based medium were able to regain their original volume within 60-120 sec. However, oocytes dehydrated in higher concentrations (2M honey, and 1.5M and 2M sucrose) were rehydrated back to their original volume within 20 sec. This study concluded that natural honey and sucrose caused similar cell dehydration. Only oocytes dehydrated in 1M honey-based media reached maximal dehydration after 60 sec and equally regained original volume. Therefore, 1M of honey-based medium is suggested for sufficient and safe oocyte dehydration during vitrification. The second study was conducted to determine in vitro maturation (IVM), in vitro fertilization (IVF) and embryonic development of bovine oocytes vitrified in honey-based vitrifcation media. In Experiment 1, bovine COCs were randomly distributed in control group (non-vitrified; G1), 0.5M sucrose group (second control; G2), and 0.5M, 1M and 1.5M honey groups (G3, G4 and G5 respectively). The COCs were exposed to equilibration solution 1 (VS1) at ~ 22 oC for 5 min and to vitrification solution 2 (VS2) for 1 min, mounted on Cryotops and plunged into LN2. COCs were warmed in TCM and honey/sucrose medium at 38.5oC for 1 min, washed, matured in vitro (IVM), denuded, and immunostained to evaluate maturation. Maturation rate was significantly higher (80.7%) in control group (G1) than in vitrified groups (56, 52, 55 and 51% in G2, G3, G4 and G5, respectively) (P<.0001), whereas there was no significant difference among the vitrified groups (P>0.05). In Experiment 2, bovine COCs distributed in control (not vitrified, G1) and vitrified groups using 1M honey and 0.5M sucrose (G2 and G3 respectively), underwent for IVM, IVF and in vitro culture (IVC) for 9 days. Cleavage rate was significantly higher (P<.0001) in the control group (74%, G1, n=183) than rates of vitrified groups (51% in G2, n=137; and 42% in G3, n=131), whereas no differences among vitrified groups (P=0.0723). Rate of blastocyst formation was significantly higher (34%) in G1 than in the vitrified groups (P<.0001); however, blastocyst formation rates in the honey group were significantly higher (P=0.0026) than in the sucrose group (13% and 3% respectively). Addition of natural honey (1.0M; or 21.7%w/v) in vitrification medium can safely and sufficiently dehydrate bovine oocytes during vitrification procedure. The vitrification of bovine oocytes in 1M honey improved their post-warming maturation abtility and embryonic development.

Copper is one of the most vital trace elements in ruminant nutrition. It is required for several metabolic activities and it is also an essential component of several physiologically important metalloenzymes. Thus copper deficiency in ruminants results in distinctive pathologies, and hence in significant economic losses to farmers. Copper deficiency results from very low copper in diet (primary copper deficiency) and interference with Cu absorption in the animal due to Mo and S in food or water (secondary copper deficiency). The molybdenum-induced copper deficiency that affects ruminants can be attributed to the formation of thiomolybdates (TMs)from molybdate and sulfide in the rumen. The TMs formed then react irreversibly with copper to form insoluble Cu-TM complex which ultimately end up being excreted, thus reducing copper bioavailability to the ruminant. <p>In this study, an attempt has been made to use computer simulations to model speciation of copper in rumen fluid in the presence of TMs with the aim of understanding the extent to which TMs affects the levels of copper in the rumen. <p>This was done by initially refining the computer model of copper speciation with respect to low molecular mass (LMM) ligands in bovine rumen with the aim of correcting the discrepancy that was observed during experimental validation of the computer model in a previous study. To this end, mass balance equations which describes the distribution of Cu(II) amongst the different ligands were encoded into a spreadsheet to calculate equilibrium concentration of all species. Formation constants obtained from literature as well as those obtained from studies in our group were used as input values in the spreadsheet. Results show that at average ruminal pH, the metal would be present mostly as carbonate and phosphate complexes. The results obtained from the computer model in the present study were validated using 1H NMR experiments on simulated rumen fluid as well as actual rumen fluid containing Cu(II); using acetic acid chemical shift as the probe for monitoring the speciation pattern. Excellent agreement was observed between the computer model and experimental results. Discrepancy was however observed upon introduction of copper lysine as copper source into the model. Incorporation of a mixed ligand complex of Cu(II), acetate and lysine into the computer model gave an excellent agreement between the computer model and experimental results. <p>The study was extended to include glycine, histidine, methionine and EDTA complexes as the copper source in both rumen saliva (McDougalls solution) and rumen fluid. Results show that only the histidine and EDTA complexes persist to any significant extent, in spite of the large number of competing ligands present in these matrices.<p>In this study, success has also been achieved in the integration of the slow (kinetically controlled) formation of TMs and copper-tetrathiomolybdate (TM4) complexation into the previously developed model for the rapidly equilibrating copper-ligand speciation. To simulate the formation of the TMs and Cu-TM4 complex with respect to time, the differential equations representing rate expressions for each chemical species were solved to obtain an analytical solution using the Laplace transform method. The analytical solutions obtained were encoded in a spreadsheet and calculated as function of time to obtain time dependent concentrations of TMs and Cu-TM4 complex. This was then integrated with previously developed model for the rapidly equilibrating copper-ligand speciation in the rumen. The kinetic data used in the simulation of the formation thiomolybdates was obtained fron literature wheras that for Cu-TM4 complexation was obtained from our lab using Cu(II) - Ion Selective Electrode. The results show that that in the presence of TM4 the, Cu(II) bound to low molecular ligands in the rumen is drastically reduced confirming the effect TM4 on Cu(II) observed in several in vitro studies.<p>The study shows that in thiomolybdate contaminated rumen environment, the bioavailability of copper is considerably reduced. Though metal bioavailabilities are hard to predict this approach could help better our understanding of this process.

Bovine adenovirus (BAdV)-3 is a non-enveloped, icosahedral virus with a double-stranded DNA genome, and is being developed as a vector for vaccination of animals and humans. Expression of viral genes is divided into early, intermediate, and late phases. The late genes of BAdV-3 are grouped into seven families (L1 to L7) based on usage of common polyadenylation site(s). The L1 region of BAdV-3 encodes the 52K protein, a non-structural protein conserved among members of the family Adenoviridae. In human adenovirus (HAdV)-5, the 52K protein is involved in packaging of the viral DNA into the capsid. The N-terminal half of the protein has been proposed to mediate serotype specificity of DNA packaging. The objective of this study was to characterize the 52K protein of BAdV-3. <p> DNA sequence analysis revealed that the BAdV-3 52K open reading frame encodes a protein of 370 amino acids rather than 331 amino acids as previously reported. Western blotting with anti-52K serum detected the expression of a 40kDa protein at 24 to 72 hrs post-infection. BAdV-3 52K localized predominantly to the nucleus in BAdV-3 infected cells and in transfected cells in the absence of other viral proteins. Analysis of mutant 52K proteins revealed that residues 102-110 were necessary but not sufficient for nuclear import. This suggests that residues upstream or downstream of the identified 52K nuclear localization signal (NLS) are required, or that the function of the NLS is dependent on its conformation within 52K. <p> The nuclear import of 52K is significantly, but not completely, dependent on soluble factors, ATP, and temperature. A peptide competing for binding to importin beta and a peptide encoding the NLS of Ycbp80 were also able to inhibit nuclear import of 52K. However, a dominant negative mutant of Ran was unable to block 52K nuclear import. These results suggest that 52K uses a classical importin alpha/importin beta pathway for nuclear import. In support of this, a specific interaction between 52K and importin alpha-3 was detected. In addition, 52K was able to accumulate in the nucleus in the absence of soluble factors and ATP when the nuclear membrane was permeabilized with detergent. This suggests that, in addition to nuclear import by the importin alpha/importin beta pathway, 52K is able to accumulate in the nucleus by binding to nuclear components. <p> A yeast two-hybrid system identified interactions between BAdV-3 52K and pV, pVI, pVII, and IVa2. However, only the interaction with pVII could be confirmed by GST pulldown. 52K and pVII also interact during BAdV-3 infection. An interaction between 52K and pVII has previously been shown in HAdV-5 infected cells. <p> Mass spectrometry analysis of proteins co-precipitating with BAdV-3 52K identified a cellular protein, NFkB-binding protein (NFBP), which interacted with 52K. The interaction between NFBP and 52K was confirmed <i>in vitro</i> and <i>in vivo</i>. NFBP has been shown to be essential for ribosomal RNA (rRNA) processing. While NFBP is normally localized in the nucleolus, co-expression with 52K results in the redistribution of NFBP from the nucleolus to other parts of the nucleus. While this suggested that redistribution of NFBP by 52K could inhibit rRNA processing during BAdV-3 infection, we were unable to detect a difference in rRNA processing in cells expressing truncated or full-length 52K in the absence of other viral proteins. Since NFBP is a multi-functional protein, future experiments should focus on other possible biological functions of the interaction of NFBP with BAdV-3 52K.

<p>In this Ph.D. dissertation, the capacity of cultured bovine polymorphonuclear neutrophils (PMNs) to passively acquire functional membrane proteins from apoptotic or necrotic cells was examined. The rapid transfer of membrane proteins from a variety of syngeneic, allogeneic and xenogeneic donor cells to PMNs was observed. In contrast to PMNs from other species, bovine PMNs did not express endogenous major histocompatability class II (MHC II) protein, either constitutively or inducibly. The entire bovine PMN population was, however, able to acquire detectable levels of surface MHC II or cluster of differentiation (CD) 3 protein following PMN co-culture with cells in conditions which permitted close contact with dieing cells. Therefore, it was hypothesized that membrane lipids and proteins were acquired by bovine PMN following fusion with microparticles (MPs) shed from either apoptotic or necrotic cells. </p> <p>It was then determined whether the lifespan of bovine PMNs could be sufficient to provide an opportunity for PMNs to interact with T cells. Lymphocyte recruitment to sites of inflammation often occurs 3-5 days after the initial PMN recruitment. PMN survival would need to span this interval to provide an opportunity for an interaction between PMNs and lymphocytes. Pro-inflammatory cytokines, such as interferon (IFN)-ã and granulocyte macrophage colony stimulating factor (GM-CSF), and bacterial lipopolysaccharide (LPS) were observed to prolong the lifespan of cultured PMNs beyond 96 hours. These observations supported the conclusion that it was biologically possible for PMNs and T cells to interact at sites of inflammation.</p> <p>Using confocal microscopy, direct evidence was provided for the formation and release of MPs from peripheral blood mononuclear cells (PBMCs) and the attachment of these MPs to bovine PMNs. A time-dependent integration of both MP membranes and integral membrane proteins into the PMN plasma membrane was also observed. The passively acquired membrane lipids and proteins then diffused throughout the PMN plasma membrane. Another observation was the formation of MPs which contained donor cell cytoplasmic proteins and subsequent transfer this cytoplasmic protein to recipient PMNs. These observations raised the possibility that MPs could also transfer genetic material. Thus, confocal microscopy provided direct evidence that MPs were one mechanism by which bovine PMNs could passively acquire membrane lipids and integral membrane proteins.</p> <p>Finally, the functional consequences of passive acquisition of membrane proteins were examined using two different approaches. A significant increase in green fluorescent protein (GFP) transgene expression was observed following PMN infection using the GFP expressing bovine adenovirus vector (BAV304). These PMNs had passively acquired membranes from an adenovirus permissive cell line. This observation provided indirect evidence for the passive acquisition of a functional viral receptor protein. Direct evidence that PMNs passively acquired functional membrane proteins was provided by the observation that the passive transfer of ovine MHC II molecules to bovine PMNs enabled these cells to induce antigen-specific proliferation and cytokine expression by xenoreactive T cell lines. Despite a reduction in amplitude and duration, T cell responses induced by PMNs were qualitatively similar to those observed following activation by the stimulator B cell line. These observations supported the conclusion that PMNs could function as antigen presenting cells (APCs) following the passive acquisition of MHC II protein.</p> <p>In conclusion, this research project provided evidence that bovine PMNs have an impressive ability to acquire membranes and functional integral membrane proteins from dead or dying cells. The implications of this transfer of immunological information are discussed within the context of the role which PMNs might play in both innate and adaptive immune responses. </p>

With the combination of computer-assisted image analysis and ultrasonographic imaging technology, it has been possible to study and increase the knowledge in different areas of medicine. Studies of ovarian development in female mammals using ultrasonography have shown a relationship between the day in the estrous cycle and the main structures of the ovary.<p>Ultrasound images of bovine ovaries were used to determine whether ultrasound-detected features can automatically determine the phase in the estrous cycle based on a single day's ultrasound examination of the ovaries. Five ultrasound-detected features of the bovine ovaries were used to determine the phase in the estrous cycle: (1) size of the dominant follicle; (2) size of the first subordinate follicle; (3) size of the second subordinate follicle; (4) size of the corpus luteum and (5) number of subordinate follicles with size ≥ 2mm. The collection of ultrasound images used for this study was formed by a group of 45 pairs of ovaries (left and right) which were imaged on day 3, day = 10 and day ≥ 17 of the estrous cycle corresponding to the metestrus, diestrus and proestrus phases respectively.<p>Four different experiments were performed to test the hypothesis. For experiments 1, 2 and 3 the bovine ovaries were classified into three different classes: day 3 of wave 1 (D3W1), day 1 of wave 2 (D1W2) and day 17 or higher (D≥17) that were related to the follicular development of the ovary and the estrous cycle phases as: metestrus, diestrus and proestrus respectively. For experiment 4 the bovine ovaries were classified into four classes: D3W1, D6W1, D1W2 and D≥17. The additional class (D6W1: day 6 of wave 1) was incorporated to represent the early-diestrus phase in the estrous cycle.<p>Two classifiers were implemented for all experiments and their performances compared: a decision tree classifier and a naive Bayes classifier. The decision tree classifier had the best performance with a classification rate of 100% for experiments 1, 2 and 3, giving a rather simple decision tree which used only two features to make a classification: size of the dominant follicle and size of the corpus luteum, suggesting these are key features in distinguishing between phases in the estrous cycle giving the most relevant information. The naive Bayes had a classification rate of 86.36% for experiment 1, 95.55% for experiment 2 and 90% for experiment 3. The results of this study supported the hypothesis that by using ultrasound detected features of bovine ovaries we can determine automatically the stage in the estrous cycle based on a single day's examination.

Copper is one of the most vital trace elements in ruminant nutrition. It is required for several metabolic activities and it is also an essential component of several physiologically important metalloenzymes. Thus copper deficiency in ruminants results in distinctive pathologies, and hence in significant economic losses to farmers. Copper deficiency results from very low copper in diet (primary copper deficiency) and interference with Cu absorption in the animal due to Mo and S in food or water (secondary copper deficiency). The molybdenum-induced copper deficiency that affects ruminants can be attributed to the formation of thiomolybdates (TMs)from molybdate and sulfide in the rumen. The TMs formed then react irreversibly with copper to form insoluble Cu-TM complex which ultimately end up being excreted, thus reducing copper bioavailability to the ruminant. <p>In this study, an attempt has been made to use computer simulations to model speciation of copper in rumen fluid in the presence of TMs with the aim of understanding the extent to which TMs affects the levels of copper in the rumen. <p>This was done by initially refining the computer model of copper speciation with respect to low molecular mass (LMM) ligands in bovine rumen with the aim of correcting the discrepancy that was observed during experimental validation of the computer model in a previous study. To this end, mass balance equations which describes the distribution of Cu(II) amongst the different ligands were encoded into a spreadsheet to calculate equilibrium concentration of all species. Formation constants obtained from literature as well as those obtained from studies in our group were used as input values in the spreadsheet. Results show that at average ruminal pH, the metal would be present mostly as carbonate and phosphate complexes. The results obtained from the computer model in the present study were validated using 1H NMR experiments on simulated rumen fluid as well as actual rumen fluid containing Cu(II); using acetic acid chemical shift as the probe for monitoring the speciation pattern. Excellent agreement was observed between the computer model and experimental results. Discrepancy was however observed upon introduction of copper lysine as copper source into the model. Incorporation of a mixed ligand complex of Cu(II), acetate and lysine into the computer model gave an excellent agreement between the computer model and experimental results. <p>The study was extended to include glycine, histidine, methionine and EDTA complexes as the copper source in both rumen saliva (McDougalls solution) and rumen fluid. Results show that only the histidine and EDTA complexes persist to any significant extent, in spite of the large number of competing ligands present in these matrices.<p>In this study, success has also been achieved in the integration of the slow (kinetically controlled) formation of TMs and copper-tetrathiomolybdate (TM4) complexation into the previously developed model for the rapidly equilibrating copper-ligand speciation. To simulate the formation of the TMs and Cu-TM4 complex with respect to time, the differential equations representing rate expressions for each chemical species were solved to obtain an analytical solution using the Laplace transform method. The analytical solutions obtained were encoded in a spreadsheet and calculated as function of time to obtain time dependent concentrations of TMs and Cu-TM4 complex. This was then integrated with previously developed model for the rapidly equilibrating copper-ligand speciation in the rumen. The kinetic data used in the simulation of the formation thiomolybdates was obtained fron literature wheras that for Cu-TM4 complexation was obtained from our lab using Cu(II) - Ion Selective Electrode. The results show that that in the presence of TM4 the, Cu(II) bound to low molecular ligands in the rumen is drastically reduced confirming the effect TM4 on Cu(II) observed in several in vitro studies.<p>The study shows that in thiomolybdate contaminated rumen environment, the bioavailability of copper is considerably reduced. Though metal bioavailabilities are hard to predict this approach could help better our understanding of this process.

Vaccination has proven a cost-effective method of managing infectious diseases, but attempts to develop an effective fetal vaccine have proven difficult due to the immaturity of the immune system and the propensity of the developing immune system to induce tolerance to immunizing antigens. This thesis is concerned with the induction of specific immunity in the second trimester ovine fetus using the oral DNA immunization method. In utero oral delivery of naked DNA plasmid was selected as the method of immunization due to previous successes in the third trimester ovine fetus and the immunostimulatory properties of the bacterial DNA backbone, which may help overcome developmental tolerance. Transfection and expression studies in the third trimester ovine fetus revealed the oral mucosal epithelium as the primary site of transgene expression and functionally active antigen was also localized to lymph nodes draining the oral cavity. Efficient transfection and expression of plasmid following oral delivery was specific to the fetus and correlated with a lesser degree of epithelial differentiation. Oral DNA delivery in the second trimester resulted in detection of transgene activity in 100% of treated fetuses and the level of transgene activity was greater than in fetuses treated in the mid-third trimester. Using a plasmid encoding the gene for bovine herpesvirus-1 truncated glycoprotein D (tgD), immunization studies were then conducted in the second trimester fetus. A new lower age limit for fetal immunization was established at 55-60 days gestation (gestation period is 148 days), which coincides with the appearance of lymphocytes in peripheral tissues. Antigen-specific antibody, interferon-× responses and/or neonatal anamnestic responses were detected in 66% of fetuses immunized between 55 and 84 days gestation. The duration of fetal primary immune responses was equivalent to that achieved in young lambs following optimized DNA vaccination, but the magnitude of fetal immune responses was limited. The persistence of immune memory from the second trimester to birth was consistent with experimental data which showed that the duration of immune memory had a stronger correlation to the duration, as compared to the magnitude, of the primary antibody response. Overall, the experiments within showed that oral DNA immunization of the early second trimester fetus is feasible and not associated with the induction of tolerance. These findings suggest that it may be possible to protect against mother-to-child transmission of infectious pathogens by targeting protection at the level of the fetus.