Identification of Cowdria ruminantium proteins that induce specific cellular immune responses
- Authors: Van Kleef, Mirinda
- Date: 2002
- Subjects: Ruminants--Pathogens Heartwater
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4061 , http://hdl.handle.net/10962/d1004373
- Description: Cowdria ruminantium (Cowdria) is an obligate intracellular pathogen that causes heartwater in ruminants. Cellular immunity and the type I cytokine IFN-γ have been implicated in protective immunity to heartwater. The aim of this thesis was to identify proteins of the Welgevonden isolate of Cowdria that induce lymphocyte proliferation and IFN-γ production. Differential centrifugation was found to be the simplest and most efficient method of Cowdria purification. Cowdria organisms were fractionated into their constituent proteins of between 11 and 168 kDa by continuous flow electrophoresis. The resulting fractions were tested for their ability to stimulate lymphocyte proliferation in vitro. In an attempt to simulate the natural infective process, peripheral blood mononuclear cells (PBMC) were obtained from two cattle rendered immune by infection and treatment and assayed in proliferation assays with the proteins fractions. In a parallel study, four cattle were immunised with inactivated Cowdria to determine whether their lymphocytes responded similarly. Cowdria-specific proliferation was detected for only a brief period after immunisation by infection with live organisms. This response was only detected again two to three years later. In contrast, PBMC from animals immunised with inactivated organisms were continuously responsive for at least three years. Only Cowdria proteins with molecular masses of 11, 12, 14 to 17 and 19 to 23 kDa induced proliferative responses in PBMC obtained from all six animals. Cell surface phenotypic analysis of Cowdria specific T-cell lines indicated that CD4⁺ lymphocytes were enriched over time with a concomitant increase in antigen-specific proliferation and IFN-γ production. Proteins of molecular masses 13 to 18 kDa induced CD4⁺ lymphocyte proliferation and IFN-γ production by T-cell lines from all the animals tested. Antibodies raised in a chicken and in rabbits to these low molecular weight proteins had low titres and specificity. Two-dimensional electrophoresis indicated that proteins within a single molecular weight range comprised several components with different pIs, revealing the complexity of the Cowdria proteome. This complicates the search for potentially protective antigens. Nevertheless, since they cause proliferation and IFN-γ production by lymphocytes from immunised cattle, these low molecular weight proteins merit further investigation as potential vaccine antigens. , Author: Mirinda van Kleef neé Rossouw
- Full Text:
- Date Issued: 2002
- Authors: Van Kleef, Mirinda
- Date: 2002
- Subjects: Ruminants--Pathogens Heartwater
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4061 , http://hdl.handle.net/10962/d1004373
- Description: Cowdria ruminantium (Cowdria) is an obligate intracellular pathogen that causes heartwater in ruminants. Cellular immunity and the type I cytokine IFN-γ have been implicated in protective immunity to heartwater. The aim of this thesis was to identify proteins of the Welgevonden isolate of Cowdria that induce lymphocyte proliferation and IFN-γ production. Differential centrifugation was found to be the simplest and most efficient method of Cowdria purification. Cowdria organisms were fractionated into their constituent proteins of between 11 and 168 kDa by continuous flow electrophoresis. The resulting fractions were tested for their ability to stimulate lymphocyte proliferation in vitro. In an attempt to simulate the natural infective process, peripheral blood mononuclear cells (PBMC) were obtained from two cattle rendered immune by infection and treatment and assayed in proliferation assays with the proteins fractions. In a parallel study, four cattle were immunised with inactivated Cowdria to determine whether their lymphocytes responded similarly. Cowdria-specific proliferation was detected for only a brief period after immunisation by infection with live organisms. This response was only detected again two to three years later. In contrast, PBMC from animals immunised with inactivated organisms were continuously responsive for at least three years. Only Cowdria proteins with molecular masses of 11, 12, 14 to 17 and 19 to 23 kDa induced proliferative responses in PBMC obtained from all six animals. Cell surface phenotypic analysis of Cowdria specific T-cell lines indicated that CD4⁺ lymphocytes were enriched over time with a concomitant increase in antigen-specific proliferation and IFN-γ production. Proteins of molecular masses 13 to 18 kDa induced CD4⁺ lymphocyte proliferation and IFN-γ production by T-cell lines from all the animals tested. Antibodies raised in a chicken and in rabbits to these low molecular weight proteins had low titres and specificity. Two-dimensional electrophoresis indicated that proteins within a single molecular weight range comprised several components with different pIs, revealing the complexity of the Cowdria proteome. This complicates the search for potentially protective antigens. Nevertheless, since they cause proliferation and IFN-γ production by lymphocytes from immunised cattle, these low molecular weight proteins merit further investigation as potential vaccine antigens. , Author: Mirinda van Kleef neé Rossouw
- Full Text:
- Date Issued: 2002
Bacterial interaction in hide biodeterioration with special reference to selected Clostridium species
- Authors: Thompson, Gillian Ann
- Date: 1995
- Subjects: Hides and skins -- Preservation Aerobic bacteria Pseudomonas aeruginosa Clostridium Halobacterium
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4041 , http://hdl.handle.net/10962/d1004102
- Description: Animal hides are the basic raw material of the leather industry and they undergo rapid putrefaction unless "cured". This study investigated the role and interactive effects of three selected bacteria, Pseudomonas aeruginosa. Clostridium histoly ticum and Clostridium sporogenes in in-situ cattle hide degradation using a model system set up for the purpose. The system consisted of 3cm diameter hide pieces contained in sealed jars and sterilised by ethylene oxide to remove resident microbes and inactivate autolytic tissue enzymes. The inocula were prepared either as individual cultures or as combinations of two inocula or all three inocula. Degradative changes during storage at 30°C were measured for up to 8 days using ten different parameters. Initial trials confirmed that the selected inocula were readily isolated from raw hides and could outcompete resident populations to produce putrefactive decomposition. Growth rates and enzyme profiles of the organisms and the effects of nutrients and reductants on their relative denaturative effects were used to standardise the system. Trials on the effects of ethylene oxide indicated the suitability of the method for hide and collagen sterilisation. The findings of in-situ trials with the selected inocula confirmed previous studies of protein putrefaction in that a bacterial succession was evident involving aerobic proteolytic bacteria, micro-aerophilic proteolytic bacteria and strictly anaerobic amino acid degrading bacteria. However, this study showed that the micro-aerophilic collagenase producing C. histolyticum degraded hides at a far greater rate when inoculated on its own than when in the presence of either or both of the other two inocula. It also demonstrated a bacterial antagonism between the two clostridia in which C. sporogenes prevented degradative changes occurring for up to 4-6 days possibly due to cysteine production by C. sporogenes. These findings have implications for hide preservation since maintenance of aerobic conditions and suppression of spore outgrowth could be used to delay growth of collagenase producing clostridia. The use of C. sporogenes as a biocontrol agent is also postulated. The model system was also used to examine salted hides during storage and these studies indicated that Halobacteriaceae do not produce collagenase but that inadequately salted hides could possibly be subject to degradation by delsulfovibrios.
- Full Text:
- Date Issued: 1995
- Authors: Thompson, Gillian Ann
- Date: 1995
- Subjects: Hides and skins -- Preservation Aerobic bacteria Pseudomonas aeruginosa Clostridium Halobacterium
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4041 , http://hdl.handle.net/10962/d1004102
- Description: Animal hides are the basic raw material of the leather industry and they undergo rapid putrefaction unless "cured". This study investigated the role and interactive effects of three selected bacteria, Pseudomonas aeruginosa. Clostridium histoly ticum and Clostridium sporogenes in in-situ cattle hide degradation using a model system set up for the purpose. The system consisted of 3cm diameter hide pieces contained in sealed jars and sterilised by ethylene oxide to remove resident microbes and inactivate autolytic tissue enzymes. The inocula were prepared either as individual cultures or as combinations of two inocula or all three inocula. Degradative changes during storage at 30°C were measured for up to 8 days using ten different parameters. Initial trials confirmed that the selected inocula were readily isolated from raw hides and could outcompete resident populations to produce putrefactive decomposition. Growth rates and enzyme profiles of the organisms and the effects of nutrients and reductants on their relative denaturative effects were used to standardise the system. Trials on the effects of ethylene oxide indicated the suitability of the method for hide and collagen sterilisation. The findings of in-situ trials with the selected inocula confirmed previous studies of protein putrefaction in that a bacterial succession was evident involving aerobic proteolytic bacteria, micro-aerophilic proteolytic bacteria and strictly anaerobic amino acid degrading bacteria. However, this study showed that the micro-aerophilic collagenase producing C. histolyticum degraded hides at a far greater rate when inoculated on its own than when in the presence of either or both of the other two inocula. It also demonstrated a bacterial antagonism between the two clostridia in which C. sporogenes prevented degradative changes occurring for up to 4-6 days possibly due to cysteine production by C. sporogenes. These findings have implications for hide preservation since maintenance of aerobic conditions and suppression of spore outgrowth could be used to delay growth of collagenase producing clostridia. The use of C. sporogenes as a biocontrol agent is also postulated. The model system was also used to examine salted hides during storage and these studies indicated that Halobacteriaceae do not produce collagenase but that inadequately salted hides could possibly be subject to degradation by delsulfovibrios.
- Full Text:
- Date Issued: 1995
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