Veterinary Science - Theses
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ItemAntigenic characterisation of M2e protein of avian influenza virus( 2011)Highly pathogenic avian influenza virus (AIV) strain H5N1 is a pathogen of significant veterinary and public health importance, particularly in countries where the virus has become endemic, such as Indonesia. One of the viral proteins, M2e, could be potentially used as part of a test to differentiate infected from vaccinated chickens in an effort to improve AIV control in endemic countries. However, the antigenic and immunogenic properties of the M2e protein are poorly understood. In this study, the genetic conservation of the M2e protein was determined in H5N1 strains isolated from vaccinated commercial flocks in Indonesia and in 104 AIV of other subtypes from sequences available in the GenBank. From these comparisons mutations specific for Indonesian strains, as well as the most frequent mutations were identified to occur in the M2e protein. The mutation occurred as either single or multiple mutations in all positions between amino acid 10 and 20, except in the positions 15 and 17 that were conserved in all strains. The influence of these mutations on the antigenicity of the M2e was analysed by generating sixteen M2e recombinant proteins in which specific mutations were introduced by mutagenesis. Eighteen synthetic M2e peptides were also prepared for subsequent antigenic comparison. Antigenicity of these M2e proteins/peptides was analysed in two immunoassays using anti-AIV sera to fifteen different strains of thirteen subtypes. Anti-M2e peptide chicken and rabbit sera, as well as monoclonal antibodies to M2e were also used in this study. The results suggest that the M2e protein antigenic site extends from amino acid position 10 to 18. Within this M2e antigenic site there are six amino acid positions that are important for antibody binding: 10, 11, 13, 14, 16 and 18. Amino acid changes at the position 10 effected the binding of most anti-AIV sera, whereas changes at other five positions effected the binding of some sera only. This limited antigenic variation is useful for designing an AIV diagnostic test that uses the most abundant antigenic sequence. All AIV chicken sera investigated in this study and obtained by immunisation with low or highly pathogenic AIV contained M2e antibodies in significant titers, indicating that the M2e protein is immunogenic in natural infections. However, not all sera could be used for M2e antigenic differentiation. Some sera were highly cross-reactive with all M2e proteins/peptides, regardless of any amino acid changes. Other sera recognised only one immunodominant amino acid change (P10L, H, S), whereas the third group of sera were able to detect amino acid changes that occurred in four to six positions in the M2e. The findings contained in this thesis form the basis of further investigations into the use of M2e as a universal marker in AIV diagnostic test.
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ItemThe role of ADAM 12 in determining skeletal muscle development and intramuscular fat deposition in cattle( 2011)The research undertaken throughout this PhD thesis aimed to better understand the cellular mechanisms that influence development of skeletal muscle and intramuscular fat in beef cattle. In particular, I investigated the role of the extracellular matrix protease disintegrin and metalloprotease protein 12 (ADAM 12) in regulating muscle development and intramuscular fat deposition. These experiments sought to determine whether ADAM12 can be used as a potential modulator to manipulate these processes to increase muscling and intramuscular fat (IMF) deposition in cattle. My intital investigation sought to determine if variation in ADAM 12 expression in skeletal muscle was associated with different muscle phenotypes observed in cattle of various breeds. Primary skeletal muscle cells were isolated from Angus (high muscling), Hereford (moderate muscling) and Wagyu cross (X) Angus (low muscling) and grown in culture. The myogenic potential was found to differ between breeds of cattle whereby myoblasts from Angus cattle were found to proliferate at a greater rate than those of Hereford and Wagyu X Angus during early stages of growth (5-20 hours in culture) in vitro. The proliferation rates of myoblasts during early stages of culture in vitro were also found to be positively related to the liveweight of cattle. ADAM 12 gene expression was not found to be related to in vitro proliferation rates, suggesting that its influence on muscling was negligible, or that its expression in in vitro culture conditions was not reflective of its role in development of muscle tissue in vivo. The role of ADAM 12 during fat development was investigated using an in vitro model of adipogenesis, 3T3-L1 cells. ADAM 12 gene expression was knocked down over a 13-day developmental timecourse using Stealth™ RNAi. DNA, RNA and protein samples were collected to closely analyse the effect of ADAM 12 knockdown on proliferation and differentiation of 3T3-L1 cells. ADAM 12 gene knockdown was found to reduce the proliferation and differentiation of 3T3-L1 cells. However, in ADAM 12 knockdown cells enzyme markers of adipogenesis G3PDH (early adipogenic marker- glucose metabolism) and FAS (late adipogenic marker- lipid filling) were found to be greater. This was consistent with the phenotypic observation and morphometric analysis that the ADAM 12 gene knockdown treated cells displayed hypertrophy, with fewer lipids of greater size. Microarray analysis found the pathway most affected by ADAM 12 gene knockdown to be regulation of insulin-like growth factor by insulin‐like growth factor binding proteins which is thought to be responsible for the effect found on proliferation. Adipocytokines such as adiponectin and adipsin were also found to be significantly down‐regulated in ADAM 12 gene knockdown cells. Absence of these adipocytokines, in particular adiponectin, have previously been shown to cause hypertrophy of mature adipocytes due to alterations in insulin‐sensitivity and fatty acid oxidation. Thus ADAM 12 appears to be an important regulator through all stages of fat development (proliferation, differentiation and energy metabolism). Following in vitro experimentation, the role of ADAM 12 in skeletal muscle and intramuscular fat development in vivo was investigated. The m. longissimus dorsi and m. semitendinosus (LD and ST) muscles from five Angus and Brahman cattle were collected for histology and ADAM 12 gene expression analysis. Muscle fibre typing was used to determine if ADAM 12 expression patterns related to differences in skeletal muscle traits, such as muscling and IMF deposition, which are often influenced by proportions of the different muscle fibres. Consistent with other findings, the ST muscle was found to contain a higher proportion of glycolytic myofibres than the LD muscle which contained a greater proportion of oxidative myofibres. ADAM 12 gene expression was also found to be greater in the LD compared with the ST muscle. Fluorescent immunohistochemical (FIHC) staining for ADAM 12 and image analysis found that the relative amount of ADAM 12 distribution to also be greater in the LD muscle. Regression analysis found a positive relationship for the distribution of ADAM 12 against the proportion of type I myofibres (r2 = 0.86, p < 0.05) and a negative relationship for the distribution of ADAM 12 against type IIX myofibres (r2 = 0.82, p < 0.05). The gene expression and regression analysis suggest ADAM 12 is type I myofibre specific in cattle. This was further confirmed by FIHC staining of ADAM 12 with slow myosin heavy chain I (slow MHC-I) with which ADAM 12 co-localised. The type I myofibre specificity of ADAM 12 may be important in the regulation and maintenance slow myofibres in the LD and of IMF deposition, to which proportion or number of type I myofibres have been related to previously. Lastly, the expression profiles of ADAM 12 was investigated in skeletal muscle across a developmental time-course in two cattle breeds with extreme muscle phenotypes, Piedmontese X Hereford (high muscling) and Wagyu X Hereford (high marbling) cattle. Muscle biopsies (from LD muscle) were collected at 3, 12 and 25 months of age. PiedmonteseX cattle contained a greater proportion type IIX, type IIAX and type IIC myofibres compared with WagyuX cattle which had a higher proportion of the more oxidative type I and type IIA myofibres. Gene and protein expression of ADAM 12 was not different between breeds of cattle. However, ADAM 12 gene expression at 3-months of age was found to be positively related to feedlot average daily weight gain (kg/d) (r2 = 0.62, p < 0.05), liveweight at 25-months of age (kg) (r2 = 0.67, p < 0.05), hot carcass weight (kg) (r2 = 0.81, p < 0.05), retail beef yield (kg) (r2 = 0.65, p < 0.05) and eye muscle area (cm2 ) (r2 = 0.77, p < 0.05). These results suggest ADAM 12 is a candidate to be used to predict muscling and beef yield in cattle from a very young age. Analysis of ADAM 12 protein and gene expression found that expression of ADAM 12 in skeletal muscle was elevated at 3-months of age during pre-weaning muscle growth, with reduced expression at 12-months of age. Expression of ADAM 12 in skeletal muscle was upregulated again at 25-months of age, possibly related to its role in IMF deposition and marbling during this stage of growth in cattle. Thus ADAM 12 proves to be an important regulator during muscle development and IMF deposition in cattle and is a candidate to predict beef yield from a very young age. Findings from this PhD thesis show that ADAM 12 may be an important regulator of muscle and marbling development in cattle. The specific association of ADAM 12 with type I myofibres, and its role in signalling adipogenesis as outlined by this PhD thesis and work by others also suggest it is a candidate to increase marbling in cattle. Greater muscling and marbling by modulation of ADAM 12 may improve the quality and value of beef and efficiency of beef production (by increasing beef yield) to enhance competitiveness of Australia’s beef export.
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ItemA comparative study of the performance of New Zealand Friesian cross Holstein-Friesian cows in Victorian commercial dairy herds( 2011)This project combines the findings of two distinct research studies. The first being a comparative study involving nine commercial Victorian dairy herds, containing both New Zealand Friesian cross Holstein-Friesian (NZF×HF) and Holstein-Friesian (HF) cows. The second involved four farmer focus groups, collecting subjective information from three distinct groups of farmers on their breeding and semen selection decisions. The information gained in both parts of this study was designed to complement a previous crossbred study by Pyman (2007) and enable a holistic look at the use of a ‘different’ type of crossbred cow and whether this may be advantageous under typical Victorian, pasture based, seasonal calving conditions. The comparative field study collected data from nine Victorian herds over one single lactation. In total, 816 cows, including 529 Holstein-Friesians and 287 NZ Friesian×Holstein-Friesian cows were used to analyse reproductive performance and production based on a 305-day lactation. This enabled a comparison of breed differences to be assessed under pasture-based, seasonal calving conditions. The milk yield and reproductive performance parameters were adjusted for herd, age group, and interval from calving to mating start date. The Holstein-Friesians produced a higher volume of milk (203 litres, p=0.015) than the NZF×HF cows, but only after the exclusion of cows with lactations less than 250 days. The NZF×HF cows had higher concentrations of milk fat (0.17%, p≤0.0001) and protein (0.09-0.10%, p<0.001) than the HF cows, regardless of lactation length. Reproductive performance of the NZF×HF cows was superior to that of the HF cows in the nine herds with 14-week ICR (77.8 vs 69.0, p=0.027) and not in calf at 21 weeks (20.5 vs 29.8, p<0.019). Of the 23 farmers involved in the four focus groups, 13 were split calving, 9 were seasonal and 1 was calving year round. For the majority of the split calving herds and for the year round calving herd their current calving system “had chosen them”, rather than “them choosing the system”, as a result of their inability to get cows in calf. All crossbred and 5 of 9 LIC farmers included fertility in their breeding objectives, despite improvements over the last few years, while only 2 of 6 HF farmers included fertility, despite no farmers noting fertility improvements and inspite of the fact that half of these farmers were seasonally calving. In general, the farmers that had remained seasonal calvers, felt that this system was easier/simpler, provided a better lifestyle and was more cost effective, as they could rely more on seasonal pasture growth. All three groups of farmers wanted to breed cows that specifically fit their system, with an “ideal” cow being one “that you did not know was in the herd”. As a result, when selecting sires, all three groups of farmers were more concerned with non-production traits; including fertility, calving ease, temperament and udder, than production traits. Most farmers consulted bull catalogues, but relied on individual relationships to assist with breeding and sire selection. Farmers were generally happy with the sires available but all three groups felt that inbreeding was an issue, especially for the minor breed groups. Farmers that were crossbreeding seemed to use one of two broad systems to maintain their herd structure: 1) Non-Specific Crossing Structure: farmers used sires on an individual cow basis to maintain a cow’s size or to maintain a cow within the herd. 2) Specific Crossing Structure: farmers used sires (three or four way cross) in a predetermined order to maximise hybrid vigor. Overall the study was able to show that daughters of NZF sires, crossed with Victorian HF’s, were better able to get in calf and were less likely to be non-pregnant at the end of joining, than Victorian HF cows. Given that fertility was the main driver “forcing” farmers to adopt split calving systems and was also found to be far more important than production, in influencing farmer’s breeding objectives and semen selection decisions, it is likely that this crossbred may suit pasture-based, Victorian farmers, especially those wishing to maintain a 12 month calving interval.
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ItemCharacterization of the chicken and duck response to H5N1 avian influenza infection( 2011)Avian influenza viruses are increasingly widespread in poultry and show varied disease severity depending on their hemagglutinin and neuraminidase structure. Whilst many influenza viruses, such as the H5N3 subtype, are of low-pathogenicity, H5N1 influenza viruses result in rapid mortality that in poultry occurs in a matter of hours. The mechanisms of disease pathogenesis are still somewhat poorly understood. Ducks often appear asymptomatic, with few strains of H5N1 causing severe pathogenesis. The gradual progression of infection in ducks contrasts the rapid nature of infection in chickens and it is not clear how this difference in virulence comes about. One possibility is that the innate immune response in chickens and ducks varies during infection and this may be critical to the clinical outcome. With this in mind, we investigated the expression of several key proinflammatory cytokines following infection of chickens and ducks with highly-pathogenic H5N1 and low-pathogenic H5N3 influenza virus. Two H5N1 strains, A/Muscovy duck/Vietnam/453/2004 (Vt453) and A/Duck/Indramayu/BBVW/109/2006 (Ind109) were compared, as well as the low pathogenic H5N3 A/Duck/Victoria/1462/2008 (Vc1462) strain. Intriguingly, in the chicken, H5N1 viruses caused fatal infections, a high viral load and increased production of proinflammatory molecules. Inflammatory molecules such as IL6, IFNγ, the acute phase reactant SAA and also NO inducing gene iNOS, were raised by up to 80 fold at 24 hours post infection. Meanwhile, infection with the Vc1462 H5N3 influenza strain induced a comparably low cytokine response in chickens. In contrast, ducks displayed only small changes in these cytokines and this only occurred later in the infection period. Only the Vt453 H5N1 strain caused mortality in ducks and this was associated with increased levels of cytokines, such as IFNγ and iNOS, as well as increased virus replication in the lung and heart. These observations support the belief that hypercytokinemia may contribute to pathogenesis in chickens, whilst the lower cytokine response in ducks may explain their resistance to disease and decreased mortality. Given the increased levels of inflammatory molecules and the observation that IL6, a pleitropic inflammatory gene, is highly upregulated in the sera of H5N1 infected patients, it appeared likely that IL6 had an impact on hypercytokinemia induced disease severity. Since IL6 signalling results in increased levels of downstream inflammatory molecules, potentially triggering hypercytokinemia, we aimed to suppress the levels of IL6 during H5N1 infection. To investigate whether a more moderated IL6 response may improve the severity of H5N1 infection in chickens, we aimed to inhibit the signalling of IL6 using Madindoline-A (Mad-A) and Galielallactone (Gal). Following H5N1 infection, chickens treated with Mad-A/Gal inhibitors had reduced levels of IL6 and IL6-stimulated genes, such as SAA and AGP as well as lower NO and iNOS levels. However, even though this decreased IL6 response was associated with reduced viral titres, chickens did not appear to have increased survival. Nevertheless, these studies provide insight into the role possible therapeutics could play to target inflammation and improve the immune response during H5N1 infection. Therefore, examining the key components of the inflammatory response during H5N1 induced hypercytokinemia in chickens and ducks may help us understand how to ameliorate the initial hyperinflammatory response and prolong survival so that an adaptive response which alleviates viral replication can commence.
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ItemCharacterization of the eg95 gene family in Echinococcus granulosus G6 genotype: implications for the efficacy of the EG95 vaccine against genotypes other than G1( 2011)Cystic echinococcosis (CE) in humans and livestock animals is caused by infection with the cestode parasite Echinococcus granulosus. The parasite has a worldwide distribution, causing human morbidity and mortality as well as economic losses. E. granulosus presents significant intraspecific variability and several different genotypes have been described varying with respect to host preference and other biological characteristics. The G1 genotype is the variant most commonly associated with human infections worldwide, with genotype G6 also causing a significant proportion of human CE cases. A recombinant vaccine, designated EG95, has been developed for use in animals to interrupt the cycle of transmission of E. granulosus, with a view to decreasing the transmission of the parasite to humans. The vaccine is based on an antigen expressed in the oncosphere of E. granulosus. EG95 is encoded by members of a family of closely related genes in the G1 genotype. Potential variability in the protein target of the EG95 vaccine in different genotypes of E. granulosus could lead to parasites of genotypes other than G1 being insusceptible to the vaccine. Currently there is no reliable information about the likely efficacy of the EG95 vaccine against genotypes other than G1. The principal aim of the research described in this thesis was to provide information about the variability of the eg95-related gene(s) and their predicted protein products in the G6 genotype of E. granulosus, and other genotypes, also providing data that could be used for assessment of the antigenicity of the EG95-related proteins in each genotype. Using genomic DNA cloning techniques, seven members of an eg95 –related gene family were characterised from the G6 genotype of E. granulosus. Three proteins are predicted to be encoded by these genes, with two genes considered to be pseudogenes. Investigations were undertaken to determine the ability of two of theEG95-related proteins, encoded by genes in the G6 genotype, to react with specific antibodies in the sera of sheep vaccinated with EG95 and shown to be protected against a challenge infection with the G1 genotype. Proteins encoded by genes in the G6 genotype displayed only limited antigenic cross-reactivity with the current EG95 vaccine antigen, suggesting that the current vaccine may not protect animals against an infection with parasites of the G6 genotype. Data presented in this thesis provides the information that would enable a G6 genotype-specific vaccine to be developed against E. granulosus, should this be considered a desirable addition to the available tools for control of CE transmission.
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ItemVascular remodelling and mast cell changes in a sheep model of chronic asthma( 2011)Remodelling of the bronchial vasculature is a recognised disease feature which contributes to the pathogenesis of chronic asthma. Similarly, mast cells are known to play critical roles in allergic airway disease. Research into these areas of asthma pathophysiology can benefit greatly from the use of sheep. This is because the microcirculation, and the distribution and phenotype of mast cells throughout the sheep’s small and large airways are similar to their respective counterparts in human airways. In contrast, the smaller airways in mice do not have an active mast cell component nor do they have an extensive microcirculation, making these species less attractive for studying these aspects of asthma. This thesis uses a sheep model of asthma to characterise the nature and extent of vascular remodelling and changes in mast cell density following repeated exposure to house dust mite (HDM) allergen. Until now, these important airway changes have not been extensively characterised in the sheep model. The thesis aims to correlate these changes with local airway function, and to determine whether they can be inhibited in vivo by administering, a selective inhibitor of the calcium-activated potassium channel KCa3.1, which is known to impede mast cell activation. Initially, a segmental allergen challenge approach was used, whereby sheep sensitised to HDM were given weekly infusions of 1 mg HDM in solution to spatial separate lobes of the lung for different durations. Lung function data shows that repeated HDM allergen exposure results in an increased resting airway resistance, development of bronchial hyperresponsiveness, and acute allergen-induced bronchoconstriction. Morphometric analyses of frozen airway tissue sections revealed that the airways of sheep chronically exposed to HDM allergen exhibit an increased mast cell density and also undergo vascular remodelling. Increases in mast cell density were observed following 16 weeks of repeated allergen exposure, while blood vessel density and vascularity increased with 24 weeks of exposure. Continual allergen exposure was needed to maintain the increased density of mast cells, with numbers decreasing following 12 weeks of allergen withdrawal. Vessel density, however, remained elevated without continual antigenic challenge. There was no correlation between lung function and increased airway vascularity; however, mast cell numbers in HDM-challenged airways correlated with improved airway responses to allergic and non-allergic stimuli. Senicapoc, is an inhibitor of KCa3.1 which has been shown to affect mast cell activation and migration, as well as vascular smooth muscle proliferation. Hence, Senicapoc treatment could potentially prevent vascular remodelling and mast cell-mediated changes in asthma. To further elucidate the role of KCa3.1 in asthma it was ascertained whether Senicapoc administration would influence function, mast cell and vascular remodelling changes in HDM challenged sheep. Twice daily oral administration of Senicapoc for a period of 14 weeks was able to significantly reduce airway responses to allergic and non-allergic stimuli, as well as prevent increases in resting airway resistance following repeated aerosolised challenges of HDM to the whole lung. KCa3.1 blockade did not prevent an increase in airway mast cell density in HDM-challenged sheep; however, it did prevent a significant increase in blood vessel density and resulted in a decrease in mean vessel size. Overall, results presented in this thesis show the usefulness of the sheep model for studying vascular and mast cell changes in small airways, and for examining a therapeutic intervention approach to impede these changes. It will be interesting to see whether intervention strategies derived from this thesis can be applied clinically to treat asthma.