No description available.

Thesis (MSc. (Vet. Trop. Diseases))--University of Pretoria, 2004. / Includes bibliographical references.

Thesis (MMedVet (Reproduction))--University of Pretoria, 2006. / Includes bibliographical references.

Thesis (MSc. (Veterinary Science))--University of Pretoria, 2006. / Includes bibliographical references.

Please read the abstract in the section 00front of this document / Dissertation (MSc (Genetics))--University of Pretoria, 2006. / Genetics / unrestricted

In this study, the genomes of both the attenuated South African lumpy skin disease virus (LSDV) Neethling vaccine strain (LW) and a virulent field isolate from a recent outbreak namely the South African lumpy skin disease virus (LSDV) Neethling Warmbaths isolate (LD) have been cloned, sequenced and analysed. The genomic sequences of the South African LSDV Neethling Warmbaths isolate (LD) and the South African LSDV Neethling vaccine strain (LW), were compared to each other. The virulent South African isolate, LD was also compared to the previously sequenced virulent LSDV Neethling strain 2490 (LK), to determine molecular differences. The LSDV genome is approximately 150 kbp in size and consists of 156 putative genes. Of the 156 potential encoded proteins of the virulent LSDV field isolates, the South African LSDV Neethling Warmbaths isolate (LD) and the LSDV Kenyan Neethling strain 2490 (LK), 120 were identical, 21 showed differences of a single amino acid, 7 showed two amino acid differences, while only one showed three amino acid differences. These were mostly found in the variable terminal regions. The LSDV Kenyan Neethling strain 2490 (LK) was isolated in Kenya in 1958 and than re-isolated in 1987 from lesions of an experimentally infected cow (Tulmanet al. 2001). The South African LSDV Neethling Warmbaths isolate (LD) was isolated from lesions of a severely infected calf in the Northern Province of the Republic of South Africa, on the farm Bothasvlei in 2001 (David Wallace, Biotechnology Division, Onderstepoort Veterinary Institute, Republic of South Africa; Personal communication, 2001). Considering the geographically distant African regions of the isolates, namely South Africa (LD) and Kenya (LK) as well as the time when these viruses were isolated, minimal genetic variation was observed thereby suggesting that lumpy skin disease virus is genetically stable. When the attenuated vaccine strain (L W) was compared with the South African field isolate LD, a total of 480 amino acid differences were observed in 121 of the 156 potential encoded proteins. These were again mainly in genes of the terminal regions and a number of these led to frameshifts that caused truncated open reading frames (ORFs) as well as deletions of up to nine amino acids and insertions of up to 42 amino acids. These modified open reading frames (ORFs) encode proteins that are involved in various aspects, such as the regulation of host immune responses [a soluble interferon (IFN)-gamma receptor, and an interleukin-l (IL-l) receptor-like protein], gene expression (mutT motif proteins), DNA repair (superoxide dismutase), host-range specificity (ankyrin-repeat protein, kelch-like proteins) including proteins with unassigned functions. These differences could lead to a reduction in immuno evasive mechanisms and virulence factors present in attenuated LSDV strains. At this stage, it is not possible to define which amino acid differences in particular are responsible for dramatic alterations in viral virulence. A good indication, however are differences occurring in functional domains. A mutation in a trans-membrane region, for example, could alter the levels of secretion of a protein involved in the regulation of the host immune response. We conclude that the attenuated effect is likely to be the sum of the altered phenotypes of the expressed proteins, although it is also likely that a few specific proteins carry more weight. Further studies to determine the functions of the relevant encoded gene products will hopefully confirm this. The molecular design of an effective vaccine is likely to be based on the strategic manipulation of such genes. / Dissertation (MSc (Microbiology))--University of Pretoria, 2006. / Microbiology and Plant Pathology / unrestricted

Please read the abstract in the section 00front of this document / Dissertation (MSc (Microbiology))--University of Pretoria, 2006. / Microbiology and Plant Pathology / unrestricted

The aim of this study was to determine the efficacy of vaccination in preventing LSDV excretion in semen and negative effects on semen quality. Lumpy skin disease (LSD) is caused by a virus in the genus Capripoxvirus of the family Poxviridae. The virus has been reported to be excreted in the semen of experimental infected nonvaccinated bulls. Nevertheless, vaccination has been the most widely used method to reduce and prevent the spread of the disease. This work was done to determine the efficacy of lumpy skin disease vaccination in preventing the excretion of lumpy skin disease virus (LSDV) in semen of experimentally infected vaccinated bulls. It also determined further the effect of vaccination and experimental infection on semen quality. Six serologically negative bulls 11-16 months of age were vaccinated with an attenuated Neethling strain of LSD vaccine, and a repeated dose of vaccine was given twenty one days later. These bulls were then experimentally infected by intravenous injection with a virulent field strain of LSDV (V248/93). Six unvaccinated bulls were similarly infected to act as controls. All animals were observed for clinical signs, blood and semen was collected and evaluated twice a week until day 40 post vaccination and every two days until day 28 post-infection when the trial was terminated. Serology was done using the serum neutralization test and viraemia was determined by virus isolation. Semen was examined by polymerase chain reaction (PCR) for the presence of virus. Semen evaluation was done visually and microscopically. Two of the unvaccinated controls developed severe LSD, two showed mild symptoms and two were asymptomatic. No clinical abnormalities were detected following vaccination, and clinical signs were limited to mild lymph node enlargement in four bulls following challenge of the vaccinated bulls. There was a significant difference (P<0.05) in semen quality after experimental infection of the unvaccinated bulls. In the vaccinated bulls, semen quality showed no significant difference (P>0.05) following vaccination and challenge. Three of the vaccinated bulls were serologically positive at the time of experimental infection and four at the end of the trial. Five unvaccinated bulls were found to be viraemic during the course of the trial. No vaccinated bulls were found to be viraemic at any stage. Four unvaccinated bulls excreted the virus in their semen during the course of the trial. Viral nucleic acid was not detected in any semen samples following vaccination or challenge in vaccinated bulls. This study provides evidence that vaccination against LSD prevented the excretion of viral particles in semen. It also illustrated that LSD vaccination prevented any effect on semen quality after experimental infection with virulent virus. / Dissertation (MSc (Production Animal Studies))--University of Pretoria, 2006. / Production Animal Studies / unrestricted

Lumpy skin disease (LSD) is a disease of significant economic importance in Africa. It causes considerable production losses and its presence in semen is a constraint to international trade. Recent findings that LSDV viral DNA can be found in the semen of artificially infected bulls for up to five months, while viable virus could be isolated 42 days after infection, indicated the need for studies into the mechanism by which this protracted shedding occurs. Six healthy, seronegative, postpubertal Dexter bulls were housed in vector-free stables and challenged with LSD virus by intravenous injection. Sheath washes, vesicular fluid and semen collection was performed every other day and subjected to PCR. On these days, blood was collected for serum neutralization tests and virus isolation, and ultrasonography of the reproductive tracts performed. Semen was centrifuged to separate cell-rich and seminal plasma fractions, and tested by PCR. Clinical parameters were recorded twice daily. Bulls shedding viral DNA 28 days after challenge were slaughtered, their reproductive tracts were harvested and diagnostic post mortem was performed. Histopathology, immunoperoxidase staining, electron microscopy, virus isolation and PCR were done on tissue samples. Of the six bulls, two showed no clinical signs, two showed mild and two showed severe clinical signs. Fever appeared five to seven days and lesions eight to ten days post challenge. Bulls were viraemic and febrile during the same time. Viral DNA was detected in all semen fractions of all bulls, but mostly from the cell-rich fraction and from the bulls showing the most severe clinical signs. Ultrasonography showed infarction in the testes and epididymides of the two bulls that were most severely affected. Necropsy of the two bulls that were still shedding after 28 days showed testicular degeneration and infarction, as well as epididymal granuloma formation. None of the accessory sex organs showed significant pathology. Histopathological changes seen were necrogranulomata in testes and peididymides. IMP staining of reproductive tissues showed that staining was restricted to areas in the testes and epididymides that were associated with necrosis. Virus could be seen on negative staining EM of sections of the testes. Our results show that LSDV is not limited to specific fractions of the ejaculate and that the testes and epididymides are most profoundly affected. Blood contamination is not responsible for the presence of viral DNA in semen, and it is unlikely that the virus is sperm-associated. Results suggest that the ejaculate is contaminated with viral DNA as it is shed from necrotic lesions in the genital tract. Further research is indicated into the ability of infected semen to produce disease as well as treatment protocols that could render semen free of viral DNA. / Dissertation (MMedVet (Theriogenology))--University of Pretoria, 2006. / Production Animal Studies / unrestricted

Capripoxviruses are of particular interest as live recombinant vectors for use in the veterinary field, since their host-range is restricted to cattle, goats and sheep. The work presented in this thesis is a preliminary study undertaken on the South African Neethling vaccine strain of lumpy skin disease virus (LSDV). As a departure point towards the eventual identification of strong promoter areas in the 143 kb genome of LSDV, a portion of its genome was cloned. Three methods for purification of LSDV DNA were compared, to determine which yielded the best quality DNA for cloning. DNA extracted directly from infected cells was excessively contaminated with bovine host-DNA, complicating the cloning of LSDV DNA. The use of pulsed field gel electrophoresis solved the contamination problem, by separating viral DNA from bovine DNA. However, insufficient amounts of viral DNA for cloning purposes, could be recovered from the gel. Sufficient amounts of good quality LSDV DNA was obtained by extraction from purified virions. Purified LSDV DNA was digested with various restriction enzymes to identify those which yielded several 4-1 0 kb fragments, for cloning into the Bluescribe plasmid transcription vector. Enrichment for large fragments (8-1 0 kb) was achieved by sucrose density centrifugation. Cloned fragments were analysed by Southern blot hybridisation to verify their viral origin. Hybridisation studies indicated that several unique regions of the LSDV genome were cloned as Pst I and Bam HI fragments respectively, i.e. the cloned fragments contained no overlapping regions. In total, 71.25 kb of the DNA of the LSDV Neethling vaccine strain has been cloned, representing approximately 50% of the viral genome. The availability of these clones now paves the way for further molecular investigations of the LSDV Neethling genome, including identification of promoter regions. A trial gene, which will be cloned and expressed in LSDV, namely the cloned VPS-gene of bluetongue virus serotype 4, was prepared and its nucleotide sequence determined. Homopolymer sequences present at the terminal ends of the gene as a result of the original cloning strategy, are known to interfere with expression and were removed by means of the polymerase chain reaction (PCR). The nucleotide sequence of the resulting PCR-tailored BTV4 VPS-genewas determined and used to deduce the amino acid sequence of the protein. The gene is 1638 bp in length and encodes a protein of 526 aa. Conserved sequences, 6 bp in length and unique to the 5'- and 3'terminal ends of all BTV genes, were detected at the termini of the tailored gene, confirming that the original clone was a full-length copy of the gene. Amplification by PCR did not mutate the open reading frame (OAF) of the gene, since it was of similar length to that reported for 5 other BTV serotypes. With a view to future investigations, including the identification of promoter sequences in the LSDV genome, a preliminary investigation of LSDV protein synthesis was undertaken, to acquire some knowledge of the growth cycle of the virus. Eighteen putative virus-specific proteins were identified by radio-labelling infected cells with [³⁵S]-methionine. By pulse-labelling infected cells with [³⁵S]methionine at various times post infection (p.i.), viral proteins were first detected at 16 hr p.i. It is, however, unlikely that the early phase of viral replication commences as late as 16 hr p.i. and these results might be attributed to various problems, such as the low multiplicity of infection used and that host protein shut-down was inefficient, thus masking the presence viral proteins. In conclusion, this investigation resulted in the cloning of 71,25 kb of the LSDV genome, the tailoring and sequencing of the BTV4 VPS gene and the identification of 18 putative LSDV proteins. This now paves the way for further research to develop LSDV as a vaccine vector.