Vaccines are a highly effective means of preventing infectious disease. However, for many diseases of livestock the available vaccines are ineffective or sub-optimal. This is partly due to challenges surrounding the specific targeting of antigen presenting cells (APCs). In order to improve the delivery of protective antigens to host APCs, a novel lentiviral vector derived from visna / maedi virus (VMV) has been developed. Initial characterisation using an enhanced green fluorescent protein (eGFP) reporter transgene found that the novel VMV vector efficiently transduced a wide range of cell lines including cells of ovine, human, murine, bovine and caprine origin. In addition, the VMV vector was found to elicit sustained transgene expression for at least 4 weeks in rapidly dividing cell lines. One of the most important factors for acceptable vaccines is their safety. Therefore, in order to increase the bio-safety of the VMV vector, integration-defective and self-inactivating forms were produced. Integration-defective VMV lentiviral vectors (IDLVs) were found to produce 1-LTR circular episomes favourably over integrated provirus following the transduction of target feline and ovine cell lines. This led to a decrease in transgene expression over time in dividing cells. In contrast, in non-dividing cells transgene expression was maintained at a similar level to integration-competent VMV vectors. Self-inactivating (SIN) VMV vectors were constructed and found to have a significant decrease in LTR activity. Transgene expression was maintained by the insertion of an internal promoter derived from human cytomegalovirus (CMV) acting directly on the transgene. When self-inactivating and integration-defective modifications were incorporated into the same vector particle, a 4-fold decrease in transduction relative to the parent vector was observed. Ovine monocyte-derived dendritic cells (MDDCs) and macrophages (MDMs) were found to be efficiently transduced by the VMV vector, whereas lentiviral vectors derived from HIV-1 poorly transduced both of these primary cell populations. Following this work, the ability to deliver pathogen genes into APCs was studied using the Chlamydia abortus (C. abortus) major outer membrane protein (MOMP) as the transgene. C. abortus is the most common infectious cause of ovine abortion worldwide and MOMP has previously been shown to stimulate strong antibody responses after vaccination. Unexpectedly, the VMV vector encoding either eGFP or MOMP was found to induce apoptosis in MDDCs and MDMs using Annexin V staining. Apoptotic cells were detectable as early as 6 hours post-transduction of cells. Furthermore, release of the pro-inflammatory cytokine IL-1β was associated with the formation of late apoptotic cells. Apoptotic bodies produced post-transduction were able to be phagocytosed by immature MDDCs and the transgene efficiently cross-presented to T-cells. The ability of the novel VMV vector to induce a suitable recall immune responses was investigated using an in vitro model. Here, an autologous population of MDDCs were cultured with the apoptotic bodies produced post-transduction before the addition of autologous PBMC. Proteins from the apoptotic bodies were presented by the MDDCs to PBMC leading to a strong, antigen specific recall immune response against C. abortus MOMP. This was proven by the detection of cytokines IFNγ and IL-10 in the co-culture supernatant from PBMC activated by the MOMP transgene cross-presented by MDDCs. No release of IL-4 or IL-17A could be detected. These data presented in this thesis show the potential for improving delivery of antigens in livestock vaccines by the use of lentiviral vectors. In addition, this vector system provides a strong base for the study of other potential protective antigens in vitro.

This thesis describes the development of serum and whole blood immunodiagnostic assays for Maedi-Visna virus (MVV). All previously described recombinant MVV ELISA assays utilised either the core p25 or transmembrane (TM) proteins alone, or combined, but as individual proteins. The p25 and TM genes of MVV were cloned individually into the pGEX-2T expression vector. Both proteins were expressed as a combined fusion protein in frame with glutathione S-transferase (GST). The purified recombinant antigens (GST-TM and GST-TM-p25) were used to develop a MVV ELISA. Sera from 46 positive and 46 negative sheep were tested using the GST-TM and GST-TM-p25 ELISAs and a commercial p25 EIA kit. A two-graph receiver operating characteristic (TG-ROC) analysis program was used to interpret the data. The GST-TM-p25 ELISA was more sensitive than the commercial assay which is based on the p25 antigen alone and more specific than the GST-TM ELISA. The GST-TM-p25 ELISA showed a sensitivity and specificity of 100%. The human AIDS lentivirus transmembrane (TM) glycoprotein portion of the envelope viral protein has been identified as the antigen most consistently recognised by antibodies. There is suggestive evidence that the same applies to MVV as the GST-TM fusion protein, expressed in E. coli, has comparable sensitivity to the GST-TM-p25 fusion protein, but lacks specificity. However, due to the hydrophobic nature of the MVV TM protein, purification of the expressed fusion protein required lengthy purification protocols. This was despite the fact that only a truncated version of the TM protein was expressed. This prompted investigating an alternative expression system that could possibly circumvent the above mentioned problems. The yeast Pichia pastoris is known to be suitable for the high-level expression of heterologous proteins which are secreted into the culture supernatant. These features made P. pastoris an attractive host for the expression of the hydrophobic TM protein of MVV. However, limited success was achieved as only low expression levels were obtained and detection and quantification was only accomplished by means of ELISA. Evaluation of the diagnostic performance of the P. pastoris expressed MVV TM-polypeptide was performed using a panel of 36 confirmed negative and positive sera, and evaluated using a TG-ROC analysis programme, which yielded an equal Se and Sp of 83%. The use of a novel rapid immunoassay system, which allows the detection of circulating antibodies in whole blood, has been investigated for use as a MVV diagnostic assay. The central feature of this immunoassay lies in a monoclonal antibody against a glycophorin epitope present on all sheep erythrocytes. A Fab'-peptide conjugate was constructed by coupling a synthetic peptide, corresponding to a sequence from MVV TM protein, to the hinge region of the Fab' fragment of the antisheep erythrocyte antibody. Within the limited number of 10 seronegative and 10 seropositive samples the autologous red blood cell agglutination assay had a sensitivity of 90% and a specificity of 80%. Despite the limitations and difficulties encountered, the use of such rapid whole blood immunodiagnostic assays for MVV holds promise. / Thesis (Ph.D.)-University of Natal, Durban, 1997.

Submitted by (edna.saturno@ufrpe.br) on 2016-11-09T12:16:52Z No. of bitstreams: 1 Whaubtyfran Cabral Teixeira.pdf: 1328857 bytes, checksum: 071dbd504f948b5d47ca8a7a13339069 (MD5) / Made available in DSpace on 2016-11-09T12:16:52Z (GMT). No. of bitstreams: 1 Whaubtyfran Cabral Teixeira.pdf: 1328857 bytes, checksum: 071dbd504f948b5d47ca8a7a13339069 (MD5) Previous issue date: 2012-02-09 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - CAPES / The aim of the present study was to determine the seroprevalence of small ruminant lentiviruses (LVPR), to characterize the zoosanitary management and describe the characteristics of the three most important sheep and goat rearing areas from Maranhão State, Brasil. 30 goats, 31 sheep and 52 mixed farms were visited, located in 23 municipalities of the Central, East and North regions of Maranhão. A questionnaire was applied on each property covering investigative details of the owner, property, herds, and hygiene, sanitary, nutritional and reproductive management adopted. The total number of animals in the herds was 3.405 goats and 2.971 sheep. To determine the prevalence of LVPR, 1.703 were analyzed blood serum samples of goats and 1.495 sheep. The animals were older than six months, various races and both sex. For the diagnosis of infection LVPR was used in the test agar gel immunodiffusion (AGID). The statistical analysis was performed using Epi Info version 6.04. The overall prevalence of CAEV infection was 2.8% (47/1703), and the prevalence in the regions North, East and Centre were 1.3% (5/385), 2.5% (18/713) and 4.0% (24/605), respectively. Significant differences between the North and Central were observed (P<0.05). Of the total number of herds sampled, 25.6% (21/82) had at least one positive animal. The prevalence of 4.4% (10/225) for males and 2.5% (37/1478) for females were significantly different (P<0.05). It was found that the prevalence did not increase with age (P>0.05). There prevalences of 11.3% (16/142), 3.5% (23/654) and 0.9% (8/907) were recorded in purebred, crossbred and SRD animals, respectively (P <0.05). It was found an overall prevalence of MVV infection of 0.7% (11/1495) and prevalence of 0.5%, 0.7% and 1.0% in the Central, East and North regions, respectively, with no statistically significant difference (P>0.05). Aditionaly, it was found that 0.5% (1/207) of males and 0.8% (10/1288) of females were seropositive (P>0.05). In relation to age was not observed significant difference (P>0.05). There was a prevalence of 1.5% (1/66), 1.0% (8/776) and 0.3% (2/653) for sheep purebred, crossbred and SRD, respectively (P>0.05). For the formation of the herds animals were purchased from the states of Piaui, Pernambuco, Ceará, Bahia and Paraíba. The main farming system adopted in both the goats (93.9%) and in sheep (92.8%) herds is the semi-extensive, where the animals are loose in the pasture during the day and are collected in the late afternoon. Regarding the type of fold used slatted (52.4% and 41.0%) and dirt (36.6% and 44.6%) were those with the highest frequencies. The sanitary practices adopted more frequently were: cleaning the premises, disinfection of the fold, cut and disinfection of the umbilical cord of the newborn, trimming, burial of corpses and separating sick animals. The most frequently clinical changes that affect goats and sheep were, respectively: worms (97.6% and 95.2%), caseous lymphadenitis (84.1% and 79.5), myiasis (79.3% and 73.5), abortion (73.3% and 67.5), pododermatitis (70.7% and 68.7%), parasitic skin disease (57.3% and 47.0%), mastitis (50.0% and 42.2%), arthritis (39.0% and 30.1%), contagious ecthyma (37.8% and 43.5%), keratoconjunctivitis (35.4% and 39.8%), pneumonia (29, 3% and 22.9%), diarrhea (23.2% and 19.3%) and neurological disorders (8.5% and 7.2%). It was found a high percentage of herds (65.8% in goat and 69.9% in sheep) that have problems with mortality, reaching up to 10% in each herd. Vaccination was adopted in 58.5% and 61.4% of goat and sheep herds, respectively. The deworming was the most frequently used practice for the control of nematode infections by 92.7% and 95.2% of goats and sheep, respectively. It is concluded that infection with LVPR is present in sheep and goats of located in the East, Central and Northern of Maranhão. In this sense, is explicit the need to implement control measures to prevent the spread of virus between flocks and new introductions in the state, by requiring negative tests for LVPR. It was observed that the health management adopted in goats and sheep properties in the Central, East and North regions of Maranhão, is poor, with serious problems that may interfere with the performance of the herds, requiring adjustments in order to maximize productivity and reduce costs. / Objetivou-se, no presente estudo, determinar a soroprevalência das lentiviroses de pequenos ruminantes (LVPR), caracterizar o manejo zoosanitário e descrever as características da caprinovinocultura nas três principais mesorregiões produtoras de caprinos e ovinos do Estado do Maranhão, Brasil. Foram visitados 30 criatórios de caprinos, 31 de ovinos e 52 mistos, localizados em 23 municípios das mesorregiões Centro, Leste e Norte Maranhense. Aplicou-se um questionário investigativo em cada propriedade abordando dados sobre o proprietário, propriedade, reabanho e os sistemas de manejos higiênico-sanitário, nutricional e reprodutivo. O número total de animais nesses rebanhos era de 3.405 caprinos 2.971 ovinos. Para determinar a prevalência de LVPR, foram analisadas 1.703 amostras de soro sanguíneo de caprinos e 1.495 de ovinos. Foram utilizados animais com idade superior a seis meses, de ambos os sexos e raças variadas. Para o diagnóstico da infecção pelo LVPR, utilizou-se o teste da imunodifusão em gel de ágar (micro-IDGA). A análise estatística dos dados foi realizada empregando-se o programa Epi Info versão 6.04. A prevalência geral da infecção pelo CAEV foi de 2,8% (47/1703), sendo as prevalências nas mesorregiões Norte, Leste e Centro de 1,3% (5/385), 2,5% (18/713) e 4,0% (24/605), respectivamente. Houve diferença significativa entre as prevalências das mesorregiões Norte e Centro (P<0,05). Do total de propriedades amostradas, 25,6% (21/82) apresentaram pelo menos um animal positivo. Com relação à variável sexo foi encontrada prevalência de 4,4% (10/225) para machos e 2,5% (37/1478) para as fêmeas, onde se evidenciou diferença significativa (P<0,05). Verificou-se que a prevalência não aumentou com a idade (P>0,05). Observaram-se prevalências de 11,3% (16/142), 3,5% (23/654) e 0,9% (8/907) para animais de raças puras, mestiços e SRD, respectivamente, apresentando diferença estatística significativa (P<0,05). Constatou-se uma prevalência geral da infecção pelo MVV de 0,7% (11/1495) e prevalências de 0,5% (3/564), 0,7% (4/539) e 1,0% (4/392) nas mesorregiões Centro, Leste e Norte, respectivamente, não sendo verificada diferença estatística significativa (P>0,05). Em relação à variável sexo, observou-se que 0,5% (1/207) dos machos e 0,8% (10/1288) das fêmeas foram soropositivos (P>0,05). Em relação à idade também não foi evidenciada diferença significativa (P>0,05). Observou-se prevalência de 1,5% (1/66), 1,0% (8/776) e 0,3% (2/653) para ovinos de raças puras, mestiços e SRD, respectivamente (P>0,05). Para a formação dos rebanhos base foram utilizados animais oriundos dos Estados do Piauí, Pernambuco, Ceará, Bahia e Paraíba. O principal sistema de criação adotado é o semi-extensivo, onde os animais permanecem soltos no pasto durante o dia e são recolhidos ao final da tarde, tanto nas criações de caprinos (93,9%) quanto nas de ovinos (92,8%). Quanto ao tipo de aprisco utilizado o ripado (52,4% e 41,0%) e o chão batido (36,6% e 44,6%) foram os que apresentaram as maiores frequências. As práticas sanitárias adotadas com maior frequência foram limpeza das instalações, desinfecção do aprisco, corte e desinfecção do cordão umbilical do recém-nascido, casqueamento, enterro dos cadáveres e separação de animais doentes. Alterações clínicas mais citadas que acometem os animais dos rebanhos caprinos e ovinos, respectivamente, foram verminose (97,6% e 95,2%), linfadenite caseosa (84,1% e 79,5), miíase (79,3% e 73,5), aborto (73,3% e 67,5), pododermatite (70,7% e 68,7%), ectoparasitose (57,3% e 47,0%), mastite (50,0% e 42,2%), artrite (39,0% e 30,1%), ectima contagioso (37,8% e 43,5%), ceratoconjuntivite (35,4% e 39,8%), pneumonia (29,3% e 22,9%), diarréia (23,2% e 19,3%) e alterações nervosas (8,5% e 7,2%). Foi verificado um percentual elevado (65,8% dos rebanhos caprinos e 69,9% nos ovinos) de propriedades que têm problemas com mortalidade, chegando a atingir até 10% em cada rebanho. A vacinação foi adotada em 58,5% e 61,4% dos rebanhos caprinos e ovinos, respectivamente, enquanto que a desverminação foi à prática mais adotada para o controle de verminoses por 92,7% e 95,2% dos criadores de caprinos e ovinos, respectivamente. Conclui-se que a infecção por LVPR está presente em caprinos e ovinos das mesorregiões Centro, Leste e Norte Maranhense. Nesse sentido, fica explícita a necessidade de implementar medidas de controle a fim de evitar a propagação dos vírus entre os rebanhos e novas introduções no Estado, através da exigência de testes negativos para LVPR. Observou-se que o manejo sanitário, adotado nas propriedades de caprinos e ovinos nas mesorregiões estudadas, é deficiente, apresentando sérios problemas que podem está interferindo no desempenho dos rebanhos, necessitando de adequações visando à maximização da produtividade e redução de custos.

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniviversitet, 2006. / Härtill 7 uppsatser.