O trabalho teve como objetivo avaliar a concentração sérica de estrógeno e progesterona em cadelas durante o ciclo estral por imunoensaio multianalito (Luminex® xMAP®), visando avaliar a aplicabilidade fisiológica e comparar os resultados obtidos com os valores já estabelecidos na literatura pelo método de radioimunoensaio (RIE). Foram utilizadas 7 cadelas da raça Border Collie, residentes do Canil Espaço Ducão (Valinhos/SP) de diferentes idades e ao primeiro sinal de sangramento vulvar (proestro/estro) foram coletadas amostras a cada 48 hs e ao seu termino (diestro), foi realizado coletas semanalmente (intervalo de 7 dias) durante aproximadamente 3 semanas. Houve correlação positiva apenas entre os valores de progesterona na fase de proestro/estro (p=0,001). Apesar de não ter ocorrido correlação entre as outras fases, as medias dos valores séricos de estradiol e progesterona foram estatisticamente significantes, em ambas as fases, sendo os valores obtidos pelo Luminex® maiores que os obtidos pelo RIE. Os valores de progesterona confirmaram os dados fisiológicos esperados em ambas as metodologias, porém os valores de estradiol não foram compatíveis com o esperado fisiologicamente, exceto quando analisados a partir das medias globais em ambas as fases. Novos estudos de validação laboratorial serão necessários para confirmar a validação fisiológica, principalmente no perfil da progesterona. Podemos concluir ainda que os valores de referência para o Luminex® devem ser diferentes dos valores já estabelecidos pelo RIE para ambos os hormônios testados / The study aimed to evaluate the serum concentration of estrogen and progesterone in female dogs during the estrous cycle by multiplex immunoassay (Luminex® xMAP®), to evaluate the physiological applicability and compare the results with the values established in the literature by radioimmunoassay (RIA). We used seven Border Collie bitches, residents in Kennel Espaço Ducão (Valinhos/SP), of different ages and for the first sign of vulvar bleeding (proestrus/estrus) samples were collected every 48 hours and its end (diestrus) were collected weekly (every 7 days) for about three weeks. There was a positive correlation only between progesterone values in the proestrus/estrus phase (p = 0.001). Despite having not occurred correlation between the other phases, the median of serum estradiol and progesterone values were statistically significant in both phases, the values obtained by Luminex® is higher than by RIA. Progesterone values confirmed the expected physiological data in both methods, however estradiol values were not physiologically compatible with the expected, except when analyzed from the global mean in both phases. A laboratory validation studies will be needed to confirm the physiological validation, especially in progesterone profile, and we can conclude that the reference values for the Luminex® should be different from the values established by RIE for both hormones tested

O trabalho teve como objetivo avaliar a concentração sérica de estrógeno e progesterona em cadelas durante o ciclo estral por imunoensaio multianalito (Luminex® xMAP®), visando avaliar a aplicabilidade fisiológica e comparar os resultados obtidos com os valores já estabelecidos na literatura pelo método de radioimunoensaio (RIE). Foram utilizadas 7 cadelas da raça Border Collie, residentes do Canil Espaço Ducão (Valinhos/SP) de diferentes idades e ao primeiro sinal de sangramento vulvar (proestro/estro) foram coletadas amostras a cada 48 hs e ao seu termino (diestro), foi realizado coletas semanalmente (intervalo de 7 dias) durante aproximadamente 3 semanas. Houve correlação positiva apenas entre os valores de progesterona na fase de proestro/estro (p=0,001). Apesar de não ter ocorrido correlação entre as outras fases, as medias dos valores séricos de estradiol e progesterona foram estatisticamente significantes, em ambas as fases, sendo os valores obtidos pelo Luminex® maiores que os obtidos pelo RIE. Os valores de progesterona confirmaram os dados fisiológicos esperados em ambas as metodologias, porém os valores de estradiol não foram compatíveis com o esperado fisiologicamente, exceto quando analisados a partir das medias globais em ambas as fases. Novos estudos de validação laboratorial serão necessários para confirmar a validação fisiológica, principalmente no perfil da progesterona. Podemos concluir ainda que os valores de referência para o Luminex® devem ser diferentes dos valores já estabelecidos pelo RIE para ambos os hormônios testados / The study aimed to evaluate the serum concentration of estrogen and progesterone in female dogs during the estrous cycle by multiplex immunoassay (Luminex® xMAP®), to evaluate the physiological applicability and compare the results with the values established in the literature by radioimmunoassay (RIA). We used seven Border Collie bitches, residents in Kennel Espaço Ducão (Valinhos/SP), of different ages and for the first sign of vulvar bleeding (proestrus/estrus) samples were collected every 48 hours and its end (diestrus) were collected weekly (every 7 days) for about three weeks. There was a positive correlation only between progesterone values in the proestrus/estrus phase (p = 0.001). Despite having not occurred correlation between the other phases, the median of serum estradiol and progesterone values were statistically significant in both phases, the values obtained by Luminex® is higher than by RIA. Progesterone values confirmed the expected physiological data in both methods, however estradiol values were not physiologically compatible with the expected, except when analyzed from the global mean in both phases. A laboratory validation studies will be needed to confirm the physiological validation, especially in progesterone profile, and we can conclude that the reference values for the Luminex® should be different from the values established by RIE for both hormones tested

Master of Science / Department of Diagnostic Medicine and Pathobiology / Raymond R. R. Rowland / Three economically important swine diseases: Porcine Reproductive and Respiratory Syndrome (PRRS), Porcine Circovirus Associated Disease (PCVAD) and Swine influenza cost the US swine industry more than a billion dollars each year. This study developed a fluorescent microsphere immunoassay (FMIA) to simultaneously detect antibodies to the causative pathogens: PRRSV, porcine circovirus (PCV2) and swine influenza virus (SIV). The results showed that the multiplex assay possessed the predicted specificities. In the case of PRRSV NA, the assay displayed higher sensitivity when compared to a commercially available ELISA. The assay was employed to measure both IgG and IgM responses. The FMIA was found to possess several advantages over standard ELISA which include reduced sample volume, time and cost and provides a new tool for veterinary diagnostics. The FMIA was applied for swine disease surveillance in Hawaiian and Texan feral swine populations. The antibodies against PCV2 showed the highest prevalence among these three pathogens in both Hawaii and Texas. Hence we consider PCV2 as the most prevalent pathogen in Hawaiian and Texan feral pigs and this pathogen poses the greatest threat to commercial pigs. SIV seroprevelance increased from 2007 to 2010 in Hawaii State, suggesting an increasing risk for commercial pigs. Moreover, yearly surveillance in Texas State shows growth in seropositive response to all pathogens, particularly PCV2. The development of FMIA for detection of antibodies to multiple swine pathogens in serum samples offers an important alternative for swine disease surveillance in commercial and feral herds.

L'hémophilie A (HA) est une maladie hémorragique héréditaire due au déficit en facteur FVIII (FVIII) de la coagulation. Le traitement préventif de l'HA est basé sur des injections répétées de FVIII. Une réponse immunitaire anti-FVIII peut se développer secondairement au traitement, mettant en jeu des anticorps (Ac) inhibiteurs (neutralisant l'activité procoagulante du FVIII) et/ou des anticorps non-neutralisants (ANN). La cartographie épitopique fine des Ac anti-FVIII permet de mieux connaître les mécanismes physiopathologiques de cette réponse immunitaire. Les travaux de cette thèse comportent deux axes principaux : un premier axe a pour objectif d'identifier des épitopes discontinus au sein des domaines C2 et A2 du FVIII, à l'aide de peptides synthétiques prédits par un algorithme informatique, utilisés ensuite dans des tests d'inhibition basés sur la technologie Luminex. Ces travaux nous ont permis d'identifier 8 peptides mimant des épitopes discontinus répartis à la surface du domaine C2 et 2 peptides correspondant à des épitopes voisins, à la surface du domaine A2. Ces études ont permis de démontrer que la combinaison de la bioinformatique et d'un outil expérimental adapté à la réponse immunitaire anti-FVIII est fructueuse. Le second axe a pour objectif d'étudier la prévalence et la spécificité épitopique des ANN à l'aide d'un test Luminex multiplexé. Cette étude a permis de mettre en évidence une prévalence d'ANN de 18,1% chez 210 hémophiles A sans inhibiteurs provenant d'une cohorte multicentrique rétrospective française. Une forte spécificité épitopique de la réponse immune pour la chaîne lourde du FVIII est observée. Les nouveaux outils que nous avons mis en place permettront d'affiner la cartographie épitopique et le suivi de son évolution chez l'hémophile A avec anticorps anti-FVIII / Haemophilia A (HA) is an inherited bleeding disorder due to factor VIII (FVIII) deficiency. The preventive treatment of HA is based on regular infusions of FVIII. Secondary to the treatment, an immune response often occurs, composed by inhibitory antibodies and by non-neutralising antibodies (NNA). Fine epitope mapping of anti-FVIII antibodies may help for a better understanding of the physiopathology of this immune response. There was two axes in this PhD thesis: the first part is dedicated to the identification of discontinuous epitopes on FVIII C2 and A2 domains, by using synthetic peptides predicted by a bioinformatic tool in inhibition tests based on Luminex technology. Results allowed us to identify 8 peptides mimicking discontinuous epitopes around the C2 domain and 2 peptides mimicking close epitopes on the A2 domain surface. These studies demonstrate that our approach combining bioinformatics with an assay adapted for the anti-FVIII immune response study is fruitful. The second part was dedicated to the evaluation of the prevalence and epitope specificity of NNA, using a multiplexed Luminex assay. A prevalence of 18.1% of NNA was thus found in 210 HA patients without inhibitors from a french multicentric retrospective cohort. An marked epitope specificity againt the heavy chain was noticed. The new tools that we developped will be helpful for refining epitope mapping and for the follow-up of the epitope specificity in HA patients with anti-FVIII Abs.

Background: As a part of a national health control program, Statens VeterinärmedicinskaAnstalt performs diagnostics to screen flocks for certain pathogens causing high mortality,morbidity and/or serious economical losses. There are several viruses in the programincluding IBDV (infectious bursal disease virus), IBV (infectious bronchitis virus) and NDV(Newcastle disease virus). Method: 96 serum samples were collected from different poultryflocks in Sweden and analyzed by ELISA, which are currently used in the health controlprogram as well as by a commercial prototype of a multiplex immunoassay manufactured byLuminex Corp., which is currently under evaluation at the United States Department ofAgriculture USDA. This 4-plex assay detects antibodies for the three above-mentionedviruses as well as antibodies of avian reovirus. In the context of this study the ELISAs run inroutine diagnostics as well as a REO ELISA were used as the standard for comparison.Result: The antibody concentration in serum from vaccinated chickens was high while theantibody concentration level in serum from not vaccinated chickens was low. There was agood correlation between the multiplex assay and ELISA. Conclusion: The results obtainedin this study indicate that the Luminex multiplex assay tested has the potential to be usedroutinely for screening flocks.

West Nile virus (WNV) and St. Louis Encephalitis (SLEV) are arthropod-borne viruses belonging to the genus Flavivirus and are classified as significant human pathogens of global epidemiological importance. Since its introduction into the United States in 1999, WNV has spread throughout most of the country and has caused major epidemics of neuroinvasive disease (Hayes and Gubler, 2005). SLEV is endemic to the United States and is maintained in an enzootic transmission cycle in Florida.The Florida Sentinel Chicken Arboviral Surveillance Network was established in 1978 following a widespread rural epidemic of SLEV in central Florida to monitor the activity of arboviruses (Day and Stark, 1996). This program ultimately impacts vector control strategies and may warrant medical alerts to warn the population. Current serological detection methods for sentinel chickens include hemagglutination inhibition antibody test (HAI), IgM antibody capture enzyme-linkedimmunosorbent assay (MAC-ELISA), and Plaque Reduction Neutralization Test (PRNT).These serological assays may take over three weeks to generate a final result. A more rapid and equally sensitive test to replace these current serological methods would be of benefit. Microsphere-based immunoassays (MIAs) are a more rapid serological option for laboratory diagnosis of many diseases (Kellar et al, 2001). The objective of this study was to develop and validate a protocol for a MIA to detect antibodies to WNV and SLEV in sentinel chicken sera. A total of 385 sentinel chicken sera from 2005 were assayed using the MIA for WNV and 424 sera from multiple years were assayed for SLEV. The capability of the MIA to multiplex allowed for simultaneous detection of antibodies to WNV and SLEV in sentinel chicken sera. The MIA was found to be more sensitive and specific than both the HAI and MAC-ELISA for the detection of antibodies to WNV, and just as sensitive and specific as the MAC-ELISA for the detection of antibodies to SLEV in sentinel chicken sera. These results indicate that there is a potential of the MIA to decrease turn-around time and allow for earlier detection and improvement to the current surveillance system.

Surveillance of domestic poultry flocks for antibodies against avian influenza and Newcastle disease to detect and differentiate between these diseases is very important. The ability to determine if the detected influenza virus antibodies belong to one of the reportable H5 or H7 subtypes is imperative. These two major viruses are continually responsible for economic loss in poultry industries all over the world. Current serological methods of detection are an effective means of detecting antibody responses to these viruses, however continually investigating improved methods of surveillance is important. Development of a serological assay using Luminex technology which involves the use of recombinantly generated influenza A nucleoprotein, hemagglutinin H5, hemagglutinin H7, and Newcastle disease nucleocapsid proteins bound to Magplex beads allowed for the simultaneous detection of antibodies against these proteins that matches the efficiency of past methods while maintaining high levels of specificity and overall accuracy. Assay development took the form of two connected projects beginning with construction of an assay that operated in duplex, detecting antibodies against influenza nucleoprotein (AIV-NP) and Newcastle disease nucleocapsid protein (APMV-1-NC). Once optimized, the second half of development involved expansion of the assay to include detection of H5 (AIV-H5) and H7 (AIV-H7) subtypes, as well as the addition of internal assay quality controls to monitor assay performance over time. Assay thresholds and overall performance of both of these functional assays were evaluated using large quantities of field and experimental sera from chickens and turkeys to maximize specificity and overall accuracy.

Encephalitis caused by the alphaherpes viruses HSV 1, HSV 2 and VZV can be devastating and rapid, accurate diagnosis is required. Whilst existing molecular techniques are invaluable in diagnosing acute disease, detection of antibody is needed to confirm infection and to make a diagnosis after the acute stage or during post-infectious encephalitis. Current immunoassays are limited by the volume of sample required. The aim of this project was to develop a rapid, accurate, low sample volume assay to improve diagnosis using Luminex technology.The immunodominant proteins of HSV and VZV, glycoprotein D (gD) and glycoprotein E (gE), were expressed in insect cells using a baculovirus expression vector. Expressed proteins were purified, characterised and used to develop in-house enzyme-linked immunosorbent assays (ELISA) to detect HSV and VZV type-specific antibodies. The performance of each newly developed in-house ELISA was compared with commercial ELISA assays using well characterised serum panels. An excellent correlation between the in-house ELISAs and the commercial ELISA assays (100% for HSV gD and 99% for VZV gE) was observed. To differentiate between HSV-1 and HSV-2 a new commercial ELISA assay (Omega) utilising a branched chain peptide (peptide 55 which provides immune selection of HSV-2 specific antibody) was evaluated against two commercially available HSV-2 ELISA assays. The Omega assay showed an overall agreement of 97.6% with Western blot and other ELISA assays. The two expressed proteins, together with peptide 55, were used to develop a triplex fluorescent microbead immunoassay for the simultaneous detection and quantitation of anti-viral antibody in human sera. Initially a monoplex assay for each analyte was developed and optimised individually and then the three assays were mixed together in a triplex assay. Results for HSV-1 gD and VZV gE obtained from the triplex assay showed a 100% agreement with HSV-1 and VZV in-house ELISA results. In the case of peptide 55, the triplex assay results showed better sensitivity than the Omega ELISA assay with an overall agreement with Western blot and other assays of 98.4%. In addition, in order to facilitate the diagnosis of alphaherpesviruses CNS infections the triplex assay was joined together with a biplex fluorescent microbead immunoassay designed for detecting and measuring human IgG and albumin in CSF and serum samples. The sensitivity and reproducibility of the resultant five-analyte multiplex immunoassay and the previous triplex assays were compared and found to have equivalent sensitivity and specificity. The sensitivity and minimal sample requirements of the new assay suggests that it will be a powerful tool for the diagnosis and study of both acute and post-infectious viral encephalitis.

Infecção congênita é aquela transmitida da mãe ao feto antes do nascimento. A transmissão vertical pode ocorrer por via transplacentária ou por contato direto com o patógeno durante o parto. A fonte de infecção é o microrganismo presente no sangue da gestante durante a infecção primária ou crônica. Estima-se que as infecções perinatais representam 2% a 3% de todas anomalias congênitas e as mais comuns são representadas pela sigla TORCH, que inclui Toxoplasmose, Outras (como sífilis e varicela-zoster), Rubeóla, Citomegalovírus e Herpes. A maioria das infecções TORCH causa morbidade materna leve, assintomática, mas tem consequências fetais graves e, em geral, o tratamento da infecção materna não tem impacto sobre o resultado fetal. Por isso, o reconhecimento do contágio materno e o monitoramento fetal são de extrema importância na prevenção de doenças congênitas. O presente projeto visa o desenvolvimento de um produto multiparamétrico utilizando a tecnologia xMAP®, criada pela companhia norte-americana Luminex Corporation, para detecção simultânea de anticorpos da classe IgG anti-Toxoplasmose, anti-Rubéola e anti- Citomegalovírus em amostras de sangue coletado em papel de filtro. O produto, inédito no mercado nacional, tem o objetivo de atender à demanda específica da triagem pré-natal no país. Como objetivos específicos pode-se citar: a melhoria da eficiência dos programas de triagem pré-natal, graças à economia de tempo, amostras e reagentes; a contribuição financeira para o Brasil, uma vez que será produzido nacionalmente, gerando emprego e renda; a possibilidade de ampliar o mercado a partir do desenvolvimento futuro de novos produtos baseados na mesma metodologia. Para tanto, utilizaram-se antígenos específicos acoplados à microesferas de poliestireno e anticorpos de detecção conjugados à estreptavidina-ficoeritrina. Foram analisadas 1499 amostras de gestantes, coletadas em papel de filtro, cedidas e previamente triadas pela APAE-Goiânia, com o objetivo de comparar os resultados obtidos através da análise com o protótipo desenvolvido com aqueles já confirmados pelo laboratório utilizando a tradicional metodologia de ELISA. Os resultados de Concordância e Sensibilidade foram superiores a 78% para todos os parâmetros. Por outro lado, os valores de Especificidade foram mais baixos, principalmente para os parâmetros Rubéola e Citomegalovírus. Importante ressaltar o pequeno número de amostras com resultado negativo para Citomegalovírus e Rubéola disponível, o que refletiu diretamente no cálculo da Especificidade do produto / Congenital infections are transmitted from the mother to the fetus before birth. Vertical transmission can occur via placenta or by direct contact with the pathogen during childbirth. The source of infection is the microorganism present in the pregnant woman\'s blood during primary or chronic infection. It is estimated that perinatal infections are responsible for 2% to 3% of all congenital abnormalities and the most common are represented by the acronym TORCH, including Toxoplasmosis, Others (such as syphilis and varicella-zoster), Rubella, Cytomegalovirus (CMV) and Herpes. Most infections TORCH causes mild maternal morbidity, asymptomatic, but has serious fetal consequences fetal and, generally, maternal infection treatment has no impact on fetal outcome. Therefore, the recognition of maternal infection and fetal monitoring are extremely important in preventing birth defects. This project aims to develop a product using the multiplex xMAP® technology, created by US company Luminex Corporation, for simultaneous detection of IgG antibodies anti-toxoplasmosis, antirubella and anti-cytomegalovirus in blood samples collected in filter paper. The product, unprecedented in brazilian market, aims to meet the specific demand of prenatal screening in Brazil. The specific objectives are: improving the efficiency of prenatal screening programs, thanks to savings in time, samples and reagents; the financial contribution for Brazil, as it will be produced nationally, generating jobs and income; the possibility of expanding the market from the future development of new products based on the same methodology. For this purpose, were used specific antigens coupled to polystyrene beads and antibodies conjugated to streptavidin-phycoerythrin. Were analyzed 1499 samples of pregnant women, collected on filter paper, pre-screened by APAE-Goiânia, in order to compare the results obtained from the analysis with the prototype developed with those already confirmed by the laboratory using traditional ELISA methodology. The results of Concordance and Sensitivity were higher than 78% for all parameters. In contrast, the Specificity values were lower, especially for Rubella and Cytomegalovirus parameters. Importantly the small number of negative samples negative for Cytomegalovirus and Rubella provided by APAE-Goiânia, which is directly reflected in the product specificity value.

La Fièvre Catarrhale Ovine est une arbovirose due à un Orbivirus touchant les ruminants. Récemment, une épizootie majeure, notamment due au sérotype 8 du virus (BTV-8), a touché les ruminants Européens. Ce sérotype a présenté plusieurs particularités telles que le spectre d’hôte original, et la transmission transplacentaire. L’impact de l’infection par le BTV-8 chez le caprin a été étudiée dans ce travail d’un point de vue clinique, virologique, hématologique et sérologique, et notamment pour ce dernier aspect à travers le développement de deux outils sérologiques. L’impact d’une infection maternelle sur le fœtus a également été étudié.Ces travaux ont confirmé que l’impact de l’infection par le BTV-8 chez les caprins est modéré, tant d’un point de vue clinique que d’un point de vue hématologique. Ce travail a également permis de faire ressortir plusieurs connaissances nouvelles: un impact modéré sur les comptages leucocytaires ; une transmission transplacentaire du virus lors d’infection en milieu de gestation ; une détection du virus dans la semence ; une possible transmission « directe », non vectorielle.Ces 3 dernières constatations n’avaient jusqu’alors pas été rapportées dans la littérature chez les caprins, mais avaient été observées chez les ovins et les bovins. Ceci confirme que, même si les caprins sont des animaux sensibles mais que l’impact clinique est limité, la plupart des caractéristiques faisant la spécificité de l’infection par la souche européenne du BTV-8 peuvent également être retrouvées dans cette espèce. Néanmoins, l’absence de description de ces particularités dans des conditions d’infection naturelle ne permet pas de conclure quant à leur impact sur le terrain.Des outils sérologiques ont été également développés afin d’étudier les propriétés antigéniques des protéines virales chez le caprin. La production des protéines recombinantes NS1, NS3, VP7 et VP2 en système baculovirus, et de la protéine NS2 en système E. coli, ont permis l’obtention de protéines recombinantes. Ces 5 protéines recombinantes ont permis le développement de tests sérologiques permettant d’étudier leurs propriétés antigéniques et la cinétique d’apparition des anticorps après vaccination ou/et infection par le BTV-8 chez le caprin.Dans un premier temps, des tests ELISA indirect NS1, NS2, NS3, VP7 et VP2 ont été développés, et la capacité des tests ELISA NS et VP7 à permettre une différenciation entre les animaux infectés et vaccinés a été évaluée. Cependant, des anticorps anti-NS2 et NS3 ont été détectés dans les sérums d’animaux vaccinés et une faible réponse obtenue en ELISA NS1 chez les animaux infectés rend difficile l’utilisation d’un test ELISA DIVA basé sur ces 3 protéines non structurales. Enfin, une réponse en anticorps anti-VP2 est détectée par le test ELISA VP2 après vaccination et après épreuve virulente, suggérant une détection d’anticorps spécifiques de type par ce test.Dans un second temps, un test Luminex multiplex, basé sur l’utilisation des protéines VP7 et VP2 a été développé. Le Luminex VP7 présente une très bonne corrélation avec l’ELISA VP7 lorsque les sérums d’animaux infectés sont testés, avec une aire sous la courbe de 0,987. Les performances de ce test paraissent cependant modérées lorsque des sérums issus d’animaux ayant reçu une unique injection vaccinale sont testés. Le Luminex VP2 présente des performances également excellentes, avec une aire sous la courbe de 0,978. Les VPP et VPN ont été calculées pour des prévalences très faibles (0,5%, correspondant à la prévalence devant être détectée par le screening sérologique d’animaux sentinelles) : la VPP est alors très faible mais la VPN est très élevée (99,99% pour VP7, 99,95% pour VP2). Le test Luminex multiplex développé, caractérisé par une VPN très élevée, permet d’exclure avec confiance la présence du BTV-8 dans une région indemne lors de résultat négatif, correspondant parfaitement aux objectifs assignés. / Bluetongue is an infectious non contagious arbovirosis caused by Bluetongue virus (BTV), belonging to the genus Orbivirus. Recently, a major epizooty, due to BTV-8, was encountered in European ruminants. This serotype presented several original features such as an original host spectrum and transplacental transmission. This work consisted in studying the impact of BTV-8 infection in goats from a clinical, virological, haematological and serological (after development of two new serological tests) point of view, because of the lack of knowledge in this specie. The impact on foetuses of infection during gestation was also studied.The different animal studies realised confirmed that the BTV-8 infection has a moderate impact in goats from a clinical and haematological point of view. These studies led to obtain new information about BTV-8 impact: moderate impact on leucocytes counts; transplacental transmission of the virus when infection occurs in mid-pregnancy; detection of BTV-8 in bucks’ semen; direct, non vectorial transmission. The last 3 results had never been described in goats with BTV-8 before but had been encountered in sheep and cattle: it proves that, even if goats are susceptible to the infection but are less affected by the virus, most of feature of BTV-8 North European strain can also be encountered in this specie. However, these features have not been described in natural conditions, making impossible to conclude on their impact in the field.In a second part of this thesis, serological tool have been developed in order to study antigenic properties of viral proteins in goats. Recombinant proteins NS1, NS3, VP7 and VP2 were produced in baculovirus system, while NS2 was produced in E. coli system. These recombinant proteins were used to develop serological test in order to study antigenic properties and the kinetic of antibodies response against this 5 proteins after vaccination against and infection by BTV-8 in goats.In a first part, indirect ELISA NS1, NS2, NS3, VP7 and VP2 were developed, and the opportunity to develop DIVA ELISA test using NS and VP ELISA was evaluated. However, detection of antibodies against NS2 and NS3 in vaccinated animals, and the difficulties to detect antibodies against NS1 in infected animals led us to conclude that a DIVA ELISA test using non-structural proteins was difficult. Finally, it was possible to detect antibodies against VP2 in infected and vaccinated animals using our VP2 ELISA, suggesting a detection of antibodies specific of serotype by this test.In a second part, a multiplex Luminex test, using VP7 and VP2, was developed. This test has, for VP7 detection, a strong correlation with cELISA VP7, with an area under the curve of 0.987. Luminex VP7 performance is moderate when sera from goats having only one vaccine administration were tested. Concerning Luminex VP2, test performance are also excellent with an area under the curve of 0.978. When a prevalence of 0.5% was applied (prevalence that should be detected by serological screening in Europe), de predictive negative value was very high (99.99% for Luminex VP7; 99.95% for Luminex VP2). The Luminex test developed, with a very high PNV, can exclude with a high level of confidence the presence of BTV-8 in a free-area.