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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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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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.
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ItemCestodes of the order Trypanorhyncha Diesing, 1863 from South-east Asia and Australia( 2013)The objective of the present thesis was to carry out a detailed morphological study of trypanorhynch cestodes from the Australasian and Indo-Malayan regions, in order to increase the knowledge of the species diversity of this particular group of tapeworms. Special focus was thereby paid to four key genera, namely Prochristianella Dollfus, 1946, Dollfusiella Campbell & Beveridge, 1994, Otobothrium Linton, 1890 and Parachristianella Dollfus, 1946. Based on newly collected material from taxonomic surveys in Borneo and Australia, it was possible to provide detailed taxonomic descriptions of species new to science, redescriptions of already described species and/or new information on host range and locality records. The geographical distribution of 35 trypanorhynch species from the four key genera that occur in South-east Asia and/or Australia was assessed and possible ecological factors, such as the host specificity and temperature preference, which may have impacts on the distributional patterns were discussed. In conclusion, the present thesis has contributed significantly to our knowledge of the species diversity of trypanorhynchs from Australia and Borneo and furthermore increased the knowledge on the geographical distribution and host range of selected trypanorhynch taxa.
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ItemThe role of Mannheimia species in ovine mastitis( 2013)Mastitis is a major animal health and welfare concern in the dairy, wool and meat sheep industries. Previous studies on ovine mastitis have predominantly focused on dairy breeds, so there is limited information on this disease in non-dairy production systems, with little known about the risk factors for intramammary infection due to the difficulty and cost of data collection. It has been suggested for some time that this disease has an adverse effect on productivity and profitability of wool and/or meat flocks. Therefore the studies described in this thesis aimed to examine subclinical and clinical mastitis in non-dairy sheep production industries. Data on individual ewe somatic cell counts, the bacteriological status of the udder, the sire breed, the year of lambing, the number of lambs born and weaned, the duration of lactation and the lamb weaning weight were collected at the time of weaning in a mixed-bred flock. This data was used to investigate the role of different determinants on ovine subclinical mastitis and lamb weaning weight. This is the largest study of its type that has been performed in meat and wool breed ewes in Australia, with over 1,500 individual animals assessed at weaning over a two-year period. The results suggested a significant association between the breed of the dam’s sire, the year of lambing and ovine subclinical mastitis, and a high prevalence of coagulase negative staphylococci in ewes with subclinical mastitis. A previous survey of Poll Dorset flocks across Australia suggested a high rate of acute and peracute clinical mastitis caused by Mannheimia species. Mannheimia haemolytica is known to be an important cause of intramammary infection in sheep. Phylogenetic analyses of two housekeeping genes, the 16s rRNA gene and rpoB, and phenotypic tests identified Mannheimia glucosida as a significant cause of ovine mastitis, in addition to M. haemolytica. Analysis of the restriction endonuclease cleavage patterns of the isolates obtained from ovine mastitis demonstrated the possibility of horizontal transmission. To test the potential role of lambs in transferring Mannheimia species to the ewe’s udder, the restriction endonuclease cleavage patterns of isolates obtained from nasopharyngeal swabs were compared with those obtained from cases of mastitis. The results suggested that the M. haemolytica isolates obtained from mastitis were more clonal than those obtained from the nasal swabs. However, some nasal isolates within both Mannheimia species had restriction endonuclease cleavage patterns identical with those obtained from mastitis, indicating that lambs might have a role in transferring these organisms to the udder. More clonality was observed between the M. glucosida isolates than between M. haemolytica isolates. Comparison of the patterns of the M. glucosida isolates associated with ovine mastitis with some of their other genotypic characteristics indicated close correlation between the restriction endonuclease cleavage patterns of the isolates and their rpoB and 16s rRNA gene sequences, while M. haemolytica isolates with identical rpoB gene sequences could have different restriction endonuclease cleavage patterns. The sequences of the leukotoxin genes of a number of distinct strains of M. haemolytica and M. glucosida were determined and a higher overall nucleotide distance was found between the leukotoxin gene sequences of M. haemolytica isolates than between M. glucosida isolates. Comparison of the cytotoxicity of leukotoxins from M. haemolytica and M. glucosida suggested that the leukotoxin from M. glucosida strains had greater activity against ovine neutrophils than that of M. haemolytica. The neutralising capacities of polyclonal antisera against different leukotoxins were tested against homologous and heterologous leukotoxins. Each antiserum had a neutralisation titre of 32 against its homologous leukotoxin, while the titre differed when tested against heterologous toxins. The antigenic similarity coefficient calculated from the cross neutralisation data revealed that the leukotoxins from the two M. haemolytica isolates had the least similarity among the strains tested, while leukotoxin from M. glucosida had highest similarity to those from the M. haemolytica isolate I1 and a haemolytic M. ruminalis. The studies described in this thesis have helped in developing our understanding of clinical and subclinical ovine mastitis and of the important role that species within the genus Mannheimia have in this disease, despite the general belief that Staphylococcus aureus is the predominant cause of clinical mastitis in sheep. The results from cross neutralisation analysis assay suggest that M. glucosida may be the best candidate for a monovalent vaccine to aid in control of ovine mastitis.
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ItemFunctional analysis of Schistosoma mansoni egg proteins( 2013)Schistosomiasis is a prevalent, socioeconomically important disease of humans worldwide. Despite major efforts, there is only one drug routinely used for effective treatment and no vaccine to combat schistosomiasis. The overall objective of this thesis was to functionally characterise some key proteins from eggs of Schistosoma mansoni and explore their involvement in the disease process in vivo. In spite of the utility of double-stranded RNA interference (RNAi) in S. mansoni, methods used to date have had severe limitations. Based on evidence from the literature (chapter 1), the lentiviral delivery of artificial miRNAs (shRNAmir) was likely to circumvent these limitations. The main aims of this thesis were to: (1) construct a lentiviral vector for the delivery of a miRNA cassette in S. mansoni, (2) validate the accessibility of the miRNA-pathway in S. mansoni for selective gene knock-down, and (3) investigate the role of selected target molecules in immune responses and immunopathology in mice. This thesis demonstrated, for the first time, successful lentiviral transduction of S. mansoni (chapter 3), enabling the targeting of dividing and non-dividing cells. Direct polymerase chain reaction (PCR)-based detection of the transgene confirmed the presence of provirus in the parasite’s genome. In addition, the transgene was transcriptionally active under the control of a mammalian (CMV) promoter, allowing the validation of the lentiviral system in mammalian cells prior to application to the parasite. Furthermore, this is the first study to show that the miRNA pathway is functional in S. mansoni and can be utilised effectively for RNAi studies (chapter 4). Semi-quantitative PCR results showed transcriptional down-regulation of target genes. A first screen for off-target effects indicated a high specificity of the shRNAmir sequences designed specifically to the target genes. Importantly, lentiviral transduction and shRNAmir-induced transcriptional gene knock-down had no effect on the vitality or maturation of larval eggs stages, enabling in vivo studies. Experimental studies in mice (chapter 5) demonstrated that this newly established technique can be employed effectively for the identification and characterisation of molecules involved in host responses. While Th2 cells are indispensable for the regulation of granuloma formation, the key mechanism underlying S. mansoni egg-induced pathological changes related to the secreted immune-modulating protein omega-1. Furthermore, a central role of macrophages and tissue cells in the initiation of pathological changes was observed, deflecting the focus from CD4+ T cells alone. Therefore, this study substantially enhances our understanding of processes leading to granulomatous disease in response to S. mansoni eggs.
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ItemInvestigation of the osteochondral response to injury in racing Thoroughbreds( 2013)For prevention and appropriate treatment of subchondral bone (SCB) injuries a thorough understanding of the prevalence, pathogenesis and repair mechanisms of the condition are mandatory. Palmar/plantar osteochondral disease (POD) is a SCB fatigue injury in racehorses that occurs with a high prevalence. There is limited quantitative data on the role of microfractures, osteocyte death, modelling, remodelling, loading intensity and unloading on SCB injury (SCBI) and repair. The association between articular cartilage and SCB injuries in this condition is not well understood. Qualitative associations have been established between SCBI, modelling and remodelling and it has been speculated that modelling and remodelling may contribute to injury by creating brittle and/or porotic bone or stiffness gradients within bone. To better understand the pathogenesis of SCBI and its association with loading intensity, the injury and bone response to injury (resorption and formation) were quantified and their associations with recent and life-time exercise intensity were investigated in a cross-sectional study. It was also intended to study if resting from training had an effect on removal of microdamage from SCB. It was hypothesized that SCBI occurs prior to cartilage damage, is consistent with bone fatigue and therefore is associated with the history of exercise duration and intensity. According to second hypothesis the response of bone to injury could contribute to the pathogenesis of SCBI. Fore- and hind limbs from two separate groups of Thoroughbred racehorse cadavers were collected at post-mortem and severity of POD was graded 0-3. Forelimbs from 38 horses were examined using high-resolution peripheral quantitative CT, back scattered electron microscopy and histomorphometry. Also, hind limbs from 46 horses were collected for histomorphometry using cryosections, 1 µm thick resin embedded sections and transmission electron microscopy. Of the forelimb cohort 65.8% of horses had some degree of POD and 30.7% of horses had at least one Grade 2-3 lesion. In hind limbs 57.8% had at least one POD lesion of any severity and 24.4% had grade 2-3 lesions. The majority of horses had some degree of SCB microfracture or osteocyte loss. SCB microfractures were present in all grades of the disease whereas significant cartilage changes were limited to grade 3 lesions. SCB lesions were characterised by accumulations of microfractures, osteocyte loss in the affected area, sclerosis of the surrounding trabecular bone, focal resorption of superficial SCB, and in grade 3 lesions, articular surface attrition (collapse). Short term rest was associated with greater focal bone resorption which in some cases was concentrated in the immediate SCB leading to loss of support of the overlying cartilage and subchondral plate. The severity of macroscopic and microscopic lesions correlated with age and indicators of the accumulation of training distance. In conclusion, SCB appears to respond to fatigue injury in a similar manner to cortical bone i.e. sclerosis in the surrounding bone accompanies localised resorption at the site of damage. It is unclear whether focal resorption at the site of fatigue injury contributes to further bone fatigue however, bone resorption accelerates when horses are rested from training and this appears to contribute to collapse of the articular cartilage into the SCB. SCB was the primary site of the injury and articular cartilage changes were only associated with severe injuries where SCB attrition had occurred. A better understanding of the effects of training and rest on bone turnover rate is required to reduce the accumulation of SCB fatigue damage that occurs throughout the careers of equine athletes.