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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

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

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.

Novel vaccine strategies for the presentation of immunologically important epitopes to the immune system are continuously being developed. Two such systems include the particulate protein and live viral vector delivery systems. In his study the long-term objective is to explore the African horsesickness virus (AHSV) serotype 9 viral protein 7 (VP7) and the Lumpy skin disease (LSDV) viral vector as two different vaccine strategies, particularly in view of the development of an HIV-1 vaccine. Consequently two very specific objectives were outlined in this study. The first was to express the HIV¬1 subtype C strain Du 151 gp41 epitopes ALDSWK and RVLAIERYLKD on the surface of the AHSV-9 VP7 particulate protein crystalline structures. A longer-term aim is to synthesise large quantities of these chimeric VP7 crystals in order to assess the immune response against the inserted epitopes. Secondly, the efficiency of the LSDV bi-directional promoter pA7LA8R in expressing chimeric VP7 proteins was to be evaluated by utilising the late element of this promoter to determine expression levels. Nucleotide sequences encoding the ALDSWK and RVLAIERYLKD epitopes were amplified from the HIV-1 subtype C strain Du 151 gp160 gene utilising PCR. These sequences were cloned individually as well as in combination into a multiple cloning site (549-566bp) present in the AHSV-9 VP7 gene. Recombinant pFASTBAC vectors PFASTBAC-VP7-MT 177-RVLAIERYLKD, PFASTBAC-VP7-MT 177-ALDSWK AND PFASTBAC-VP7-MT-177-RVLAIERYLKD-ALDSWK were identified, sequenced and used in the generation of recombinant baculoviruses utilising the BAC-to-BAC™ Baculovirus expression system. Expression of all three chimeric proteins, VP7-ALDSWK, VP7-RVLAIERYLKD and VP7- RVLAIERYLKD-ALDSWK was detected in infected Sf9 insect cells utilising SDS-PAGE. Further investigations will involve high-level expression of these proteins, which in turn will allow their characterisation as well as solubility, scanning electron and immunogenicity studies. In order to evaluate the efficiency of the LSDV bi-directional promoter, the AHSV-9 VP7 gene was cloned under the control of the late element (pA7L) of this promoter. The recombinant pHSsgpt-VP7 transfer vector was subsequently transfected into lamb testis cells infected with wild type LSDV in order to generate recombinant LSDV-VP7. Several rounds of recombinant virus selection in the presence of mycophenolic acid resulted in the loss of the LSDV-VP7 recombinant. Due to this unforeseen result, the expression of the VP7 protein from the late element of the pA7LA8R bi¬directional promoter could not be quantified and the efficiency of this promoter was not determined. The loss of LSDV recombinants, which contain a gene under the control of the late promoter element pA7L, has occurred previously and is suspected to be because of the instability of these recombinants. Due to the difficulties inherent in working with the LSDV viral vector delivery system, it has subsequently been decided to explore an alternate poxviral vector system. The focus in this study is now being shifted onto the promising Modified Vaccinia Ankara (MVA) viral vector system. / Dissertation (MSc (Genetics))--University of Pretoria, 2006. / Genetics / unrestricted

Lumpy skin disease (LSD) is an economically important and debilitating disease of cattle caused by the lumpy skin disease virus (LSDV), a poxvirus in the genus Capripoxvirus. The disease is of economic importance to farmers in endemic regions and is a major constraint to international trade in livestock and their products. It is characterised by fever, enlargement of superficial lymph nodes, loss of weight, inappetence, salivation, lachrymation and formation of eruptive circumscribed skin lesions. The quality of meat and milk are reduced; there is infertility due to reduced sperm quality, abortions and reduced calving rates. The hides are permanently scarred, thereby reducing their quality and trade may be affected following movement restrictions from affected areas. v Lumpy skin disease has the potential to become an emerging disease because of global climate change and changes in patterns of trade in animals and animal products. The disease has become endemic in Africa except in countries like Libya, Algeria, Tunisia and Morocco, where the disease has never been reported. It has also spread to the Middle East where outbreaks were first reported in Israel (1989), Kuwait (1991), Saudi Arabia (1990) , Lebanon (1993), The United Arab Emirates (2000) and Oman (2010). In endemic areas, LSD outbreaks are common in summer. The persistence of LSDV between inter-epidemic periods has not been determined and there is no carrier state reported in either cattle or wild animals. Transmission of the disease has been associated with a high incidence of biting insects such as in wet conditions. The spread of LSD from Egypt to Israel e.g. was associated with movement of the stable fly, Stomoxys calcitrans. The virus has been recovered from S. calcitrans and Biomya fasciata, caught while feeding on infected animals and transmission by insects is suspected to be mechanical, which has been demonstrated in Aedes aegypti mosquitoes. During the 1957 outbreak of LSD in Kenya, affected animals were observed to have high tick infestations, especially of Amblyomma species. In a pilot trial in 2008 at the University of Pretoria (UP), Department of Veterinary Tropical Diseases (DVTD), Amblyomma hebraeum, Rhipicephalus appendiculatus and R. (B) decoloratus ticks were implicated in the transmission of LSDV. The overall objective of this study was to investigate the vector competence of three common sub-Saharan tick species (R. (B) decoloratus, R. appendiculatus and A. hebraeum) and their potential roles in the epidemiology of LSD. This was achieved by testing for persistence of LSDV in ticks and its subsequent transmission to recipient animals following interrupted feeding, transstadial and transovarial development of the ticks. The over-wintering of LSDV was also investigated during transstadial passage in A. hebraeum and transovarial passage in R. (B) decoloratus. During the study, seven cattle were artificially infected with LSDV to serve as source (donors) of infection to ticks. To test for mechanical / intrastadial transmission and persistence in ticks, adult ticks (A. hebraeum and R. appendiculatus) were partially fed on donor animals and then transferred to recipient animals or collected for testing. To test for transstadial transmission/passage, nymphal stages of A. hebraeum and R. appendiculatus were fed on donor animals until they engorged and dropped. Engorged nymphs were incubated to moult to adults. The emergent adults were placed on recipient animals and also tested for the virus. To test for transovarial transmission and passage R. (B) decoloratus (one- host tick) larvae were fed on donor animals until engorged adults. For R. appendiculatus and A. hebraeum (three-host ticks), adults were fed to repletion on the donor animals. Engorged females were collected and incubated to lay eggs and the eggs were allowed to hatch. The emergent larvae were placed to feed on recipient animals to test for transovarial transmission, while larvae were tested for the presence of the virus. Over-wintering of LSDV in ticks was tested by transstadial passage in A. hebraeum and transovarial passage in R. (B) decoloratus under fluctuating reduced temperatures, simulating wintery climatic conditions. Engorged A. hebraeum nymphs and R. (B) decoloratus females were infected by intracoelomic injection. The presence of the virus in LSDV- infected animals was tested by real-time PCR, virus isolation (VI), and the serum neutralisation test (SNT). Tick saliva was tested by real-time PCR and VI while ticks were tested by immunohistochemistry, transmission electron microscopy, VI and real-time PCR. Mechanical/intrastadial and transstadial transmission is reported in A. hebraeum and R. appendiculatus. Transovarial transmission was reported in A. hebraeum, R. appendiculatus and R. (B) decoloratus. The virus was demonstrated in saliva and tick organs of A. hebraeum and R. appendiculatus adults following both mechanical/intrastadial and transstadial persistence. Transovarial passage of LSDV was demonstrated in R. (B) decoloratus, R. appendiculatus and A. hebraeum larvae. The virus also persisted through cold temperature exposure during transstadial passage in A. hebraeum and transovarial passage in R. appendiculatus. This study confirms the vector competency of A. hebraeum, R. appendiculatus and R. (B) decoloratus ticks for LSDV. It also shows the potential for LSDV to over-winter in ticks and demonstrates that LSDV may persist in ticks during inter-epidemic periods. / Thesis (PhD)--University of Pretoria, 2013. / gm2014 / Veterinary Tropical Diseases / unrestricted