Veterinary Science - Theses
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ItemMolecular basis of attenuation of Mycoplasma synoviae( 2013)Comparative genomic analysis of M. synoviae strains including temperature-sensitive (ts+) vaccine strain MS-H, non-temperature-sensitive (ts–) vaccine parent strain 86079/7NS and two of the ts– MS-H reisolates, revealed a large number of single nucleotide polymorphisms (SNPs) some of which were non-synonymous mutations in essential genes of MS-H. One such non-synonymous mutation causing Gly123Arg substitution in a highly conserved domain of GTP binding protein Obg was further investigated. In-silico analysis of M. synoviae Obg 3D structures, obtained by homology modeling, revealed that Gly123Arg substitution was likely to destablise the Obg structure whilst another substitution, Ala210Val, mapped in G3 motif of the conserved GTP binding domain, was likely to compensate the destablising effect of Gly123Arg change in the Obg domain. Plasmid shuttle vectors, based on the origin of replication (oriC) of M. synoviae, were developed and used to transform MS-H by electroporation. One plasmid vector, pMAS-LoriC, containing complete oriC region, was found to replicate autonomously in the MS-H and integrated at the chromosomal oriC locus. In order to complement the MS-H with the wild-type obg, an oriC-based vector, pKS-VOTL, was developed and used to transform MS-H. Variable lipoprotein haemagglutinine A (vlhA) promoter region was used to drive expression of the wild-type obg. The pKS-VOTL plasmid readily integrated at the chromosomal oriC with no homologous recombination event other than at oriC locus was observed. Over-expression of wild type obg was confirmed using Northern blot, SDS-PAGE, and Western blot analyses. The MS-H clones transformed with pKS-VOTL retained the ts+ phenotype of MS-H; however unlike MS-H, these MS-H transformants could grow, albeit to a very low titer, at 33°C after exposure to nonpermissive temperature of 39.5°C. It was postulated that overproduction of wild-type Obg exerted growth inhibitory effect at the nonpermissive temperature whilst promoted the growth at the permissive temperature. High-resolution melting curve analysis, targeting the informative SNPs in obg, was used to develop rapid genotyping technique which could reliably differentiate MS-H from its ts–reisolates and also from all field strains used in this study. The technique was also applied to clinical specimens taken from SPF chickens vaccinated with MS-H and commercial field chicken flocks infected with a field M. synoviae strain. Findings of this thesis indicated that, like many other mycoplasmas, genetic manipulations of M. synoviae are now technically feasible. Mutations in obg are most likely responsible for the temperature-sensitivity/attenuation of MS-H while mutations in other essential genes, especially those encoding aminoacyl-tRNA synthetase and tRNA (guanine-N1)-methyltransferease, might have a role to play in MS-H attenuation. Using oriC vectors, expression of foreign proteins is now also possible in MS-H. Such strategy can serve as foundations for production of diagnostic mycoplasma antigens and for the development of recombinant vaccines.
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ItemA study investigating the incidence and associated risk factors of heifer mastitis in the Macalister Irrigation District( 2013)The project aimed to assess the incidence, causative pathogens and risk factors for mastitis in primiparous heifers in the Macalister Irrigation District (MID). Additional aims included analysis of the effect of mastitis on production, lactation length and risk of removal from the herd. Data was collected from 19 farms over a period of two years. This included a retrospective analysis of data collected in 2009, and prospective data collection in 2010. Information such as management practices, treatment records, individual cow cell counts, production data, cull records and bulk milk cell counts were collected in both years. Additional data gathered in 2010 included microbiological culture results from both clinical and subclinical cases of mastitis, and recording of health events. Overall 11.3% of the heifer cohort in this study suffered from at least one case of clinical mastitis (CM) during the first lactation. Heifers were particularly at risk of mastitis early in lactation, with 65.7% of cases occurring within 30 days in milk, compared to 37.7% in mature cows in the same period. Strep. uberis was the bacteria most commonly isolated from quarter cases of clinical mastitis (48.1%) and from heifers diagnosed with subclinical mastitis (15.6%). The use of an internal teat sealant pre-partum had the greatest effect on the odds of CM within 30 days of calving (CM0-30d) (OR 0.44 CI 0.28-0.68 P<0.001) followed by calving in 2010 (OR 1.71 95% CI 1.31-2.33 P<0.001). Herd size as a continuous variable was found to have a quadratic effect on CM0-30d. Adverse health events such as dystocia and lameness were assessed as risk factors for mastitis within a smaller subset of herds, and no relationships were found between CM0-30d and any of these events. Similarly no significant associations were detected between CM0-30d and production outcomes, lactation length or the odds of removal from the herd. This research increases our understanding of heifer mastitis within an Australian context, and validates certain risk mitigation strategies specific to heifers in Australian pasture based systems.
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ItemComparative analyses of key parasitic helminths through transcriptomic data integration using bioinformatics( 2013)Parasitic helminths (worms) of humans and animals cause diseases of major socioeconomic impact worldwide. Considering their impact, the emergence of drug resistance and the absence of commercial vaccines, there is a growing demand for alternative intervention strategies to control such worms. Clearly, improved knowledge and understanding of the molecular biology and biochemistry of parasitic helminths should underpin the development of new methods for the treatment and control of parasitic helminth infections. Advanced molecular and informatic technologies are providing unique opportunities to explore the systems biology of helminth at substantially reduced cost and time. Despite these advances, the amount of functional information that can be inferred from sequence data is still limited. The studies described in this thesis create a framework for the integration of sequence data from a range of parasitic helminths, allowing for improved annotation and comparative analyses and leading to new insights into the molecular and system biology of selected parasitic helminths. This framework enabled comparative studies of two key lungworms of ruminants, with a focus on parasite-host interactions, and provides a foundation for many future studies of parasites.
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ItemRadiographic bone morphology of the equine metacarpophalangeal joint and relationships to the incidence of pathology in thoroughbred( 2013)Pathological conditions of the bones of the metacarpophalangeal joint (MCPJ) are common amongst racehorses. These conditions are of a great economic cost to the horse industry as well as being detrimental to the welfare of the horses themselves. This study aimed to: 1. develop reliable and repeatable radiological measurements of the MCPJ bones in Thoroughbreds; 2. investigate possible relationships between bone morphometry and the pathological incidences in this breed; and 3. identify correlations between these measurements and specific performance requirements in the breed. The centre of a circle best fit onto lateromedial radiographs of the most dorsal region of the lateral condyle of three MCPJs was assumed to approximate the centre of MCPJ motion and was found to be located on the lateral eminence of the lateral collateral ligament. Using this anatomical landmark, MCPJ angles were measured in cadaver limbs and then in live horses before and after lifting the contralateral limb. Angles were significantly less with the contra-lateral limb lifted. The goniometer repeatability coefficient was 4.7° (95% CI 3.9° to 6.0°) and was similar in horses standing squarely and with the contra-lateral limb lifted. Marked MCPJs, filmed in different head positions showed no significant effect of head position on the MCPJ angles. MCPJ angles measured before and after treadmill exercise showed the right MCPJ angle decreased significantly with exercise. Gross and radiographic studies established a set of MCPJ parameters that could be consistently identified on radiographs. Nine individual angular, 3 derived angular, and 16 linear parameters were developed, and used to derive 18 ratios. There was almost perfect agreement, with a CCC of more than 0.99, among all the parameters in tests of repeatability. In order to assess the effect of the MCPJ angle on MCPJ parameters, forelimbs from adult horses were loaded in a load frame to specific MCPJ angles within the normal range. Parameters measured on standard dorsopalmar and lateromedial radiographs showed that the ratio parameters were less affected than the angular parameters when the MCPJ angle was changed. An 11° change in the MCPJ angle was considered potentially acceptable for all these parameters. The effect of the orientation angle was determined on measurements obtained from 3D models of CT scan images of forelimbs from adult horses. Generally, the ratio parameters were less affected than the angular parameters during MCPJ rotation. An MCPJ rotation of 5° was considered as potentially acceptable for all these parameters. Radiographs from horses with MCPJ problems were collected from clinics along with their radiological reports. DP and LM radiographs were also taken from 18 experienced racehorses with no MCPJ problems. Significant differences were reported between normal and damaged horses in four parameters measured from those radiographs including two angles and two ratios. These parameters were B13, B14, H1/H3 and H2/H3. The study identified specific measures of the equine MCPJ bones that may be made using radiological methods. In addition, it identified some basic morphological measures that were associated with specific pathology in the MCPJ in Thoroughbred racehorses.
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ItemStructural and functional investigations of a chemokine binding protein from equine herpesvirus-1( 2013)Equine herpesvirus-1 (EHV-1) and equine herpesvirus-4 (EHV-4) are members of the Alphaherpesvirinae subfamily in the Herpesviridae family. Both EHV-1 and EHV-4 are endemic equine viruses worldwide. Infection with EHV-1 has significant health effects for equine populations by causing respiratory disease, abortion, perinatal mortality and severe neurological disease. Equine herpesvirus-4 predominantly causes respiratory disease. Abortion, perinatal mortality and neurological disease are rarely associated with EHV-4 infection. Equine herpesvirus-1 and EHV-4 both encode glycoprotein G (gG). Homologues of this protein are also present in most other alphaherpesviruses. Glycoprotein G encoded by EHV-1 is a chemokine binding protein, although no chemokine-binding activity has been demonstrated for EHV-4 gG. This project studied the ability of EHV-1 gG to bind to various recombinant chemokines, cytokines and defensins. The binding activity of full length and truncated foms of recombinant EHV-1 gG were compared using enzyme linked immunosorbant assays (ELISAs). The study also investigated the ability of recombinant EHV-1 gG to function as an immunomodulator in a mouse model using influenza virus. The immunomodulatory properties of recombinant EHV-1 gG were compared to those of recombinant EHV-4 gG and recombinant infectious laryngotracheitis virus (ILTV) which is an alphaherpesvirus of chickens. Equine herpesvirus-1 gG bound to recombinant chemokines from various species (human, mouse, equine), as well as recombinant human and mouse cytokines, and recombinant equine alpha-defensin-1. The chemokine binding of the truncated form of EHV-1 gG was similar to that of the full length recombinant EHV-1 gG. In mice, no significant difference in immunoglobulin levels was observed in animals that received infectious or inactivated influenza, compared to those that received infectious or inactivated influenza along with recombinant EHV-1, EHV-4 or ILTV gG. Recombinant ILTV gG co-delivered with infectious influenza resulted in a significant decrease in CD8 cells compared to infectious influenza alone. The co-delivery of EHV-4 gG resulted in a significant decrease in CD8 cells in infectious influenza studies and a significant increase in CD8 cells in inactivated influenza studies. No inflammatory cytokine or chemokine responses were detected in these studies using cytometric bead arrays. The results showed an influence on the cell mediated immune response. This project improves our understanding of the role of gG and the effect of its ability to bind to chemokines, cytokines and defensins. This project contributes to our understanding of the disease pathogenesis of EHV-1 and other alphaherpesviruses that encode gG. This project also brings potential benefits through the characterisation of an immunomodulatory compound that may have therapeutic application.
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ItemAnti-inflammatory effects of clenbuterol hydrochloride on leukocyte activation in the horse( 2013)The work reported in this thesis aimed to investigate the potential anti-inflammatory effects of clenbuterol hydrochloride in the treatment of equine endotoxaemia. Beta adrenergic agonists, such as clenbuterol hydrochloride reduce leukocyte activation and cytokine production through the up regulation of cyclic adenosine monophosphate. Investigations measured pro-inflammatory cytokine production (IL-1β and TNFα) by equine leukocytes, following stimulation with endotoxin, peptidoglycan and lipoteichoic acid and treatment with clenbuterol hydrochloride during in vitro and in vivo studies. The study also aimed to develop a model enabling correlation of pharmacokinetic and pharmacodynamic drug properties in order to predict an optimum dosing regimen for novel anti-inflammatory agents. The results of the study indicated that clenbuterol hydrochloride has significant anti-inflammatory effects on equine leukocytes challenged with Gram negative and Gram positive bacterial toxins in in vitro assays. Clenbuterol hydrochloride was most potent at inhibiting TNFα production in response to endotoxin, with the effects on peptidoglycan and lipoteichoic acid only being significantly inhibited at much higher clenbuterol hydrochloride concentrations (>10-6 M). Also a marked reduction in lipopolysaccharide stimulated IL-1β cytokine production following treatment with clenbuterol hydrochloride was recognised. In in vivo models of equine endotoxaemia, pre-treatment with oral clenbuterol hydrochloride caused a significant reduction in the peak rectal temperature and peak plasma TNFα concentrations. A model correlating clenbuterol hydrochloride pharmacodynamics and pharmacokinetics was developed. Clenbuterol may have a beneficial role alongside non-steroidal anti-inflammatory drugs and other previously investigated therapies in the treatment of inflammation associated with sepsis in horses. Further investigations in clinical cases of equine endotoxaemia are essential prior to the recommendation of clenbuterol hydrochloride as an adjuvant anti-inflammatory agent in the treatment of systemic inflammation.
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ItemEpithelial remodelling in a sheep model of asthma( 2013)The airway epithelium is the barrier to inhaled allergens and other agents and thus plays a central role in the restriction of, and response to, such insults. In asthma, ineffective barrier function and aberrant epithelial signalling are believed to be important in both the development and exacerbation of asthma. A review of the literature suggests that rodent models of asthma might not accurately represent the healthy or consequently the diseased airway epithelium of humans. Furthermore, little is known about small airway epithelial remodelling or the long-term effects of allergen exposure in the absence of continued antigenic stimulation. This thesis sought to firstly characterise epithelial remodelling following house dust mite (HDM) allergen challenge in a sheep model of experimental asthma (Chapters 3 and 4). In this model, chronic allergen challenge resulted in the induction of asthmatic features including goblet cell hyperplasia, epithelial hypertrophy, increased EGFR expression and allergen-induced proliferation of airway epithelial cells. Also provided is the first evidence for allergen induced goblet cell degranulation in a non-rodent system, and for degranulation induced via a relevant human allergen (HDM). The long-term effects of allergen exposure on the epithelium were also examined. Epithelial remodelling induced through chronic exposure to HDM remained for three months following the cessation of direct allergen challenge (Chapter 4). Goblet cell hyperplasia, epithelial hypertrophy and epithelial growth factor receptor (EGFR) expression remained in the absence of continued direct allergen challenge, indicative that the epithelium does not require continued allergen exposure to retain an asthmatic phenotype. Microarray analysis of epithelial brushing biopsies (Chapter 5), again demonstrated allergen driven epithelial cell proliferation and a general asthmatic phenotype including the down regulation of cellular tight junction mRNA. Subsequent qPCR validation demonstrated the transcriptional downregulation of the Bone morphogenetic protein (BMP) inhibitor noggin and the tight junction gene occludin in the more severe asthmatic sheep but not in those animals with a less severe phenotype. The downregulation of occludin mRNA was evident both prior to and throughout the establishment of chronic epithelial remodelling. In conclusion this thesis has demonstrated the induction of common asthmatic epithelial changes through chronic allergen challenge in a large animal model, and the retention of this asthmatic phenotype in the absence of continued stimulation. The persistence of epithelial remodelling is an interesting finding given its role as the first line of defence to the external environment. The knowledge gained in this thesis may have useful implications for future therapeutic strategies.