Veterinary Science - Theses
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ItemMolecular and functional characterisation of membrane proteins of Mycoplasma bovis( 2017)Mycoplasma bovis is a pathogen of considerable economic importance that has been associated with pneumonia, arthritis, mastitis and otitis in cattle, reducing productivity and causing or predisposing to mortality. The control of M. bovis infections has typically relied on antimicrobial therapy, which is expensive and has variable success. Understanding the basic biology of this pathogen and the mechanisms it uses for colonisation and survival could lead to development of cheaper and more effective control strategies. Although the complete genomic sequences of four strains of M. bovis have been available for some time, the use of these data to increase our understanding of the pathogenesis of M. bovis has been limited and the annotations of a large proportion of the genes in these sequences remain hypothetical. This thesis focuses on exploring the function of membrane–associated proteins of M. bovis, as they are likely to be most directly involved in interactions with the host. The studies utilised a combination of bioinformatic analysis, three-dimensional ab initio structural modeling and exploration of biochemical functions of purified recombinant proteins expressed from genes that were previously uncharacterised to attribute a range of roles to several of these proteins. The genes encoding these membrane proteins were first codon optimised by mutating the TGA codons to TGG to allow full-length expression of the proteins in E. coli. These proteins were expressed in E. coli JM109 and the soluble GST-tagged recombinant proteins were then purified by affinity chromatography and their immunogenicity established by Western immunoblotting. Monospecific antisera were raised against each recombinant protein and the growth inhibitory effects of the antisera were tested on running drop cultures of M. bovis. All the genes targeted were successfully expressed and purified, with the exception of the product of MBOVPG45_0318. The recombinant product of MBOVPG45_0353, MbLolA, MbFg-bA, MbFn-bA and MilA were recognised by antibodies in sera from calves experimentally infected with M. bovis, while antisera raised in rats against the recombinant product of MBOVPG45_0353, MbLolA and MilA-EF could inhibit the growth of M. bovis cells in vitro. Previous studies on M. bovis demonstrated that the amino terminal end of an immunogenic 226-kDa (P226) protein, encoded by milA (the full length product of which has a predicted molecular weight of 303 kDa) had lipase activity. The predicted sequence of MilA has glycosaminoglycan binding motifs, as well as multiple copies of a domain of unknown function (DUF445) that is also found in apolipoproteins. Recombinant regions of MilA bound to 1-anilinonaphthalene-8-sulfonic acid and a variety of lipids, with the amino-terminus binding more tightly. MilA was also able to bind ATP, suggesting a potential autotransporter function. Other ATP binding proteins identified in this study included the products of MBOVPG45_0305, MBOVPG45_0353 and MBOVPG45_0779, which may play important roles in cellular processes. The pathogenesis of disease caused by infection with mycoplasmas depends in part on interactions between their cellular components and the host’s extracellular matrix, which aid their adherence to host cells and thus colonisation and the subsequent development of disease. The results of this study identified novel proteins of M. bovis that bound heparin (the product of MBOVPG45_0232, MbFn-bA, MbLolA and MilA) and fibronectin (the product of MBOVPG45_0232, MbFn-bA, MbFg-bA and MbLolA). A novel fibrinogen binding protein of M. bovis (MbFg-bA) was also identified and the binding was shown to be regulated by divalent cations, with magnesium ions increasing binding, while calcium and manganese ions inhibited binding. MilA and MbFn-bA were shown to be surface exposed and accessible to the proteolytic effect of trypsin. MilA was shown to be post-translationally processed into 226-kDa and 50-kDa polypeptides, while MbFn-bA was detected as a 48-kDa polypeptide in all strains and also as the predicted full length 70-kDa polypeptide in some strains. Sequencing of mbFn-bA in several strains of M. bovis detected the deletion of the region encoding the carboxyl terminal 147 amino acids in the mbFn-bA of the PG45 type strain, which could account for the variation detected in immunoblots of MbFn-bA in these strains. The significance of M. bovis infection has become increasingly apparent in recent times and the increasing resistance of this bacterium to antimicrobials necessitates a search for better methods of control. Serological diagnosis is an alternative strategy for the control of M. bovis infection and offers the opportunity to separate infected from uninfected cattle. Current diagnostic assays are suboptimal necessitating the search for antigens that are specific for M. bovis and desirable for the development of such diagnostic assays. The studies described in this thesis have identified additional immunogenic membrane proteins of M. bovis, including the product of MBOVPG45_0353, MbFg-bA, MbFn-bA and MbLolA, that may warrant further analyses as possible diagnostic antigens. In conclusion, the results of these studies have increased our understanding of the functions of previously hypothetical membrane proteins of M. bovis. In some instances individual proteins were shown to have a number of functions and thus can be predicted to play multiple roles in the pathogenesis of disease caused by M. bovis. These results complement genetic studies that have identified genes required for colonisation and persistence in animals infected with M. bovis.
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ItemNipah viruses from Malaysia and Bangladesh: comparisons of viral shedding and pathogenesis, and investigations of transmission in the ferret model for infection( 2014)Nipah virus (NiV) has caused human disease with a high fatality rate in Malaysia, Singapore, India, and Bangladesh. Isolates from Malaysia (NiV-MY) and Bangladesh (NiV-BD) are associated with markedly different patterns of transmission in people. In Malaysia, symptomatic human infection was attributed to pig-to-human transmission. In Bangladesh, where outbreaks continue to occur on almost an annual basis, human-to-human transmission is a regularly identified pathway for infection. Despite observed epidemiological differences, no comparisons of NiV transmission have been reported in a mammalian infection model under controlled conditions. This project compared infections of NiV-MY and NiV-BD in ferrets (a human surrogate) with respect to tissue tropism, shedding and transmission. Following oronasal exposure to NiV-MY or NiV-BD, increased viral loads were observed in oropharyngeal secretions from NiV-BD-infected ferrets sampled over the course of infection, consistent with enhanced replication of NiV-BD at sites relevant to transmission via respiratory secretions. In a comparative time-course study, NiV in respiratory secretions was demonstrated to originate primarily from the lower respiratory tract at early time points following infection, and significantly higher viral loads were detected in respiratory tissues from NiV-BD-infected ferrets, compared to those of NiV-MY-infected ferrets. A transmission study was carried out to investigate whether these findings were reflective of increased transmissibility of NiV-BD, and to document the nature of contact required to achieve NiV transmission in ferrets. For both viral isolates, ferrets inoculated with respiratory secretions from infected donor ferrets were more likely to acquire infection than animals whose exposure was limited to cohabitation. Critically, exposure of ferrets to secretions from NiV-MY-infected donors resulted in similar outcomes of infection to those exposed to secretions of NiV-BD-infected donors. Findings from these studies suggest that, although NiV-BD appears to replicate to higher levels in respiratory tissues of infected ferrets, differences observed in natural NiV outbreaks are likely to be driven by environmental and/or host factors, rather than differences between viral strains.
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ItemMolecular pathogenesis of mycoplasma gallisepticum in chickens( 2010)Mycoplasma gallisepticum (MG) is an important poultry pathogen that causes respiratory disease and loss of production worldwide, and is currently controlled by vaccination with live attenuated vaccines. These vaccines have limitations as they vary in their pathogenicity, the protection they afford, and their transmissibility, but each has been shown to effectively reduce losses associated with challenge in the field. A live attenuated vaccine, ts-11, has been used for the control of MG in several countries. This strain infects for extensive periods, but is confined to the upper respiratory tract, resulting in life-long immunity after a single administration, but not colonisation of the air sacs or disease. However it is highly dose dependent and the flock antibody response is weak. GapA is the primary cytadherence molecule in MG and absence of GapA expression has been observed in the vast majority of cells in the ts-11 vaccine strain. The immunogenicity of a GapA+ MG ts-11 vaccine was investigated by eye-drop administration to specific pathogen free chickens. Chickens were monitored for antibodies against MG using the rapid serum agglutination (RSA) test, for air sac lesions and for damage to the tracheal mucosa before and after challenge. Birds vaccinated with GapA+ MG ts-11 were protected against clinical signs of disease following challenge with virulent MG and GapA+ MG ts-11 was non-pathogenic and more immunogenic than the currently available MG ts-11 vaccine. The capacities of GapA+ MG ts-11 to express an enzymatic marker and deliver a chicken cytokine were also examined. To achieve this, mycoplasma expression vectors were constructed; in which the Escherichia coli alkaline phosphatase (phoA) gene was fused to the chicken interleukin-6 (ChIL-6) gene with or without the cleavage signal sequence of MG vlhA1.9 and placed under the control of the MG ltuf promoter with a vlhA export signal sequence. The expression of PhoA was detected by colony and Western blotting using antibody specific for PhoA and the release of ChIL-6 in the media was detected by bioassay. Using a similar approach the gene for a heterologous protective antigen, the S1 glycoprotein of infectious bronchitis virus (IBV), and the chicken interleukin-6 (ChIL-6) gene were used to construct recombinant GapA+ MG ts-11 strains to evaluate the potential of ts-11 as a vaccine vector and thus to induce a protective immune response against other diseases of poultry. The capacity of GapA+ MG ts-11 expressing the S1 glycoprotein of IBV and ChIL-6 to induce protective immunity in the respiratory tract of chickens was assessed by eye- drop administration in SPF chickens, followed by challenge with the virulent IBV strain N2-75. Chickens were monitored for antibodies against MG using the RSA test and for antibodies against the S1 protein by Western blotting. After IBV challenge, birds vaccinated with recombinant GapA+ MG ts-11 vaccines were compared to birds vaccinated with parental strain of MG. Chickens vaccinated with GapA+ MG ts-11 expressing the S1 and releasing ChIL-6 had a detectable immune response against MG and were protected against damage by pathogenic IBV without any effect on weight gain.Thus the work reported in this thesis has identified a GapA+ MG ts-11 strain with potential as a vaccine candidate and an optimal enzymatic marker for foreign gene expression in MG and has shown that this can be used to develop recombinant respiratory tract vaccines for poultry.