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The experiments reported in this dissertation explored the effects of selection for antibody response on other immunological measures and on production traits. The role of thyroid hormones in antibody response was also studied. Selection for high (HA) and low (LA) antibody response to sheep erythrocytes altered subclasses of antibodies in different ways. In line LA antibody response was primarily mercaptoethanol-susceptible (IgM), while the line HA response was primarily mercaptoethanol-resistant antibody (IgG). Sublines of HA and LA were developed with all possible combinations of major histocompatibility complex haplotypes B¹³ and B²¹. An experiment was conducted to test Marek’s disease resistance of these haplotypes in line LA. Mortality from a natural exposure was high for all three groups, and there was no difference among haplotypes. Correlated responses of growth and reproductive traits in lines HA and LA were due to genetic correlations with antibody response. These genetic correlations were generally negative and are suggestive of differential allocation of resources. Phenotypic correlations were generally very small. Changes in allelic frequencies at alloantigen systems were also observed in response to selection. Experiments designed to study the role of thyroid hormones on antibody responses showed no direct relationship. Chickens from lines HA and LA fed thiouracil exhibited hypothyroidism but did not differ from controls in antibody response. Differences in thyroid hormone concentrations between lines of dwarf and non-dwarf White Rocks selected for high and low juvenile body weight bore no relationship to differences in antibody responses. / Ph. D.

Infectious bursal disease virus (IBDV) has a tropism for the lymphoid tissue of poultry and infects actively dividing and differentiating B-lymphocytes in the bursa of Fabricius. This results in a high mortality rate and severe immunosuppression. These immunodepressed chickens are highly susceptible to secondary infections and have a reduced capacity to respond to vaccination. The principal method to control IBDV is through extensive vaccination using either attenuated live or inactivated IBDV vaccines. However, in recent years due to the emergence of new virulent strains, risk of reversion to pathogenicity, cost considerations and intervention by maternal antibodies, the effectiveness of these vaccines in the veterinary field is being reduced. An alternative approach to prevent infection is by interfering with the binding of IBDV to its receptor protein on the surface of bursal cells. Hence this study was undertaken on the characterisation of a possible IBDV receptor on bursal membranes. Infectious bursal disease virus was isolated from infected bursal tissue using CsCl density gradient centrifugation and visualised with Tris-Tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transmission electron microscopy. Following purification of double stranded RNA from infected bursal tissue and commercially available live IBDV vaccines, a polymerase chain reaction (PCR)-based diagnostic assay based on sequences from the highly conserved viral protein (VP2) region was performed. The presence of the virus was demonstrated by the amplification of a 150 bp band in 2% agarose and 15% nondenaturing PAGE gels. The correctness of this product was confirmed byrestriction digestion with a specific restriction endonuclease (BamHI) that resulted in the predicted digestion fragments of 93 and 57 bp. Following preparation of bursal membrane proteins from uninfected bursal tissue, using sucrose density gradient centrifugation, isolation of IBDV receptor protein was carried out by immobilising IBDV on a Sepharose 4B chromatography matrix. After affinity purification, two prominent protein bands around 40 kDa were visualised using a silver stained Tris-Tricine SDS-PAGE gel. Previous work in this laboratory identified two possible IBDV receptor proteins on bursal membranes of 32 and 40 kDa. Antibodies against peptide sequences derived from the 32 kDa receptor protein were raised in rabbits in the present study. These anti-IBDV receptor peptide antibodies recognised the affinity purified native 40 kDa IBDV receptor proteins in an enzyme-linked immunosorbent assay (ELISA). However, due to the possible epitope denaturation by the reducing treatment buffer prior to Tris-Tricine SDS-PAGE such as SDS and 2-mercapthethanol or detergent (Na-deoxycholate) used during the affinity purification of the IBDV receptor protein, the anti-IBDV receptor peptide antibody did not recognise the receptor protein on a western blot. An inhibition assay was performed in an ELISA format by coating the 40 kDa IBDV receptor protein to see if the anti-IBDV receptor peptide antibody could inhibit IBDV binding to the receptor. The result showed that the anti-IBDV receptor peptide antibody effectively inhibited the binding of IBDV to the receptor. This result could pave the way for reducing IBDV infection by interfering at the viral attachment stage prior to crossing the bursal cell membrane barrier. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2004.

published_or_final_version / abstract / toc / Zoology / Master / Master of Philosophy

Vaccination against Marek's disease (MD) is greatly enhanced by host genetic resistance. Genes of the growth hormone (GH) axis have been reported to affect the ontogeny and effector functions of cells of the immune system. Two strains of White Leghorn chickens bred for contrasting homozygous markers in the GH and GH receptor (GHR) genes were challenged with MDV. Contrasts for the significant interaction between marker genotype and tissue indicated that the GH/GHR marker genotype caused a shift in the distribution of the viral load in lymphoid tissues in the two strains. The analysis suggests that genetic variations in genes of the GH axis may differentially affect the host response to MDV replication in lymphoid tissues. Regarding the early time course of infection, at day 6 the viral load was highest in the thymus, while at day 10, it was highest in the spleen, indicating that the virus may have accumulated in the spleen or was continuing to replicate in this tissue. (Abstract shortened by UMI.)

Infectious bursal disease virus (IBDV) is a chicken pathogen that infects the bursa of Fabricius, an organ involved in the development of the immune system in chickens. Infection by the virus leads to destruction of the bursa and immunosuppression. Infection by virulent strains may result in mortality. Current methods to combat the virus involve the use of vaccines. These are usually a mixture of live attenuated and oil inactivated virus. Variant strains of the virus are able to escape the vaccine-generated antibodies. In addition, the vaccines result in damage to the bursa. Identification of a receptor for IBDV could result in the development of either treatment for the virus or superior vaccines by interfering with the attachment of the virus to host cells. Several methods for identifying IBDV binding proteins from the membranes of cells from the bursa of Fabricius were examined. Affinity chromatography of IBDV binding proteins with a matrix consisting of IBDV cross-linked to Sepharose 4B allowed separation of a number of virus binding proteins. In contrast, virus overlay protein blot assay (VOPBA) and reversible cross-linking with 2-iminothiolane proved less; conclusive. Predominant proteins in the affinity-separated fraction were of 40 and 32 kDa. These were further examined by N-terminal amino acid sequencing of the whole protein and N-terminal sequencing of peptides produced by endoproteinase Lys-C digestion of the protein respectively. The 40 kDa protein showed homology with human synovial stimulatory protein involved in the formation of autoantibodies in rheumatoid arthritis. Virus was also shown to bind to a 440 kDa protein complex. This 440 kDa protein complex appeared to consist primarily of a 40 kDa protein when examined by reducing Tris-Tricine SDS-PAGE. Analysis of bursal membrane proteins by Western blots using sera from rheumatoid arthritis patients revealed interactions between several IBDV proteins and the antibodies from rheumatoid arthritis patients. Using serum from one of the five patients showed a strong interaction at approximately 80 kDa and a weaker interaction at approximately 40 kDa. This may indicate an immune reaction between a chicken homolog of the synovial stimulatory protein and antibodies in rheumatoid arthritis sera. The 32 kDa protein showed homology to a Pseudomonas fluorescens protein. A section of this sequence was amplified by PCR from chicken DNA and RT-PCR from chicken RNA using degenerate primers constructed from the established N-terminal amino acid sequences and chicken codon usage tables. The fragment produced upon amplification from chicken DNA and RNA did not correspond to the predicted size of 177 bp. In contrast, when the RT-PCR product was heated and snap cooled before examination by agarose gel electrophoresis, the product consisted of two fragments, one of approximately 400 bp in size and one of approximately 200 bp in size. The establishment ofthe 40 and 32 kDa chicken bursal membrane proteins as possible receptors for the virus could allow for the development of vaccines and/or treatment strategies for the virus. Treatment strategies or vaccines would be based on blocking of the interaction between IBDV and chicken host cells. Peptide mimics of the epitopes involved in such interactions could possibly achieve this. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 2000.

Marek's disease (MD) is a highly contagious and economically important disease in the poultry industry. It is caused by an oncogenic avian herpes virus. The ability of the virus to evolve into new strains is a continual threat. Vaccination, proper management and genetic resistance are required to completely eliminate the pathogen. The discovery of several markers associated with MD resistance shows that genetic selection for resistance is feasible. Our objective was to identify markers in QTL regions that are associated with MD viraemia. The markers analysed were in the ODC gene, the GH gene and two chemokine genes, all of which are candidate genes for immune responsiveness. A database in a commercial strain of White Leghorn chickens was created. Heterozygous males and homozygous females were identified. The offspring were challenged with MD virus and spleen and thymus samples were collected six days after infection. The viral titre was quantified using competitive PCR. The data was analysed using non-parametric statistics. We found that the paternal alleles of a Hindlll RFLP in the ODC gene were associated with differences in MD viraemia in one of the six sire families analysed. In addition, a Sacl RFLP located in the GH gene also segregated for alleles, which affected MD viraemia. The analysis of the ODC gene was extended to include a second RFLP at a Msp\ site. Together with the Hindlll RFLP it defines three different haplotypes. One genotypic class AB (Hindlll (+/-), Mspl (+/+)) was associated with low vireamia in the thymus and the genotype BB (Hindlll (-/-), Mspl (+/+)) with high viraemia in the spleen. The result suggests that genetic variations in the ODC and GH gene affect MD viraemia. However, we cannot exclude that the observed effects might be due to linkage disequilibrium with adjacent genes. In the latter case, chromosome 3 and chromosome 1, which harbour the ODC and GH gene respectively, must segregate for regions that affect viraemia. The markers identified in this analysis can be used in marker assisted selection.

No description available.

A model of growth and plasmid transfer between strains of Escherichia coli and Salmonella typhimurium was developed with reference to the literature. This was the organising principle for the collection of a complete set of in vitro life history parameters of one S. typhimurium and one E. coli strain. In the course of estimating these parameters two results of note were obtained. Fits of the Lotka-Volterra competition model were obtained for data on S. typhimuiurm growing in competition with E. coli. The first noteworthy discovery was the failure of this model to account for several characteristics of growth of these strains under competition. The growth rates of plasmid-bearing and plasmid-free strains were obtained. The second main result came from examination of the results of the growth rate data, which revealed that the cost to S. typhimuiurm 576 of bearing the resistance plasmid was low (4%). The model was also used to simulate the effect of antibiotic dose on the density of the donor, recipient and transconjugant populations over time. These simulations predicted that there would be a convex relationship between antibiotic dose and transconjugant density (i.e. that the density would first rise, then fall, with increasing dose). Following from this result, laboratory experiments and in vivo experiments in chickens were directed towards obtaining information on the relationship between these two variables. This convex relationship was not demonstrated within a single experiment, although some experimental environments produced an increase in transconjugant density with dose, and others, a decrease. Few transconjugants were formed in vivo. In order to investigate the low cost of resistance and low rate of in vivo transconjugant production, cost of resistance and plasmid transfer rate of this plasmid in several strain combinations of E. coli and S. typhimuiurm was evaluated.