A demodicidose é uma das principais dermatopatias parasitárias dos cães, contudo muitos aspectos da doença ainda são pouco compreendidos. A doença no cão adulto é menos frequente do que a forma juvenil e parece ocorrer devido à presença de condição imunodepressora/imunossupressora que leve à perda da capacidade de controle, pelo sistema imune, da população de ácaros Demodex spp. na pele do animal. O objetivo deste trabalho foi avaliar se hiperadrenocorticismo (HAC) espontâneo, neoplasia e quimioterapia, classicamente aceitos como “gatilhos” para esta doença parasitária, seriam fatores de risco para o aumento na presença de ácaros Demodex spp. na pele de cães adultos. Foram selecionados cães sem histórico de demodicidose juvenil e estes, distribuídos em cinco grupos. O grupo um (G1) foi formado por 33 cães clinicamente saudáveis; o grupo dois (G2), por 27 cães com afecções crônicas; o grupo três (G3), por 14 cães diagnosticados com HAC espontâneo; o grupo quatro (G4), por 30 cães com neoplasia e o grupo cinco (G5), por 27 cães submetidos a protocolos quimioterápicos antineoplásicos. O exame parasitológico de pele (EPP) foi realizado, pontualmente, por meio de tricograma, impressão por fita adesiva e raspado cutâneo profundo em cinco pontos anatômicos pré-determinados. Regiões com lesões cutâneas também foram coletadas e hemogramas, realizados no intervalo de um mês prévio ou posterior à inclusão no estudo, foram avaliados. Em 16 cães foi possível demonstrar a presença de ácaros Demodex spp. Seis cães apresentaram lesões de pele e em apenas dois foi observada a presença do parasita, sendo que não houve diferença significativa na presença do ácaro entre cães com e sem alterações no hemograma. Os grupos de risco G3, G4 e G5 apresentaram maior frequência de EPP positivo, porém diferença estatisticamente significativa foi mostrada apenas entre os cães com HAC espontâneo e os com afecções crônicas, com odds ratio de 10,4. Concluiu-se que HAC espontâneo, neoplasia e quimioterapia parecem influenciar no aumento da população dos ácaros Demodex spp. na pele de cães adultos. Além disso, não há relação entre a presença de leucopenia, neutropenia e/ou linfopenia e o aumento na presença dos ácaros na pele desses animais. Por fim, as lesões cutâneas em cães com HAC espontâneo, neoplasia ou em quimioterapia são pouco frequentes e apresentam causas distintas, não sendo a proliferação excessiva de ácaros Demodex spp. a principal etiologia identificada. / Demodicosis is one of the most common parasitic skin diseases of dogs, but many aspects of canine demodicosis are still poorly understood. Adult onset demodicosis is less frequent and seems to occur due to the presence of an immunosuppressive condition leading to immune system loss of ability to control the Demodex mite population in the skin. The aim of this study was to evaluate if spontaneous hyperadrenocorticism (HAC), cancer and chemotherapy, classically accepted as "triggers" for this parasitic disease, would be risk factors for the increased presence of Demodex mites in the skin of adult dogs. Dogs without a history of juvenile demodicosis were selected and then divided into five groups. Group 1 (G1) was formed by 33 clinically healthy dogs; group two (G2), by 27 dogs with chronic conditions; group three (G3), by 14 dogs diagnosed with spontaneous HAC; group four (G4), by 30 dogs with cancer and group five (G5), by 27 dogs submitted to antineoplastic chemotherapy protocols. Trichogram, acetate tape impression and deep skin scrapes were performed, just once, in five predetermined anatomical points. Skin lesions are also evaluated by these techniques and complete blood count, performed within one month prior or after the inclusion in the study, were evaluated. In 16 dogs it was possible to demonstrate the presence of Demodex mites. Six dogs had skin lesions and only in two of them it was possible to observe the presence of the parasite; there was no significant difference in the presence of the parasite between dogs with and without changes in complete blood count. Risk groups G3, G4 and G5 had a higher frequency of positive samples, but significant difference was shown only between dogs with spontaneous HAC and those with chronic diseases (odds ratio 10.4). It was concluded that spontaneous HAC, cancer and chemotherapy seem to influence the increase of the parasite population in the skin. Furthermore, there is no relationship between the presence of leukopenia, neutropenia and/or lymphopenia and the increased presence of mites in the skin of adult dogs. Finally, skin lesions in dogs with spontaneous HAC, cancer or chemotherapy are uncommon and present different causes, not being excessive proliferation of Demodex mites the main identified etiology.

Abstract Transmembrane collagen XVII (BP180) is a structural component of hemidesmosomes that connects the two layers of skin. Collagen XVII is associated with both autoimmune and inherited bullous skin diseases. Mutations in collagen XVII gene cause junctional epidermolysis bullosa, and in the diseases of the pemphigoid group autoantibodies target collagen XVII. In this work, collagen XVII was studied in both junctional epidermolysis bullosa and gestational pemphigoid. Two novel glycine substitution mutations were found in the largest collagenous domain of collagen XVII. Analysis of recombinantly produced mutated proteins showed that these novel mutations and previously described glycine substitution mutations decrease the thermal stability of collagen XVII ectodomain. In addition, these mutations were found to cause intracellular accumulation of the mutated proteins and affect the post-translational modifications of collagen XVII. Meanwhile, an in-frame deletion of nine amino acids had no effect on the thermal stability or secretion of the collagen XVII ectodomain. Gestational pemphigoid autoantigen collagen XVII has been mainly studied in the skin, and its expression and function during pregnancy are so far largely unknown. For the first time, collagen XVII was shown to be expressed by cytotrophoblasts of the first trimester human placenta and by cultured cytotrophoblasts. Transmigration assay of cytotrophoblasts indicated that collagen XVII promotes trophoblast invasion, and may thus have a role in placental formation. In addition, significant amounts of in vivo produced collagen XVII were found in the amniotic fluid throughout pregnancy. Collagen XVII expression was also observed in hemidesmosomes of amniotic membranes and in cells cultured from amniotic fluid. These findings suggest that collagen XVII could have a function, albeit so far unknown, during pregnancy.

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

Chronic venous insufficiency (CVI) is characterized by valve dysfunction and venous hypertension, leading to erythrocytes’ extravasation into the tissue over time. CVI patients present dermal manifestations as; hyperpigmentation of the leg due to iron accumulation, histological changes regarding the dermal layer and fat (lipodermatosclerosis), and a high risk of developing a leg ulcer. For decades, researchers have studied CVI, and chronic venous ulcer (CVU) and iron have been considered critical pathological factors in this context, especially concerning oxidative stress and ROS formation. However, a clear understanding of the pathogenic effects of iron on the tissue network and the cross-talk of resident immune and skin tissue cells in the course of CVI is still missing. Therefore, in this project, we aim to investigate the pathological effect of iron overload on the cross-talk of resident immune and tissue cells in the skin. In the current thesis work, biopsies from CVI patients were analysed. Immunohistochemistry (IHC) and spectrometric assay confirmed a massive iron accumulation in their dermis and hypodermis. To dissect the different skin cells’ responses to iron overload, in vitro techniques were exploited. Mimic the erythrocyte overload in a macrophage (M2-like) culture revealed a shift in the gene signature towards inflammatory activation states of these cells. Hence, cytokine analysis confirmed an evident pro-inflammatory activation of macrophages (Ma). A mouse model with skin iron overload was generated to investigate the effect of iron overload in a more complex picture. Here, it was confirmed that iron induces an expansion of immune cells and a pro-inflammatory activation in the skin with a shift in resident macrophage subtypes, which was coupled to the adipose layer. Indeed, the dWAT of these mice shirked and showed clear signs of lipolysis. Moreover, IHC and IF staining of iron-mice skin showed increased cellularity of the lower dermis, which was linked to an expansion of the fibroblast population (dermis and stromal vascular fraction of the dWAT). Consistent with in vitro data, ECM genes were downregulated in the dermis, which may explain changes in the skin architecture of these mice. In this thesis, the newly established mouse model made it possible to understand how iron may affect skin cells in CVI patients and can be extremely useful for future research to develop a new therapeutic approach. This work wants to highlight the significance of iron overload in the skin, which affects cellular cross-talk, altering skin homeostasis and possibly leading to an ulcer.

No / To date there is ample evidence that patients with vitiligo accumulate millimolar concentrations of hydrogen peroxide (H2O2) in their epidermis as well as in their blood lymphocytes/monocytes. Several enzymes are affected by this H2O2 including catalase, glutathione peroxidase, and 4¿-carbinolamine dehydratase. The latter enzyme disrupts the recycling of the essential cofactor (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (6BH4) for the aromatic amino acid hydroxylases as well as the nitric oxide synthases. In this report we have elucidated the influence of H2O2 on dihydropteridine reductase (DHPR), the last enzyme in the 6BH4-recycling process. Here we show for the first time that concentrations of less than 30 ¿M H2O2 increase DHPR activities, whereas levels greater than 30 ¿M H2O2 deactivate the enzyme based on the oxidation of Met146 and Met151 in the sequence, consequently leading to disruption of the NADH-dependent enzyme active site. This oxidation was confirmed by Fourier transform-Raman spectroscopy yielding the expected SO band at 1025 cm characteristic of methionine sulfoxide. Hence these results unmasked a novel regulatory mechanism for DHPR enzyme activity. Moreover, we also demonstrated that DHPR activities in whole blood of patients with vitiligo are significantly decreased in untreated patients, whereas activities are normalized after removal of epidermal H2O2 with a topical pseudocatalase (PC-KUS). Taken together, these new data add more evidence to a systemic involvement of H2O2 in the pathomechanism of vitiligo.