published_or_final_version / abstract / Zoology / Master / Master of Philosophy

published_or_final_version / abstract / Zoology / Master / Master of Philosophy

published_or_final_version / abstract / Biological Sciences / Doctoral / Doctor of Philosophy

Monoclonal antibodies were developed against USDA challenge strain of infectious laryngotracheitis virus (ILTV). Indirect immunofluorescence test was used to detect antibodies in supernatants of hybridomas. Hybridoma cells were developed by fusing Sp 2/0 myeloma cells with spleen cells obtained from mice immunized four times with partially purified USDA challenge strain of infectious laryngotracheitis virus. The supernatant of three hybridomas, designated as 2D1D8, 2E11G2, 2C6C7 were found positive for antibody activity against USDA challenge strain of ILTV. Hybridomas producing antibodies were cloned by the limiting dilution method. All three monoclonal antibodies reacted with USDA challenge strain of ILTV, S 88 00224 strain of ILTV, and 86 1169 strain of ILTV in an indirect immunofluorescence test. None of the monoclonal antibodies reacted with avian adenovirus 301 or parrot herpes virus in an indirect immunofluorescence test. The monoclonal antibodies were isotyped, and all three monoclonal antibodies were found to be IgM. / Graduation date: 1993

published_or_final_version / Zoology / Doctoral / Doctor of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy

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.

Monoclonal antibodies (MCA) produced against infectious laryngotracheitis virus (ILTV) of chickens reacted in western blotting experiments with several different ILTV protein bands in the absence of tunicamycin which inhibits carbohydrate synthesis. Most of the MCA lost their reactivity in western blotting experiments when extracts of tunicamycin-treated ILTV CELC were used, suggesting their specificity for carbohydrate-based epitopes. In an indirect immunofluorescence test most of the MCA bound primarily to cytoplasmic antigens except some MCA which bound primarily to nuclear antigens. Additivity ELISA was also performed to study whether MCA are against the same epitope or different epitopes. The polymerase chain reaction (PCR) was developed as a diagnostic technique for detection of ILTV using primers made from a portion of the ILTV thymidine kinase gene. The 647-basepair amplified ILTV PCR product was labeled to create a non-radioactive, biotinylated DNA probe. Hybridization was performed using the probe to detect ILTV. Both PCR and hybridization detected ILTV, and neither hybridization nor PCR gave positive results with any other pathogen. Hybridization was specific for ILTV, However, slight hybridization occurred with CELC DNA when relatively relaxed conditions were used. In another experiment, diagnostic tests to detect ILTV in tracheas of experimentally-infected chickens, including the indirect fluorescent antibody test (IFAT), immunoperoxidase (IP), virus isolation (VI), histopathology, PCR, and hybridization, were performed and compared. Using virus isolation as a reference, the sensitivity and specificity of the tests were calculated. The IP test and IFAT performed better than any other test used in this study. / Graduation date: 1995

published_or_final_version / Biological Sciences / Doctoral / Doctor of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy