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.

The statistical evaluation of molecular imaging approaches for detecting, diagnosing, and monitoring molecular response to treatment are required prior to their adoption. The assessment of fluorescence-enhanced optical imaging is particularly challenging since neither instrument nor agent has been established. Small animal imaging does not address the depth of penetration issues adequately and the risk of administering molecular optical imaging agents into patients remains unknown. Herein, we focus upon the development of a framework for OAIQ which includes a lumpy-object model to simulate natural anatomical tissue structure as well as the non-specific distribution of fluorescent contrast agents. This work is required for adoption of fluorescence-enhanced optical imaging in the clinic. Herein, the imaging system is simulated by the diffusion approximation of the time-dependent radiative transfer equation, which describes near infra-red light propagation through clinically relevant volumes. We predict the time-dependent light propagation within a 200 cc breast interrogated with 25 points of excitation illumination and 128 points of fluorescent light collection. We simulate the fluorescence generation from Cardio-Green at tissue target concentrations of 1, 0.5, and 0.25 µM with backgrounds containing 0.01 µM. The fluorescence boundary measurements for 1 cc spherical targets simulated within lumpy backgrounds of (i) endogenous optical properties (absorption and scattering), as well as (ii) exogenous fluorophore crosssection are generated with lump strength varying up to 100% of the average background. The imaging data are then used to validate a PMBF/CONTN tomographic reconstruction algorithm. Our results show that the image recovery is sensitive to the heterogeneous background structures. Further analysis on the imaging data by a Hotelling observer affirms that the detection capability of the imaging system is adversely affected by the presence of heterogeneous background structures. The above issue is also addressed using the human-observer studies wherein multiple cases of randomly located targets superimposed on random heterogeneous backgrounds are used in a “double-blind” situation. The results of this study show consistency with the outcome of above mentioned analyses. Finally, the Hotelling observer’s analysis is used to demonstrate (i) the inverse correlation between detectability and target depth, and (ii) the plateauing of detectability with improved excitation light rejection.

Please read the abstract in the section 00front of this document / Thesis (PhD (Genetics))--University of Pretoria, 2007. / Genetics / unrestricted

Lumpy skin disease (LSD) and Rift Valley fever (RVF) are transboundary viral diseases occurring in Africa and the Middle East (e.g. Israel, Saudi Arabia and Yemen) with increasing potential for global spread. Although the role of wildlife in the epidemiology of these diseases is still not clearly understood, the African buffalo (Syncerus caffer) is thought to play a role in the epidemiology of these diseases. This study sought to expand our understanding of the role of buffalo in the maintenance of RVF and LSD by determining seroprevalence to these viral diseases in buffalo during the inter-epidemic period. Lumpy skin disease is endemic in Africa, and has spread to the Middle East (e.g. Israel); consequently there is a high risk of lumpy skin disease virus (LSDV) expanding its geographical distribution to other areas and due to its economic importance it is included in the list of Notifiable Diseases of the World Organization of Animal Health (OIE). The African buffalo is also suspected to play a role in the epidemiology of RVF. Like LSD, RVF was, until recently, only endemic in Africa. However, it spread to the Arabian Peninsula (Saudi Arabia and Yemen) in 2000 exacerbating concerns that it will extend to other regions of the world. Studies have already established that competent mosquito vectors for RVFV exist in North America and other parts of the world. A total of 248 buffalo sera was tested for antibodies to capripoxviruses and neutralising antibodies against LSDV and RVFV using an indirect enzyme-linked immunosorbent assay (I-ELISA) as well as the serum neutralisation test (SNT). The samples were obtained from the Kruger National Park (KNP) and Hluhluwe-iMfolozi Park (HiP) in South Africa. The prevalence of antibodies to LSDV and RVFV in the sera tested was 70/248 (28.2%) and 15/248 (6.1%), respectively using an I-ELISA. The LSDV I-ELISA, using a sheeppox virus as antigen, has not been validated for use in African buffalo. The high percentage of LSDV positive antibody results obtained in this study is however a concern. Results obtained is in contrast with other published results as well as results obtained with the SNT for antibodies against LSDV. The SNT is currently the gold standard for LSDV antibody testing. Using this test for LSDV in this study, 5/66 (7.6 %) samples tested positive. The results of the RVF I-ELISA, which had previously been validated for use in the African buffalo, correlated with the SNT results. From 12 SNT RVFV-positive sera, 3 (25%) had very high SNT titres of 1:640. Neutralising antibody titres of more than 1:80 were found in 80% of the positive sera tested. Eleven buffaloes (4.4% of the total samples) also showed evidence of antibodies to both LSDV and RVFV. The results obtained in this study complement other reports indicating the role of African buffalo in the epidemiology of these diseases during inter-epidemic periods. / Dissertation (MSc)--University of Pretoria, 2012. / Veterinary Tropical Diseases / unrestricted

La dermatose nodulaire contagieuse (DNC) est une des maladies virales les plus importantes économiquement chez les bovins en Ethiopie. Elle est causée par le virus LSD (Lympy skin disease virus) appartenant au groupe des Capripoxvirus. L'objectif de cette thèse est de mieux comprendre l'épidémiologie de cette maladie afin de proposer des méthodes de contrôle et de prévention efficaces et applicables sur le terrain. Cette thèse est construite en cinq chapitres. Le premier chapitre fait une description générale du système de production agricole en Ethiopie et présente nos connaissances actuelles sur ce virus et cette maladie. Le second chapitre est consacré à l'évaluation d'un test d'immunofluorescence indirecte (IFI) pour le diagnostic sérologique à l'aide de méthodes sans gold standard. Le test de séroneutralisation virale a été utilisé comme second test de comparaison. L'analyse à l'aide d'un modèle bayesien a montré que l'IFI présentait une bonne sensibilité (92%) et une bonne spécificité (88%) ce qui suggère que ce test peut être utilisé pour le diagnostic et le dépistage de masse de la Dermatose Nodulaire Contagieuse avec une relativement faible proportion d'erreurs. La possibilité de tester un grand nombre de sérums en IFI est un autre avantage de cette technique pour conduire des études épidémiologiques de grande envergure. La sensibilité et la spécificité de la séroneutralisation virale (SNV) étaient respectivement de 78% et de 97%. En conséquence, le test IFI sera préféré pour un dépistage de masse en raison de sa meilleure sensibilité tandis que le test SNV sera réservé à la confirmation. Une étude épidémiologique transversale a été menée pour estimer la prévalence de la Dermatose Nodulaire Contagieuse Bovine à l'échelle du troupeau et de l'individu et pour définir les facteurs de risque associés à cette maladie dans le contexte particulier de l'Ethiopie. C'est l'objet de la troisième partie de cette thèse. Un total de 330 questionnaires d'enquêtes a été collecté de 44 associations paysannes situées dans 15 districts. La prévalence moyenne de la DNC à l'échelle du troupeau était de 42,8% (IC à 95% : 37,5 – 48,3). Elle était significativement plus élevée dans les zones d'altitude moyenne 55,2% (IC à 95% : 47,5 – 62,6) que dans les zones de basse altitude (22,3%) ou les zones de haute altitude (43,5%). La prévalence de la DNC et la mortalité due à cette maladie, observées à l'échelle de l'animal, étaient de 8,1% et de 2,12% respectivement. A nouveau, elles étaient plus élevées dans les zones d'altitude moyenne (10,4% et 3,2% respectivement) que dans les zones de basse et haute altitude (P < 0,05). L'analyse de facteurs de risque a montré que trois variables étaient significativement associées avec la prévalence de la DNC : l'effet de la zone agroclimatique, la conduite de troupeaux différents sur les mêmes pâtures et les mêmes lieux d'abreuvement et l'introduction de nouveaux animaux. L'incidence maximale de la DNC était concomitante de l'augmentation des populations d'insectes hématophages : cette association dans le temps était significative (coefficient de Spearman de 0,88 ; 0,79 et 0,79 respectivement pour les zones de haute, moyenne et basse altitude). L'évaluation de la faisabilité financière et des bénéfices espérés de la vaccination ont constitué la quatrième partie de la thèse. / Lumpy skin disease (LSD) is one of economically important viral diseases of cattle in Ethiopia caused by Lumpy skin disease virus in the member of the genus Capripox viruses. The objective of this thesis is to better understand the epidemiological features of the disease in order to propose practical and applicable control and prevention options. The thesis is classified in five chapters. The first chapter describes the general agricultural production system in Ethiopia and relates the current knowledge on the virus and the disease as given by the literature.The second chapter deals with the performance of indirect fluorescence antibody test (IFAT) as a serological diagnostic and screening tool that was evaluated using methods without gold standard. Virus neutralization test (VNT) was used as the second test for comparison. The analysis of conditional dependent Bayesian model showed that the IFAT had good accuracy both in sensitivity (92%) and specificity (88%) parameters indicating that it could be used for LSD diagnosis and screening (epidemiological studies, epidemiosurveillance) with less misclassification. Its capacity to run large number of samples per plate just like ELISA could be also taken as an advantage for large epidemiological studies. The sensitivity and specificity of VNT was 78%, 97% respectively. The two tests IFAT and VNT were found conditionally independent on the disease status of the animal. Thus, higher sensitivity and throughput for IFAT would render the test being selected for screening purposes and higher specificity performance of VNT would qualify it to be used as a confirmation test. A cross sectional study was then conducted to estimate the prevalence of LSD at herd and animal-levels and to analyze the risk factors associated with the disease occurrence in Ethiopia. It is presented in the third chapter. A total of 330 questionnaire surveys were collected from 44 peasant associations (PA) distributed in 15 districts. The average herd level LSD prevalence was 42.8% (95% CI: 37.5–48.3) and it was significantly higher in the midland agro-climate 55.2% (95% CI: 47.5–62.6) than in lowland and highland agro-climate zones (22.3% and 43.5%, respectively). The observed LSD prevalence and mortality at animal level were 8.1% and 2.12% respectively which were still higher in the midland zone (10.4% and 3.2%, respectively) than in lowland and highland zones (P< 0.05). The risk factor analysis showed that three variables: the effect of agro-climates, communal grazing/watering management and introduction of new animals were significantly associated with LSD occurrence. The temporal association between LSD occurrence and increase in the biting-fly population was also positively correlated by Spearman rank correlation coefficient (0.88, 0.79 and 0.79 for highland, midland and lowland zones, respectively) and statistically significant. The need to evaluate the financial feasibility and benefit possibly expected of vaccination led us to the fourth component of the thesis.

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

Lumpy skin disease virus (LSDV), a poxvirus that belongs to the genus Capripoxvirus is an important pathogen that can be shed in the semen of infected bulls. The screening of semen for infectious virus prior to artificial insemination requires a sensitive diagnostic method. The isolation of the virus on cell cultures and/or the use of polymerase chain reaction (PCR) are sensitive diagnostic tests which can be used to screen semen for LSD viral DNA prior to artificial insemination. Although cell culture is a sensitive method and detects infectious virus, its use has major limitations due to the toxic effect of semen on the cells. This study was therefore aimed at finding a method that decreases the toxic effect of semen on cell culture and enhances LSDV isolation. Secondly, the efficiency of this method in enhancing the isolation of LSDV in field samples was tested. In order to eliminate the toxic effect of semen on cell culture, a pilot study was conducted in which semen samples from LSDV sero-negative bulls were collected and infected with a field isolate of LSDV, strain 248/93 with a titre of 6.5 log TCID50. The semen samples were subjected to one of four different methods, viz centrifugation, serial dilution, filtration and chemical treatment with kaolin. The centrifugation, serial dilution, and filtration methods were supplemented with additional amounts of gentamycin. The toxic effects of semen on cell culture were completely eliminated when supernatants of semen samples, centrifuged at 2000 rpm for 1, 3 and 5 mins and serial diluted was used to inoculate confluent monolayers of bovine dermis cells. Semen diluted in MEM with or without additional antibiotics was the most sensitive method of demonstrating virus at higher dilutions, followed by pellets of samples centrifuged for 1 and 3 minutes. The toxicity recorded when the pellet fraction of semen samples were centrifuged for 5 mins at 2000 rpm was comparable to results obtained from serially diluted samples supplemented with gentamycin. The use of filtration and kaolin treatment of semen samples could not remove the toxic effect of semen on cells. To evaluate the presence of LSDV in semen of experimentally infected bulls, six seronegative post-pubertal bulls housed in an insect proof facility were infected with LSDV via the intravenous route. The experimentally infected bulls were monitored for clinical sign of the disease. Two bulls showed severe, two a mild and two an inapparent infection. Blood samples were collected for virus isolation and semen samples for virus isolation and PCR. Vesicular fluid and preputial washes were also investigated for the presence of LSD viral nucleic acid using PCR. The infectious titre of the virus shed in semen of these bulls was also calculated. The incubation period in infected bulls varied from 7 to 14 days. The length of viraemia varied between groups and did not correlate with the severity of clinical disease. The virus was isolated from blood samples of bulls in the severely infected group on several occasions. Bulls in the mildly infected group had the lowest rate of isolated virus when compared to those with inapparent infection. The use of supernatants of centrifuged serial diluted semen samples, as shown in the pilot study, have considerably reduced the toxic effect of semen on cell culture. This method was used to test field samples for its sensitivity to isolated LSDV in semen of experimentally infected bulls with PCR as a gold standard. In all the semen samples tested using supernatants of semen samples LSDV was isolated in 53.1% of the samples on cell culture while in the serial diluted samples, only 28.1% of samples were positive with a median time of detection on cell culture of 4 and 8 days, respectively. The use of the supernatant fraction was able to detect infectious LSDV in semen samples for prolonged periods with reduced time of development of cytopathic effect, than previously reported. In order to compare the sensitivity of PCR and virus isolation, PCR positive and a few negative samples were subjected to virus isolation using the centrifugation method developed in the pilot study. The PCR was able to detect LSD viral nucleic acids in some semen samples even when virus could not be isolated on cell culture. The PCR was also able to detect viral nucleic acid in vesicular fluid and preputial washes of infected bulls. The titre of the virus shed in the semen at a certain stage of the infection was calculated to be 3 log TCID50. In conclusion, this study provides evidence of a complete reduction of the toxic effect of semen on cell culture and increase chances of LSDV isolation with reduced detection time when semen samples are processed using the centrifugation method as described in the pilot study. Furthermore, it showed PCR was more sensitive than virus isolation in the detection of LSD viral nucleic acid in semen samples and can be used for routine diagnosis. However, virus isolation must be used when the infective nature of virus shed in semen is desirable. This study provides the first evidence of the shedding of LSDV nucleic acid in vesicular fluid and preputial washes of experimentally infected bulls. It also represents the first report that a considerable amount of LSDV is shed in semen of experimentally infected bulls, which may be infective at certain stages of clinical disease. Lumpy skin disease virus (LSDV), a poxvirus that belongs to the genus Capripoxvirus is an important pathogen that can be shed in the semen of infected bulls. The screening of semen for infectious virus prior to artificial insemination requires a sensitive diagnostic method. The isolation of the virus on cell cultures and/or the use of polymerase chain reaction (PCR) are sensitive diagnostic tests which can be used to screen semen for LSD viral DNA prior to artificial insemination. Although cell culture is a sensitive method and detects infectious virus, its use has major limitations due to the toxic effect of semen on the cells. This study was therefore aimed at finding a method that decreases the toxic effect of semen on cell culture and enhances LSDV isolation. Secondly, the efficiency of this method in enhancing the isolation of LSDV in field samples was tested. In order to eliminate the toxic effect of semen on cell culture, a pilot study was conducted in which semen samples from LSDV sero-negative bulls were collected and infected with a field isolate of LSDV, strain 248/93 with a titre of 6.5 log TCID50. The semen samples were subjected to one of four different methods, viz centrifugation, serial dilution, filtration and chemical treatment with kaolin. The centrifugation, serial dilution, and filtration methods were supplemented with additional amounts of gentamycin. The toxic effects of semen on cell culture were completely eliminated when supernatants of semen samples, centrifuged at 2000 rpm for 1, 3 and 5 mins and serial diluted was used to inoculate confluent monolayers of bovine dermis cells. Semen diluted in MEM with or without additional antibiotics was the most sensitive method of demonstrating virus at higher dilutions, followed by pellets of samples centrifuged for 1 and 3 minutes. The toxicity recorded when the pellet fraction of semen samples were centrifuged for 5 mins at 2000 rpm was comparable to results obtained from serially diluted samples supplemented with gentamycin. The use of filtration and kaolin treatment of semen samples could not remove the toxic effect of semen on cells. To evaluate the presence of LSDV in semen of experimentally infected bulls, six seronegative post-pubertal bulls housed in an insect proof facility were infected with LSDV via the intravenous route. The experimentally infected bulls were monitored for clinical sign of the disease. Two bulls showed severe, two a mild and two an inapparent infection. Blood samples were collected for virus isolation and semen samples for virus isolation and PCR. Vesicular fluid and preputial washes were also investigated for the presence of LSD viral nucleic acid using PCR. The infectious titre of the virus shed in semen of these bulls was also calculated. The incubation period in infected bulls varied from 7 to 14 days. The length of viraemia varied between groups and did not correlate with the severity of clinical disease. The virus was isolated from blood samples of bulls in the severely infected group on several occasions. Bulls in the mildly infected group had the lowest rate of isolated virus when compared to those with inapparent infection. The use of supernatants of centrifuged serial diluted semen samples, as shown in the pilot study, have considerably reduced the toxic effect of semen on cell culture. This method was used to test field samples for its sensitivity to isolated LSDV in semen of experimentally infected bulls with PCR as a gold standard. In all the semen samples tested using supernatants of semen samples LSDV was isolated in 53.1% of the samples on cell culture while in the serial diluted samples, only 28.1% of samples were positive with a median time of detection on cell culture of 4 and 8 days, respectively. The use of the supernatant fraction was able to detect infectious LSDV in semen samples for prolonged periods with reduced time of development of cytopathic effect, than previously reported. In order to compare the sensitivity of PCR and virus isolation, PCR positive and a few negative samples were subjected to virus isolation using the centrifugation method developed in the pilot study. The PCR was able to detect LSD viral nucleic acids in some semen samples even when virus could not be isolated on cell culture. The PCR was also able to detect viral nucleic acid in vesicular fluid and preputial washes of infected bulls. The titre of the virus shed in the semen at a certain stage of the infection was calculated to be 3 log TCID50. In conclusion, this study provides evidence of a complete reduction of the toxic effect of semen on cell culture and increase chances of LSDV isolation with reduced detection time when semen samples are processed using the centrifugation method as described in the pilot study. Furthermore, it showed PCR was more sensitive than virus isolation in the detection of LSD viral nucleic acid in semen samples and can be used for routine diagnosis. However, virus isolation must be used when the infective nature of virus shed in semen is desirable. This study provides the first evidence of the shedding of LSDV nucleic acid in vesicular fluid and preputial washes of experimentally infected bulls. It also represents the first report that a considerable amount of LSDV is shed in semen of experimentally infected bulls, which may be infective at certain stages of clinical disease. / Dissertation (MSc (Veterinary Science))--University of Pretoria, 2006. / Veterinary Tropical Diseases / unrestricted