Pasture feeding depresses adipose tissue development in beef cattle whereas grain feeding, enhances adipogenesis. Therefore, we hypothesized that specific fatty acids would differentially affect lipogenesis in explants of bovine subcutaneous (SC) and intramuscular (IM) adipose tissues. Angus steers were harvested at 12, 14, and 16 mo of age, and IM and SC adipose tissue explants from the 8-11th thoracic rib region were dissected and cultured in media. Media contained no supplemental fatty acids or 40 microM of five fatty acids, stearic acid (18:0), oleic acid (18:1 n-9), trans-11 vaccenic acid (18:1 trans-11), conjugated linoleic acid (CLA, 18:2 trans-10, cis-12), or alpha-linolenic acid (18:3 n-3). After 48 h of culture, lipogenesis using [U-14C]glucose and [1-14C]acetate was measured. Lipogenesis from glucose decreased between 12 and 16 mo of age in SC adipose tissue (from 8.9 to 4.0 nmol per 2 h per 100 mg; P = 0.001) and IM adipose tissue (from 4.4 to 2.7 nmol per 2 h 100 mg ; P = 0.08). Lipogenesis from acetate did not change over time in SC adipose (approximately 56 nmol per 2 h per 100 mg; P = 0.23), but increased over time in IM adipose tissue (from from 11.3 to 17.1 nmol per 2 h 100 mg; P = 0.02). Oleic acid increased lipid synthesis from glucose 125 percent (P = 0.04) in IM adipose tissue, whereas stearic acid and trans-vaccenic acid increased lipogenesis from glucose in SC adipose tissue by approximately 50 percent (P = 0.04). In SC adipose tissue only, trans-vaccenic and increased, lipogenesis from glucose (P < 0.02). Lipogenesis from acetate was depressed by CLA nearly 50 percent in SC adipose tissue. PPARγ gene expression increased between 14 and 16 mo of age in control IM and SC adipocytes. The increase in activity was also observed in AMPK gene expression. C/EBPβ and SCD gene expression did not increase in control samples until 16 mo of age. SC adipose tissue responded to stearic acid by increased GPR43 and AMPK gene expression at 12 mo of age. We conclude that fatty acids differentially affect lipid synthesis in IM and SC adipose tissues, which may account for the effects of pasture and grain feeding on adiposity.

During early mammalian embryogenesis, the embryonic genome undergoes critical reprogramming events that include changes in both DNA methylation and histone modifications necessary to control chromatin structure and thus, gene expression. Improper reprogramming of the epigenome during this window of development can lead to a vast number of imprinting anomalies, which are increased in children and livestock conceived in vitro. In the bovine, which closely resembles human preimplantation development, epigenetic changes occur from fertilization through the blastocyst stages. In particular, and concurrent with embryonic genome activation (EGA), de novo DNA methylation begins at the 8-cell stage. In order to explore the roles of histone-modifying enzymes during this crucial period of development, we characterized the transcript expression of several enzymes key enzymes across in vitro bovine preimplantation development using quantitative real-time PCR. Two of the 7 genes analyzed (Suz12 and Lsh) exhibited notable increases at the 8-16 cell stages, with basal levels observed both before and after this. These increases coincided with both EGA and de novo DNA methylation. We further explored their roles in bovine preimplantation embryos by knocking down expression via the use of gene-specific targeting siRNAs. Independent suppression of either Suz12 or Lsh via cytoplasmic microinjection of targeting siRNAs resulted in lower development rates (p < 0.0001), and poorer embryo quality of the morulas and blastocysts that survived. In addition, Suz12 suppression led to reductions in both H3K27 (p < 0.0001) and H3K9 (p = 0.07) trimethylation, and an increase in DNA methylation levels (p < 0.0001), as compared to the null-injected controls. Lsh suppression did not change H3K27, but led to a reduction in H3K9 trimethylation (p = 0.006) and an increase in DNA methylation (p < 0.0001). Clearly our data demonstrate that these epigenetic modifiers play a critical role in formation of the embryonic epigenome, but further research would be necessary in order to fully characterize gene activities during this developmental window.

Bovine herpesvirus 1 (BHV-1) is a member of the Alphaherpesvirinae subfamily and is the prototype ruminant herpesvirus. BHV-1 causes a number of complications in cattle including upper respiratory tract disorders, conjunctivitis, genital disorders, abortions, and immune suppression. Like all herpesviruses, reactivation from latency can occur throughout the animal’s life. Of particular economic importance is the bovine respiratory disease complex (BRDC) or ‘shipping fever’, in which BHV-1 plays a major role. BRDC is an enormous economic concern as it costs the US cattle industry approximately one billion dollars annually. In order to generate improved gene-deleted vaccines against BHV-1, there is a need to understand the contributions of viral gene products during infection. US3 is a serine/threonine kinase present in BHV-1 and is thought to play major roles during viral infection. As in other herpesviruses, US3 in BHV-1 is expected to phosphorylate several cellular and/or viral proteins. We recently presented evidence that BHV-1 US3 phosphorylates both VP8 and VP22; however, further functional characteristics of BHV-1 US3 during viral infection have not been elucidated. The hypothesis of this project is that the deletion of the US3 gene leads to reduced BHV-1 fitness. To explore this hypothesis, we generated a US3-deleted (ΔUS3) and subsequent US3-rescued (US3R) BHV-1 virus. Using these viral mutants, we characterized the growth properties of the viruses, evaluated the effect of the US3 deletion on major structural BHV-1 proteins, characterized the protein composition of the mature virions, and, identified viral processes that were impaired in the deletion mutant. Initially, the ∆US3 virus was generated through a 3-step PCR strategy which replaced the gene of interest with an antibiotic resistance cassette. Following this, the US3 gene was rescued via a two-step en passant mutagenesis strategy which has been previously used to generate insertions, deletions, and substitutions in herpesvirus-containing bacterial artificial chromosome (BAC) DNA. In vitro characterization of ∆US3 BHV-1 has demonstrated that US3 deletion affects BHV-1 growth characteristics, expression kinetics of major structural proteins, mature virion composition, cell to cell spread, and the subcellular localization of key viral proteins during infection. Growth kinetics of ∆US3 BHV-1 were impaired compared to wild-type (WT) BHV-1, especially at late times post-infection. Plaque sizes formed by ∆US3 BHV-1 were significantly smaller than those formed by either WT or US3R BHV-1, demonstrating that US3 is important for cell to cell spread. The expression kinetics of major structural and regulatory BHV-1 proteins were different between cells infected with ∆US3 or WT BHV-1, and incorporation of these proteins into the mature viruses differed, demonstrating that US3 is instrumental in ensuring proper protein expression and mature virus composition in vitro. Of particular importance, glycoprotein B (gB), was shown to be expressed in higher quantities earlier during infection in the absence of US3, and that this protein was incorporated in significantly higher amounts in mature virions which lacked US3. Qualitative analysis of ∆US3 BHV-1 infected monolayers suggested the abolishment of cell to cell projections characteristic of WT BHV-1 infection. Finally, the disruption of gB in ∆US3 BHV-1 infected cells was confirmed by confocal microscopy and fluorescence-activated cell sorting (FACS) analysis. Through confocal microscopy, evidence was provided that infection with ∆US3 BHV-1 possibly results in earlier expression of gB on the surface of cells and less intracellular accumulation of this protein during late stages of infection. The observed effect on the localization of intracellular gB in ∆US3 BHV-1 infected cells was quantified by flow cytometry. ∆US3 BHV-1 infected cells had approximately 25% higher gB expression on the surface of cells and a corresponding 25% decrease in intracellular gB. Although these differences have not yet been demonstrated to be statistically significant and not confirmed through infection with US3R BHV-1, this suggests that US3 may influence the synthesis and cellular trafficking of gB in vitro.

Les lentivirus sont un groupe de virus appartenant à la famille des Retroviridae. Le modèle par excellence pour l'étude des lentivirus est le virus de l'immunodéficience humaine (VIH) qui est l'agent causant le syndrome d'immunodéficience acquise ou SIDA. Les études réalisées sur les protéines du virus telles que Nef, Vpu, Tat et Rev ont permis de mieux connaître la biologie et les mécanismes de pathogenèse du VIH de type 1 (VIH-1). Le virus de l'immunodéficience bovine (VIB) est un lentivirus qui a servi de modèle d'étude de la protéine Tat du VIH. Bien que le VIH-1 et le VIB appartiennent aux lentivirus, les maladies qu'ils causent sont très différentes. Le potentiel pathogénique du VIB est controversé et les mécanismes pathogéniques et la biologie du VIB ne sont pas encore bien connus. Seule la protéine Tat a été bien caractérisée. L'étude approfondie des autres protéines virales nécessaires à la réplication du virus comme la protéine Rev permettront de donner des outils nécessaires à la compréhension de la biologie du VIB et des mécanismes impliqués dans la pathogenèse des lentivirus. La protéine Rev a une fonction essentielle dans la réplication des lentivirus. Le rôle de Rev est d'exporter les ARNs viraux non épissés et partiellement épissés du noyau au cytoplasme pour produire en outre les protéines nécessaires à l'assemblage des nouvelles particules virales. La protéine Rev du VIH-1 contient minimalement quatre domaines fonctionnels importants : un domaine basique riche en arginines nécessaire à la liaison de l'ARN ou RBD qui contient des signaux de localisation nucléaire et nucléolaire (NLS et NoLS), un domaine riche en leucines nécessaire à l'exportation du complexe Rev-ARN (NES) et deux régions nécessaires à la multimérisation de Rev. Le but de ce projet est de caractériser moléculairement et biologiquement la protéine Rev du VIB. Dans ces travaux, nous rapportons la caractérisation des NLS et NoLS de la protéine Rev du VIB. Grâce à la transfection d'une série de protéines mutantes de Rev du VIB fusionnées à la enhanced green fluorescent protein (EGFP). Nous avons démontré que le NLS de la protéine Rev du VIB est bipartite. Ce type de NLS est le premier à être identifié dans la famille des protéines Rev des lentivirus/rétrovirus. De plus, nous avons déterminé que le NoLS était localisé entre les deux motifs basiques du NLS bipartite de Rev du VIB. Le NoLS de Rev du VIB est aussi unique et diffère de la séquence consensus rapportée pour d'autres protéines virales et/ou cellulaires, toute nature confondue. Pour l'importation au noyau, nous avons démontré que la protéine Rev du VIB est importée par la voie classique (importines α:β) et que les importines α3 et α5 sont impliquées, fait qui contraste avec l'importation au noyau de la protéine Rev du VIH-1. Nous avons aussi caractérisé le domaine d'exportation ou NES de Rev du VIB en déterminant que le mécanisme d'exportation était CRM1-dépendant de façon similaire que la proteine Rev du VIH-1. Nous avons aussi identifié les résidus qui composent le NES de la protéine Rev du VIB qui appartient au groupe de PKI NES et non à celui de Rev VIH-1 NES, ce qui constitue une première chez les lentivirus. Pour compléter la caractérisation des domaines fonctionnels de la protéine Rev du VIB, les domaines de multimérisation et de liaison à l'ARN furent identifiés. Nous avons observé que Rev du VIB était capable de multimériser in vitro et in vivo dans le cytoplasme et deux régions impliquées dans cette fonction ont été identifiées. En plus, nous avons cartographié un domaine RBD bipartite chez Rev du VIB. Nous avons montré que le premier motif du RBD était situé dans la région centrale de la protéine et chevauchait le NoLS et le NLS bipartite alors que le deuxième motif était situé à l'extrémité C-terminale de la protéine Rev du VIB. En conclusion, les principaux domaines fonctionnels de la protéine Rev du VIB ont été caractérisés. Les résultats obtenus ont clairement démontré que la protéine Rev du VIB est unique chez les lentivirus et les rétrovirus. Les caractéristiques de la protéine Rev du VIB pourraient aider à expliquer en partie les profondes différences de pathogénicité observées chez les lentivirus. ______________________________________________________________________________ MOTS-CLÉS DE L’AUTEUR : VIB, Rev, NLS, NES, VIH

Decline in fertility with age has been well documented in women. There are ethical limitations to use humans as a model for basic research, and there is a lack of well characterized animal model. The objective was to characterize and validate a bovine model for the study of age-associated subfertility. All experiments were conducted on the same group of 13-14 year old cows (n=10), and their 1-4 year old young daughters (n=10). Mother-daughter pairs were used to reduce genetic variations. <p>Follicular wave pattern in a natural reproductive cycle was maintained in old cows similar to that in daughters. We hypothesized that aging in cattle is associated with elevated circulating concentrations of FSH, and reduced concentrations of steroid hormones. As stated, circulating FSH concentrations were higher (P=0.009) during follicular waves in old than young cows. The ovulatory follicle in 2-wave cycles was smaller in old cows (P=0.04), but plasma estradiol concentrations were higher (P=0.01). Luteal phase progesterone tended to be lower in old than young cows (P=0.1). The number of 4-5 mm follicles recruited into a follicular wave was lower (P<0.05) in old cows than in their daughters.<p>The response to ovarian synchronization and superstimulatory treatments was compared between old and young cows. We hypothesized that aging in cattle is associated with decreased synchrony of an induced follicular wave after steroid treatment. Conversely, the emergence of an induced follicular wave was synchronous between age groups. The preovulatory LH surge was delayed in old compared to young cows (P=0.01), but the detected ovulation times were not different. Old cows had fewer (P<0.01) follicles equal or greater than 6 mm after superstimulation, and tended (P=0.1) to have fewer ovulations than their daughters (32±4 versus 40 ±3, respectively). The response of individual cows to successive superstimulatory treatments was correlated (r>0.8; P<0.0001). <p>The hypothesis of reduced oocyte developmental competence in old cows was tested by comparing embryo production and pregnancy rates between old and young cows. Fewer (P=0.04) embryos were recovered from old cows (6±2) than their daughters (12±2). A higher proportion (P<0.01) of unfertilized oocytes and/or uncleaved zygotes were recovered from old cows (222/312, 71%) than their daughters (119/316, 38%). The recovery of fewer embryos in old cows suggests reduced oocyte developmental competence. The survival of embryos after transfer into unrelated young recipients was similar between age groups. <p>The effects of advanced age on oocyte meiotic maturation and oocyte chromosome numbers abnormalities were studied in old and young cows. Our hypothesis of compromised oocyte meiotic maturation with age was not supported; similar or higher proportion of metaphase II oocytes were recovered from old than young cows. The abnormalities of oocyte chromosomal numbers were similar between age groups. <p>To conclude, endocrine, follicular and oocyte developmental changes in old cows are consistent with those reported for women approaching menopause. Therefore, our results validated the use of a bovine model to study age-associated subfertility in women. Unlike women, we did not detect an age-related increase in abnormalities of oocyte chromosome numbers in cattle.

Bovine cysticercosis is a zoonotic disease for which cattle are the intermediate hosts of the human tapeworm <i>Taenia saginata</i>. Routine inspection measures are implemented in Canada by the Canadian Food Inspection Agency (CFIA), and similarly elsewhere, for the postmortem detection of larval parasite cysts (cysticerci) in beef destined for human consumption. Detection is based on the gross examination of traditional carcass predilection sites, although it is recognized that the parasite has no true predilection for a particular tissue or site. In order to evaluate the efficacy of the inspection protocol currently implemented in Canada, a study was undertaken to determine the distribution of <i>T. saginata</i> cysticerci in tissues of experimentally infected cattle. Forty-two cross-bred beef cattle were divided into five groups of 5-12 animals each and inoculated orally with either 10000, 5000, 1000, 100 or 10 <i>T. saginata</i> eggs obtained from cases of human taeniosis in Thailand. From 47 to 376 days post-inoculation (DPI), ten animals inoculated with 5000 eggs were killed and the carcasses partitioned into 31 tissue sites. These consisted of the traditionally inspected tissue sites of heart, masseter and pterygoid muscles, tongue, oesophagus, and diaphragm (membranous and crura); as well as non-traditional sites of lung, liver and 20 additional muscles or muscle groups. After the routine inspection for cysticerci of traditional tissue sites, tissues from all sites were each cut into approximately 0.5 cm thick slices and the total number of parasitic cysts and cyst density (cysts/g of tissue) were determined for each site. Traditional sites were similarly evaluated for the remaining 32 animals that were killed between 117 and 466 DPI. Sites were ranked based on cyst density. In the animals for which non-traditional sites were also evaluated, no sites had higher cyst densities than those traditionally inspected. When only traditional sites for all animals were compared, the heart ranked highest overall, although not significantly different from masseter, and was the most frequently affected site. The traditional site of oesophagus was among the poorest of all sites for detection of cysticerci. The heart was confirmed as the site of choice for detection of bovine cysticercosis based on high cyst density and frequency of infection. There was also enhanced visibility of parasite lesions in the heart due to the relatively early degeneration and resultant gross pathology that occurs in cardiac muscle. More thorough examination of the heart is recommended during post-mortem inspection for this parasite, particularly when examining animals from an infected herd. <p>Currently, confirmation by CFIA of suspect cysticerci recovered during meat inspection relies on gross, stereomicroscopic, or standard histological examination. Although degenerating cysticerci are more likely to be detected and submitted for diagnosis, they often cannot be definitively identified by these methods. A recently developed monoclonal antibody-based immunohistochemical (IHC) assay for post-mortem diagnosis of this parasite was optimized and standardized. The IHC method was compared to the currently used histological assay using 169 degenerated known-positive <i>T. saginata</i> cysticerci collected from the experimental infections in the first study and from field submissions, and known-negative specimens and lesions of various etiologies from non-infected cattle. The use of the IHC assay identified significantly more known-positive bovine cysticerci (91.7%) than the histological method (38.5%), and non-specifically reacted only with the other cestode species examined. Since <i>T. saginata</i> is the only larval cestode typically found in the muscle of cattle, this cross-reactivity is not significant and the IHC assay will be a useful tool for the identification of lesions caused by degenerated bovine cysticerci.<p>This research provided evidence to support changes to the current post-mortem inspection, detection and diagnostic procedures and will contribute to more effective and efficient control of bovine cysticercosis.

Mastitis is the most prevalent and major cause of economic losses in dairy farms. Bovine mastitis caused by strains of <i>S. aureus</i> is a major economically important disease affecting the dairy industry worldwide. <i>S. aureus</i> is one of the most common udder pathogens that cause either clinical or sub-clinical mammary gland infections. Different treatment regimes have failed to cure <i>S. aureus</i> intramammary infections. Most mastitis vaccination strategies have focused on the enhancement of systemic humoral immunity rather than strengthening local intramammary immunity. Vaccines aimed at enhancing intramammary immunity of dairy cows against <i>S. aureus</i> mastitis have had limited success. Commercially available vaccines show various degrees of success and work in research laboratories with experimental vaccines suggest that in part, the failure of these vaccines lies in the limited antigenic repertoire contained in the vaccine formulations. Moreover, not only does variation in the antigenic composition but also presence of capsular polysaccharide in most pathogenic strains and decreased activity of immune effectors in milk affect the success of vaccines. In addition to these, the ability of <i>S. aureus</i> to attach and internalize into mammary epithelial cells, enables bacteria to escape from the effect of immunity and antibiotics by being hidden in the intracellular niche and thereby causing chronic recurrent intramammary infection. <i>S. aureus</i> also has the ability to become electron-transport-defective and to form slow-growing small colonies that are non haemolytic and less virulent. These small colony variants might hide from the immune surveillance in the intracellular area and revert to the parental strain causing chronic recurrent infections. If immunization targets antigenic molecules that are conserved throughout all pathogenic strains, even the small colony variants can be controlled since the immune system will clear the parental strain which causes lethal infection. Thus, immunization trials should focus on conserved immunogenic antigen molecules among pathogenic strains formulated with an adjuvant and delivered by a route of immunization to induce maximum stimulation of the immune system. Moreover, immunization should focus on inducing Th1 responses, which is protective against <i>S. aureus</i> mastitis. It has been reported that proteins with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity might be used as such antigens to induce protection against parasitic and microbial infections. Previous study in our laboratory on mastitis-causing streptococci indicates that GapC proteins of <i>S. uberis</i> and <i>S. dysgalactiae</i> have potential as vaccine antigens to protect dairy cows against mastitis caused by environmental streptococci. Two conserved cell wall associated proteins with iii glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity, GapB and GapC have been identified from <i>S. aureus</i> isolates from bovine intramammary infections. The overall goal of this study was to improve our understanding on intramammary immunity using the GapC and GapB proteins of <i>S. aureus</i> as model antigens for mastitis and to determine the regulation of expression of <i>gapB</i> and <i>gapC</i> genes and their roles in the pathogenesis of bovine <i>S. aureus</i> mastitis. We hypothesized that strengthening local intramammary immunity using GapB and GapC proteins of <i>S. aureus</i> as antigens will protect against bovine <i>S. aureus</i> mastitis. To test this hypothesis we took the approach of using the <i>gapB</i> and <i>gapC</i> genes and constructed plasmids encoding GapB, GapC and GapB::GapC (GapC/B) chimeric proteins. We set six objectives to test our hypothesis using these proteins to enhance the intramammary immunity. In aim 1 we constructed plasmids encoding the GapB, GapC proteins and also constructed a chimeric gene encoding the GapC and GapB proteins as a single entity (GapC/B chimera) as the basis for a multivalent vaccine. In this objective the humoral and cellular immune responses to GapC/B were compared to the responses to the individual proteins alone or in combination in C57 BL/6 mice. Our results showed that the GapC/B protein elicited strong humoral and cellular immune responses as judged by the levels of total IgG, IgG1, IgG2a, IL-4 and IFN-ã secretion and lymphocyte proliferation. These results strongly suggest the potential of this chimeric protein as a target for vaccine production to control mastitis caused by <i>S. aureus</i>. In aim 2 we continued our studies on GapC/B by testing the effects of DNA vaccination with plasmids encoding the individual gapB and gapC genes as well as the gapC/B protein gene with or without a boost with the recombinant proteins. The results showed that DNA vaccination alone was unable to elicit a significant humoral response and barely able to elicit a detectable cell-mediated response to the recombinant antigens but subsequent immunization with the proteins elicited an excellent response. In addition, we found that DNA vaccination using a plasmid encoding the GapC/B chimera followed by a boost with the same protein, although successful, is less effective than priming with plasmids encoding GapB or GapC followed by a boost with the individual antigens. In aim 3 we optimized immune responses in cows by comparing route of vaccination (subcutaneous versus intradermal), site of vaccination (locally at the area drained by the supramammary lymph node versus distantly at area drained by parotid lymph node. Our results showed that both subcutaneous and intradermal immunizations with the GapC/B protein at the area drained by the supramammary and parotid lymph nodes resulted in significantly increased serum and milk titers of total IgG, IgG1, IgG2, iv and IgA in all vaccinated groups as compared to placebo. The anti-GapC/B IgG1 serum and milk titers were significantly higher in all vaccinated group as compared to the placebo group. These results indicated that vaccination at the area drained by the supramammary lymph node resulted in better immune responses. In aim 4 we tested different formulations of the GapC/B antigen with adjuvants such as PCPP, CpG, PCPP + CpG and VSA-3. We found that the VSA-3 formulation induced the best immune responses in cows. In this objective we also monitored immune responses longitudinally over one lactation cycle to determine the duration of immune responses by measuring IgG, IgG1, IgG2, and IgA on monthly blood and milk samples. We found that the duration of immune responses was about four months. In aim 5 we tested the role of GapC in the virulence of <i>S. aureus</i> mastitis using the <i>S. aureus</i> wild type strain RN6390 and its isogenic GapC mutant strain H330. Our results from both in vitro adhesion and invasion assays on MAC- T cells and in vivo infection of ovine mammary glands showed that GapC is an important virulence factor in <i>S. aureus</i> mastitis. In aim 6 we examined the role of sar and agr loci on the expression of <i>gapC</i> and <i>gapB</i> genes by qRT- PCR using <i>S. aureus</i> RN6390 and its isogenic mutants defective in agrA, sarA and sar/agr (double mutant) at exponential and stationary phases of growth. Our results showed that both <i>gapB</i> and <i>gapC</i> expression were down regulated in the mutant strains, indicating that the expression of the <i>gapB</i> and <i>gapC</i> genes is controlled by the universal virulence gene regulators, agr and sar. We also checked the role of environmental factors such as pH, growth media, and oxygen tension on the expression of <i>gapB</i> and <i>gapC</i> using q-RT-PCR. Our results showed that the expression of <i>gapB</i> and <i>gapC</i> genes in different strains of <i>S. aureus</i> was not consistent under the above-mentioned environmental conditions.

Bovine adenovirus type 3 (BAdV-3) is a non-enveloped icosahedral particle which contains a double stranded DNA genome. The genome of BAdV-3 is organized into early, intermediate and late regions. The late region is organized into seven regions L1-L7 (Reddy et.al., 1998). The L6 region of late transcription unit of BAdV-3 encodes one of the non structural protein named 33K protein. The objective of the present study was to characterize the 33K protein and to identify the viral/cellular proteins involved in the interaction with 33K protein.<p> The RT-PCR analysis revealed the presence of spliced and unsliced mRNAs encoding 33K and 22K proteins respectively in BAdV-3 infected cells. The 33K and 22K proteins share a N-terminus region of 138 amino acids. To determine the specificity of these two proteins, rabbit polyclonal antiserum was raised against peptides representing unique C- terminal regions of the proteins. Anti-33Kp serum detected two major proteins of 42 kDa and 22 kDa and five minor proteins of 39kDa, 35kDa, 29kDa, 25kDa and 19kDa in BAdV-3 infected cells or 33K transfected cells. Similarly, anti-22Kp serum detected three proteins of 41kDa, 39kDa and 37kDa in BAdV-3 infected cells. However, a protein of 39kDa and 37kDa was detected in 22K (having splice sites removed) transfected cells. The 33K protein is predominantly localized to the nucleus of BAdV-3 infected cells and is involved in stimulating the transcription from major late promoter. Analysis of mutant 33K proteins demonstrated that amino acids 201-240 and amino acid 204-231 are required for nuclear localization and MLP transactivation.<p> The adenovirus 33K protein appears to be a multifunctional protein performing different role in viral infection. Earlier study has shown that the 33K protein plays a role in viral capsid assembly and efficient capsid DNA interaction in BAdV-3 (Kulshreshtha et.al., 2004). The involvement of 33K protein in different steps of adenovirus replication may require protein protein interaction. Using 33K protein as bait in yeast two hybrid system, open reading frames (ORFs) of BAdV-3 were screened for the potential interactions with 33K protein. The 33K protein showed specific interactions with two late viral proteins- 100K and protein V (pV). The yeast two hybrid findings were validated by in vitro binding using <i>in vitro</i> synthesized transcription-translation products. It was demonstrated that the interaction of 33K with 100K and pV takes place during BAdV-3 infection. The stretch of amino acids 81-120 and 161-200 in 33K protein were involved in the interaction with pV and 100K protein.<p> For screening the cellular interactions, the 33K protein was used as a bait to screen bovine retina cDNA library. The yeast two hybrid screening revealed that the 33K protein appears to interact with bovine presenilin-1-associated protein / mitochondrial carrier homolog 1 (BoPSAP / BoMtch1) and bovine microtubule associated protein (BoMAP). However, subsequent analysis by various <i>in vitro</i> and <i>in vivo</i> assays could only confirm the interaction between 33K protein and BoPSAP/BoMtch1. In addition, the 33K protein was also shown to be colocalized with BoPSAP in mitochondria. Based on these observations, it may be possible that 33K protein may play an anti-apoptotic by interacting with BoPSAP since the human homolog of PSAP has been known to induce apoptosis.

The objectives of this project were: 1) to determine if live animal lung biopsy could be used to characterize early pathologic changes in the bovine lung associated with bovine respiratory disease (BRD), 2) determine if specific infectious respiratory pathogens can be identified in association with early pathological changes, and 3) determine whether pulmonary pathology characterized by live animal lung biopsy at arrival and at the time of initial BRD diagnosis was associated with health and production outcomes of feedlot steers in a commercial feedlot.<p> A live animal percutaneous lung biopsy technique was developed to obtain a lung sample from the right middle lung lobe in intercostal space (ICS) 4 using a Bard® Magnum® reusable biopsy instrument and a modified 4-mm (8g) biopsy needle. The lung biopsy procedure was limited to 2 attempts per biopsy time. In the technique development, 34 animals chronically affected with BRD were utilized, 20 animals in the preliminary development followed by 14 additional animals in a commercial feedlot setting. The technique resulted in 1 fatality of 34 steers (2.9%) and lung parenchyma was harvested in 19 of 34 steers (55.9%) chronically affected with BRD. In addition, in the commercial feedlot setting this procedure was determined to take about 20 minutes per animal.<p> The final study was performed on one hundred feedlot steers considered at high risk of developing BRD from twenty pens within a commercial feedlot. Study animals were enrolled in three different groups: sick on arrival (ARR-SA) consisting of 27 study animals and 13 matched control animals; pen pulls with no fever (PP-NF) consisting of 14 study animals and matched 7 controls; and pen pulls with an undifferentiated fever (PP-UF) consisting of 26 study animals and 13 matched controls. Live animal percutaneous lung biopsies were collected from the right middle lung lobe at 3 different times within the first 30 days of the feeding period, about 2 weeks apart. All samples were histopathologically evaluated and were assessed for the presence of <i>Mycoplasma bovis</i>, <i>Mannheimia haemolytica</i>, Histophilus somni and bovine viral diarrhea virus with immunohistochemistry.<p> A total of 295 lung biopsies were performed yielding 210 (71.2%) lung samples that were sufficient for histopathological evaluation. A histopathology score was awarded to each biopsy based on certain histopathological lesions being present. Only 20 lung biopsy samples from 19 animals received a histopathology score (ie, pulmonary lesions were present) with the most common score being a 1 (maximum score is 20). There were too few lung biopsy samples with a histopathology score to reveal any association with subsequent health events.<p> Immunohistochemistry (IHC) was performed on all lung biopsies recovered yielding one lung sample to be positive for both <i>Mannheimia haemolytica</i> and <i>Mycoplasma bovis</i> from the PP-UF group. There were too few positive samples to reveal any association between IHC and histopathology score.<p> A post mortem evaluation was performed by a study veterinarian on all study animals who died or were humanely euthanized due to poor treatment response. In this study only 4 steers died or were euthanized due to poor treatment response and 3 control steers were humanely euthanized. There were too few animals to reveal any association between histopathology score and post mortem diagnosis.<p> On entry into the feedlot, weights between ARR-SA and the PP-UF and PP-NF groups were significantly different (p<0.05). This is likely an effect of the different processing groups of cattle. At study allocation, the body weights of ARR-SA and PP-UF, PP-UF and their matched controls, and PP-NF and their matched controls were also significantly different (p<0.05). This is likely due to the PP-UF and PP-NF groups experiencing illness for a longer period of time resulting in greater weight loss than the ARR-SA animals as well as the control animals, who were not clinically sick.<p> The live lung biopsy procedure utilized in this study did not appear to cause any long lasting adverse effects as the BRD case fatality rates from the study animals were comparable to the overall case fatality rates reported by the feedlot for fall placed calves. In fact, the study animals experienced a decreased fatality rate compared to the feedlots overall fatality rate. This may be due to the study animals inadvertently being monitored more closely as the pen checkers were aware of and participating in the study. On post mortem evaluation there was no evidence of adhesions at the biopsy site. This procedure was performed on 134 feedlot steers resulting in only 2 acute deaths as a direct result of the live animal percutaneous lung biopsy procedure.<p> The results of this study indicate that live animal, percutaneous lung biopsy can be performed safely on feedlot steers in a commercial feedlot with few clinical side effects. In this study there were only 2 fatalities in 134 steers (1.5%) due to the biopsy procedure or 2 fatalities per 349 sampling times (0.6%) This technique did not prove useful either as a diagnostic tool for the determination of early lung pathology in BRD or as prognostic indicator for health and production outcomes. However, this lung biopsy technique may be a useful diagnostic tool for chronic pneumonia assessment.

Somatic cells in semen are a potential source of nuclei for cloning animals bysomatic cell nuclear transfer. Culture of the cells from frozen semen, if possible, wouldbe extremely valuable for preservation or restoration of endangered, exotic, and extinctanimals when other ways of obtaining somatic cells are unavailable. In the present study,somatic cells isolated from ovine and bovine semen samples were characterized, culturesystems were evaluated for attachment and proliferation of these cells, and usefulness ofthese cells for somatic cell nuclear transfer was determined.Semen samples were collected from eight rams representing three breeds:Dorper, Suffolk, and Hampshire and nine bulls representing three breeds: Charolais,Brahman, and a crossbred Brahman. Somatic cells were isolated immediately postcollection by centrifuging through percoll columns and the epithelial cells wereidentified by immunofluorescence analysis. Culture systems were evaluated for theirability to support attachment and proliferation of the cells. A supplemented mediumcomposed of DMEM/F12, 10% fetal bovine serum, 10 ng/ml epidermal growth factor, 30 g/ml bovine pituitary extract, 5 g/ml insulin, 10 ng/ml cholera toxin, and 50 g/mlgentamicin significantly improved cell proliferation over sheep fetal fibroblastconditionedmedium, 3T3 cell-conditioned medium, and basic medium (p<0.05). Cellproliferation and attachment were further improved when Matrigel-coated culturesurfaces were used (p<0.05). However, the system was not adequate for obtaining cellgrowth from frozen semen.To check the chromosome anomalies, metaphase chromosomal complements ofthe cells cultured from 4 rams were evaluated. The predominant chromosome number ofcells from three of the rams (Dorper 18-month-old; Suffolk 17-month-old; Suffolk 18-month-old) was 2n = 54, which is the normal modal number for sheep. However, thenumbers of chromosomes of cells cultured from the fourth ram (Hampshire, 18-monthold)were near-triploid. These results indicate the need for chromosome analysis of cellsbefore using them for cloning experiments. In our attempts to clone animals, blastocyststage embryos were successfully produced using epithelial cells cultured from semen ofthree different bulls. However, no compact morulae or blastocysts were obtained whensomatic cells isolated from frozen semen but not cultured were used as donor cells.