A vinca (Catharanthus roseus) é uma planta perene, arbustiva, pertencente à família Apocinaceae, cujas folhas e raízes possuem propriedades medicinais. A presença de sintoma de mosaico e deformação foliar em plantas dessa espécie, associados com a presença de partículas alongadas e flexuosas, característica de vírus pertencentes ao gênero Potyvirus, conduziu a estudos complementares para a identificação e caracterização desse vírus. No estudo da gama parcial de hospedeiras foram testadas 28 espécies, envolvendo oito famílias botânicas. Catharanthus roseus e Nicotiana benthamiana apresentaram sintomas de mosaico foliar e Chenopodium amaranticolor e C. quinoa apresentaram lesões locais cloróticas nas folhas inoculadas. A transmissão do vírus com afídeos foi avaliada com as espécies Aphis gossypii, Myzus nicotianae e Toxoptera citricidus. Apenas Aphis gossypii e Myzus nicotianae transmitiram o vírus. O antissoro policlonal produzido contra este potyvirus reagiu com o vírus homólogo e com o Passionfruit woodiness virus (PWV) e Cowpea aphid-borne mosaic virus (CABMV), mas não com o Lettuce mosaic virus (LMV), Papaya ringspot virus - type W (PRSV-W), Potato virus Y (PVY) e Zucchini yellow mosaic virus (ZYMV). O peso molecular da proteína capsidial (CP) foi de aproximadamente 34 kDa. A reação de PCR realizada com os oligonucleotídeos universais de potyvirus e oligonucleotídeos específicos posteriomente confeccionados amplificaram três fragmentos de aproximadamente 0,8, 1,0 e 1,4 Kb, os quais após o seqüênciamento geraram um fragmento de 1654 nucleotídeos (nt) da região 3' terminal do genoma, que inclui parte do gene da replicase viral (Nib), a região codificadora completa do gene da proteína capsidial (CP), seguida de 286 nt da região 3' não traduzida (3'NTR). A identidade da seqüência de nucleotídeos do gene da CP variou de 67,0 a 76,0%, quando comparada com as de outros membros da família Potyviridae. A maior identidade foi com o Omphalodes virus Y (76,0%). A identidade dos aminoácidos deduzidos da proteína capsidial variou de 62,0 a 71,0%, sendo a maior com East Asian Passiflora virus (71%). Para a região não traduzida (3'NTR) a identidade variou de 16,8 a 28,6%. Em conjunto esses dados indicam que este vírus é uma nova espécie dentro do gênero Potyvirus, para o qual se propõe o nome de Vírus do mosaico do Catharanthus (Catharanthus mosaic virus - CatMV). / Catharanthus roseus is known as the common periwinkle or Madagascar periwinkle. It is a perennial, evergreen herb in the family Apocynaceae, which was originally native to the island of Madagascar, although both name and classification are contradictory in some literature. The plants grow up to 80 cm high; have glossy, dark green leaves and bloom during summer. The flowers range from white to hot pink to purple. The species has historically been used in popular medicine to treat a wide assortment of human diseases, as it contains more than 150 useful alkaloids. Plants of C. roseus exhibiting mosaic symptoms followed by malformation of the leaf blades and flower variegation were collected from a garden at the University of São Paulo, School of Agriculture (Piracicaba, State of São Paulo, Brazil). Preliminary electron microscopy exams of negatively stained leaf sap revealed that the symptoms were associated with potyvirus-like particles. The objective of the present work was to obtain further biological, immunological and molecular data to better characterize this species of the genus Potyvirus, family Potyviridae. Of 28 plant species from eight botanical families inoculated mechanically with this potyvirus, only C. roseus and Nicotiana benthamiana developed systemic mosaic, whereas Chenopodium amaranticolor and C. quinoa exhibited only chlorotic local lesions. The virus was transmitted by Aphis gossypii and Myzus nicotianae, but not by Toxoptera citricidus. Polyclonal antiserum raised against this potyvirus reacted with the homologous virus, Passion fruit woodiness virus (PWV) and Cowpea aphid borne mosaic virus (CABMV) in PTA-ELISA. The molecular mass of the coat protein (CP) was approximately 34 kDa. RT-PCR from viral RNA amplified a fragment of approximately 1654 nucleotides (nt) at the 3'-terminal of the viral genome, containing portion of the replicase gene (Nib), the entire CP gene and the 3' untranslated region (3'UTR) (286 nt). When the nucleotide sequence of the CP gene was compared with other members of the Potyviridae family, identities varied from 67.0 to 76.0%. The highest identity was with Omphalodes virus Y. Identity of the deduced amino acid of the CP varied from 62.0 to 71.0%, with the highest for East Asian Passiflora virus. For the 3' UTR, identities varied from 16.8 to 28.6%. The name Catharanthus mosaic virus (CatMV) is proposed for this new potyvirus.

O vírus do mosaico amarelo da abobrinha (Zucchini yellow mosaic virus - ZYMV) é provavelmente um dos mais dinâmicos e prejudiciais vírus emergentes de plantas. Desde sua primeira detecção na Itália e na França em 1973 e 1979, respectivamente, o ZYMV tem sido reportado em mais de 50 países variando de condições climáticas tropicais a temperadas em todos os continentes, exceto na Antártida. No Brasil esse potyvirus foi constatado primeiramente nos Estados de São Paulo e Santa Catarina, no início da década de 90 e posteriormente nos Estados do Ceará, da Bahia, do Rio Grande do Norte, do Pará, do Mato Grosso do Sul e do Maranhão. É provável que atualmente ocorra em todos os estados brasileiros onde se cultivam cucurbitáceas. Embora ocorra no Brasil há mais de 20 anos, pouco se conhece sobre as características biológicas, sorológicas e moleculares dos isolados até então encontrados no país. Esse projeto de pesquisa teve por objetivo cobrir parcialmente essa lacuna para fornecer subsídios para programas de melhoramento genético. Foram utilizados 11 isolados coletados em diversos estados brasileiros. A caracterização biológica consistiu no estudo da reação de diversas espécies vegetais inoculadas mecanicamente. A caracterização sorológico consistiu na marcação da proteína capsidial dos isolados com o antissoro policlonal contra o vírus. A caracterização molecular foi feita por meio da análise das sequências de nucleotídeos e aminoácidos deduzidos do gene da proteína capsidial dos isolados. A reação das espécies variou de acordo com o isolado inoculado. Análises de RFLP mostraram que a enzima de restrição Hpa II permitiu diferenciar o isolado fraco ZYMV-M da maioria dos isolados severos. O peso molecular da proteína capsidial dos isolados analisados foi em torno de 36 KDa, típico da proteína capsidial desse potyvirus. A identidade de nucleotídeos do gene da proteína capsidial entre os isolados brasileiros do ZYMV variou de 93% a 100% e a de aminoácidos deduzidos variou de 97% a 100%. Comparados com as sequências correspondentes de isolados do ZYMV de diferentes origens geográficas a identidade de nucleotídeos variou de 82% a 99%, enquanto a de aminoácidos deduzidos ficou entre 87% e 99%. Na árvore filogenética os isolados brasileiros do ZYMV pertencem ao grupo A, subdivisões I ou II, indicando uma variação entre os isolados. Nenhum evento de recombinação foi detectado nos isolados brasileiros. / Zucchini yellow mosaic virus (ZYMV) is probably one of the most dynamics and economically important emerging plant viruses. Since it was first report in Italy and France in 1973 and 1979, respectively, ZYMV has been found in over than 50 countries ranging from tropical to temperate climate conditions in all continents, except in Antartida. In Brazil this potyvirus was first reported in the beginning of 90´s in São Paulo and Santa Catarina States, and then in Ceará, Bahia, Rio Grande do Norte, Pará, Mato Grosso do Sul and Maranhão states. Probably it occurs in all Brazilian states which grow cucurbit species. Although ZYMV occurs in Brazil for more than 20 years, there is little information about the biological, serological and molecular characteristics for the isolates found in the country. The purpose of this work was to cover partially this gap to provide subsidies for genetic breeding programs. Eleven isolates collected in several Brazilian states were used. The biological characterization consisted in the study of the reaction of several vegetal species mechanically inoculated with the virus. The serological characterization consisted in the identification of the molecular weight of the coat protein through western blot analysis. The molecular characterization was done by the analyses of nucleotides and deduced amino acids sequences for the coat protein gene. The host reaction showed slight variation according to the virus. RFLP analyses showed that restriction enzyme HpaII allowed differentiate the mild ZYMV-M isolate from the majority of severe isolates. The coat protein molecular weight for all studied isolates was about 36 KDa, typical of the coat protein of this potyvirus. The nucleotide identity of the coat protein gene among the ZYMV Brazilian isolates ranged from 93% to 100%, and the deduced amino acids sequences ranged from 97% to 100%. Compared to corresponding sequences of ZYMV isolates from different geographical locations the nucleotide sequences identity ranged from 82% to 99%, while the deduced amino acids sequences ranged from 87% to 99%. Phylogenetic analysis place the Brazilian isolates of ZYMV into the group A, subdivision I or II, showing some diversity between the isolates. No recombination event was detected in the Brazilian isolates.

Submitted by isabela.moljf@hotmail.com (isabela.moljf@hotmail.com) on 2017-05-18T13:19:42Z No. of bitstreams: 1 josegeraldodecastrojunior.pdf: 7182456 bytes, checksum: 31d33bc1bedcad933b465924da4098e3 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-05-18T13:40:17Z (GMT) No. of bitstreams: 1 josegeraldodecastrojunior.pdf: 7182456 bytes, checksum: 31d33bc1bedcad933b465924da4098e3 (MD5) / Made available in DSpace on 2017-05-18T13:40:17Z (GMT). No. of bitstreams: 1 josegeraldodecastrojunior.pdf: 7182456 bytes, checksum: 31d33bc1bedcad933b465924da4098e3 (MD5) Previous issue date: 2013-12-04 / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / No Brasil, a leishmaniose visceral (LV), também conhecida como calazar, é uma zoonose, de transmissão vetorial, causada pelo protozoário Leishmania chagasi. Esta doença, anteriormente descrita como rural vem passando por um processo de urbanização. Em 2008, foram diagnosticados no município de Juiz de Fora, os primeiros casos considerados autóctones de leishmaniose visceral canina (LVC) e estudos sobre epidemias urbanas da LV têm indicado que a LVC vem precedendo a infecção humana, visto que os cães são os principais reservatórios domésticos. Assim, o presente estudo teve como objetivo pesquisar a infecção de LVC no município de Juiz de Fora, bem como avaliar os fatores de risco associados à doença. O trabalho foi realizado com animais do canil municipal e ONGs e a partir do soro destes foram realizadas três técnicas sorológicas: o imunocromatográfico “TR DPP® e ELISA (“EIE-Leishmaniose visceral canina®), ambos fornecidos pela FIOCRUZ/Bio-Manguinhos e ELISA in house. A amostra totalizou 781 animais e a prevalência da LVC variou de acordo com a técnica empregada: o teste DPP apresentou soropositividade de 4,87% (IC 95% de 3,5-6,7%); o ELISA Bio-Manguinhos de 2,18% (IC 95% de 1,3-3,5%) e ELISA in house de 13,73% (IC 10,8-17,3%). Em relação à variabilidade observada entre as técnicas, vale a pena destacar que as mesmas apresentam antígenos diferentes e isto pode refletir nos resultados. A amostra foi composta, em sua maioria por fêmeas adultas, sem raça definida, porte médio e pelo curto. Na análise univariada, utilizando-se o ELISA Bio-Manguinhos como confirmatório para a LVC, foi observada associação estatística (p< 0,05; IC 95%) com sintomatologia clínica segundo Quinnell et al. 2003, origem dos animais (canil municipal) e grupo racial; além disto, houve sugestão de associação com sexo masculino e porte médio/grande dos animais. Na análise multivariada, o fato de ser procedente do canil e sintomatologia clínica mantiveram associadas ao desfecho, sendo também sugestiva o sexo masculino. Este foi o primeiro inquérito da LVC no município de Juiz de Fora e a presença da doença relatada neste trabalho, reforça a idéia de que a leishmaniose está em processo de expansão e urbanização no Brasil, apontando a necessidade de vigilância epidemiológica ativa. / In Brazil, the visceral leishmaniasis (LV), also known as calazar, is a zoonotic disease, with vector transmission caused by the Leishmania chagasi protozoan. This disease, before described as rural is going through a process of urbanization. In 2008, were diagnosed in Juiz de Fora municipality, the first cases considered autochthones of canine visceral leishmaniasis (CVL) and research about urban epidemic of LV has showed that the CVL is preceding the human infection, since that the dogs are the main domestic reservoirs. Then, the present study aimed to investigate the CVL infection in Juiz de Fora, as well as to value the risk factors associated to the disease. This work was done with the animals of the public kennel and ONGs and with the serum were realized three serological techniques: the immunochomatographic “TR DPP® and ELISA (EIE-leishmaniose visceral canine®), both given by FIOCRUZ/BIO-MANGUINHOS and ELISA in house. The samples totalized 781 animals and the prevalence of CVL varied according with the technique used: the test DPP showed soropositivity of 4.87% (IC 95% of 3.5-6.7%); the ELISA Bio-Manguinhos of 2.18% (IC 95% of 1.3-3.5%) and ELISA in house of 13.73% (IC 10.8-17.3%). In relation to variability observed among the techniques, as worth pointing out that same showed different antigens and this can reflect in the results. The sampIes were compound in majority by adult females, without definite race, medium-sized and short hair. In the univarious analysis using the ELISA Bio-Manguinhos as confirmatory for the CVL, was observed statistic association (p<0.05; IC 95%) with clinic sintomatology according by Quinnell et al. (2003), origin of animals (public kennel) and racial group; and also, there was suggestion of the association with masculine sex and medium/big port animals. In the multivarious analysis, the fact of being precedent from the kennell and clinic sintomatology kept associated to the result, being suggestive the masculine sex. This was the first enquiry of CVL in Juiz de Fora municipality and presence of the disease showed in this research, reinforce the idea that the leishmaniasis is in the process of expansion and urbanization in Brazil, pointing out necessity of an active epidemiologic vigilance.

Made available in DSpace on 2014-08-20T14:31:32Z (GMT). No. of bitstreams: 1 dissertacao_ana_lucia_recuero.pdf: 1024878 bytes, checksum: c121b797ab6a81b00fcaa6a96bab6b13 (MD5) Previous issue date: 2007-06-29 / The objectives of this study were to describe the prevalence and risk factors for G. lamblia infection in calves up to 12 months of age and children up to 12 years old in 30 dairy farms of Municipality from Capão do Leão,RS,as well as to compare analytically three diagnostic methods.Fresh fecal samples were randomly collected from 148 calves and 22 children.Feces were examined for the presence of G.lamblia by using two conventional tests of microscopic examination as Faust and Ritchie and a commercially available immunoenzymatic assay (RIDASCREEN ® Giárdia,R- Biopharm AG)to detect G.lamblia specific coproantigen.Data describing herd management practices,age,gender,breed and fecal consistency were gathered to assess potential risk factors associated with shedding.The overall prevalence for G. lamblia was 70.0%to the farms;23.6%to the calves and 13.6%to the children. Calves that were 1 4 months of age were 13.87 (95%CI,2.48 296.38;=0.001) times more likely to be shedding G.lamblia than calves with more than 4 months of age.Farms that were the waste water to less than 40 m to the well,were 3.38 (95% CI,1.15 9.86;P =0.01)times more likely to be calves with G.lamblia than farms that were the waste water to more than 40 m to the well.Farms that were the waste water in a more elevated plane that the well,were 2.35 (95%CI;1.08 5.24;=0.02) times more likely to be calves with G.lamblia than farms with waste water in a inferior plane to the well.The others parasites found in calves were Eimeria 83.1%;Strongylus 64.9%;Moniezia 10,8%,Toxocara 4.7%and Trichuris 2.0%.In children,only Entamoeba coli 18.2%and Endolimax nana 4.5%.Based on the results of these three tests,using ELISA as gold standard,analysis indicated that in calves,Faust Sensitivity (Se) 65.5%,Specifcity (Sp)95,0%;Predictive positive value (Ppv)76.0%;Predictive negative value (Pnv)91.9%;Accuracy (A)89.2%and a substantial Kappa (K) coefficient of correlation of 0.638.The Ritchie s technique was:(Se=48.3%, Sp=100,0%;Ppv=100.0%;Pnv=88.8%;A=89.9%)and a moderated K coefficient of correlation of 0.60.If accepted the positives results to Faust and/or Ritchie: (Se=72.4%;Sp=95.0%;Ppv=77.8%;Pnv=93.4%;A=90.5%)and a substantial K coefficient of correlation of 0.691.It was conclude in this study that the use of in parallel tests,increased the sensibility in detection of G.lamblia and If consideration is to be given to reducing the risk of infection with this parasite,management must be a top priority,especially in water quality,waste water careful with appropriated localization and health practices with younger animals and children / Os objetivos deste trabalho foram descrever a prevalência e fatores de risco para a infecção por Giardia lamblia em terneiros até 12 meses de idade e em crianças até 12 anos em 30 propriedades leiteiras do Município do Capão do Leão,RS,bem como comparar analiticamente três métodos de diagnóstico.Amostras de fezes recentes foram aleatoriamente coletadas de 148 terneiros e 22 crianças.As fezes foram examinadas para a presença de G.lamblia usando dois testes convencionais de exame microscópico como Faust e Ritchie e um ensaio imunoenzimático comercial (RIDASCREEN ® Giardia ,R-Biopharm AG)para detectar coproantígeno específico de G.lamblia .Foram recuperados dados descritivos de práticas de manejo,idade, gênero,raça e consistência fecal para identificar os principais fatores de risco associados com a disseminação.A prevalência geral para G.lamblia foi de 70,0% para as propriedades,23,6%para os terneiros e 13,6%para as crianças.Os terneiros que se encontravam na faixa etária de 1 a 4 meses foram 13,87 (95%CI, 2,48 296.38;=0.001)vezes mais prováveis de disseminarem G.lamblia do que terneiros com mais de 4 meses.Propriedades onde a fossa se localizava a menos de 40 m do poço tiveram 3,38 (95%CI,1.15 9.86;P=0.01)vezes mais probabilidade de possuírem terneiros com G.lamblia do que propriedades onde a fossa se localizava a mais de 40 m do poço.Propriedades onde a fossa se localizava em um plano mais elevado do que o poço apresentaram 2,35 (95%CI;1.08 5,24;P=0.02) vezes mais chances de contarem com terneiros disseminadores de G.lamblia do que propriedades onde a fossa se localizava em um plano inferior ao poço.Outros parasitos encontrados em terneiros foram Eimeria 83.1%;Strongyloidea 64.9%;Moniezia 10,8%,Toxocara 4.7%e Trichuris 2.0%.Em crianças,somente Entamoeba coli 18.2%e Endolimax nana 4.5%.Baseados nos resultados dos três testes,usando o ELISA como padrão ouro,as análises indicaram para terneiros que os parâmetros do método de Faust foram de 65,5%de Sensibilidade (S);95,0%de Especificidade (E);76.0%de Valor Preditivo ositivo (Vpp);91.9%de Valor Preditivo negativo (Vpn);89.2%de Acurácia (A)e um substancial coeficiente de correlação K de 0.638.O método de Ritchie apresentou:(S=48,3%,E=100,0%;Vpp=100.0%;Vpn=88.8%;A=89.9%)e um moderado coeficiente de correlação K de 0.60.Considerando os resultados positivos para Faust e/ou Ritchie,encontrou-se:(S=72.4%;E=95,0%;Vpp=77.8%; Vpn=93.4%;A=90.5%)e um substancial k de 0,691.Concluiu-se com este estudo que a utilização de testes em paralelo aumentou a sensibilidade na detecção de G lamblia e que na busca de considerações para reduzir os fatores de risco para a infecção deste parasito,as maiores prioridades são qualidade da água,localização apropriada da fossa e práticas de saúde para as crianças e animais jovens.

Made available in DSpace on 2014-08-20T14:31:33Z (GMT). No. of bitstreams: 1 dissertacao_amanda_fernandes_pinheiro.pdf: 1197006 bytes, checksum: f76aeabc9ec7ff7c3ad7cdaa92c92c7f (MD5) Previous issue date: 2010-10-22 / The neosporosis is a disease caused by intracellular protozoa Neospora caninum, which is of great importance, especially in cattle, because it may cause abortion in infected animals, causing big economic losses for the cattle industry of many countries in the world. In the present study it was reported the expression, purification, and characterization of the protein NcSRS2 of N. caninum in the methylotrophic yeast Pichia pastoris. The gene ncsrs2 was cloned in the vector of expression pPICZαB, followed by the integration in the genome of yeast. The recombining protein NcSRS2 was shown in the supernatant of the culture, where later it was concentrated and purified. An indirect immunoenzymatic assay (ELISA) was developed, using seronegative and seropositive of cattle and sheep, naturally field-infected. It showed that the recombinant protein presented the antigenic characteristics of native protein, which allowed its recognition by the blood serum of different species of animals with neosporosis. The results were compared with the indirect immunofluorescence (IFI). The diagnosis test ELISA- NcSRS2 described in the present work is a specific and sensitive method for the detection of N. caninum in cattle and sheep. The results indicate that the recombinant protein produced in this work, can be used for the development of diagnosis methods with low-cost production, and in industrial scale, because they are necessary and important for the introduction of proper controlling measurements for this parasitosis in cattle flocks. / A neosporose é uma enfermidade causada pelo protozoário intracelular Neospora caninum esta é de grande importância principalmente em bovinos, pois pode ocasionar abortos nos animais infectados, causando grandes perdas econômicas para indústria pecuária de vários países do mundo. No presente estudo, foi relatada a expressão, purificação e a caracterização da proteína NcSRS2 de N. caninum na levedura metilotrófica Pichia pastoris. O gene ncsrs2 foi clonado no vetor de expressão pPICZαB seguindo de integração no genoma da levedura. A proteína recombinante NcSRS2 foi demonstrada no sobrenadante da cultura onde posteriormente foi concentrada e purificada. Um ensaio imunoenzimático indireto (ELISA) foi desenvolvido utilizando soros negativos e positivos de bovinos e ovinos naturalmente infectados a campo por N. caninum. Este demonstrou que a proteína recombinante apresentou as características antigênicas da proteína nativa o que permitiu o seu reconhecimento por soros de diferentes espécies de animais com neosporose. Os resultados foram comparados com a imunofluorescência indireta (IFI). O teste diagnóstico ELISA-NcSRS2 descrito no presente trabalho constitui um método específico e sensível para a detecção de N. caninum em bovinos e ovinos. Os resultados indicam que a proteína recombinante produzida neste trabalho pode ser utilizada para o desenvolvimento de métodos diagnósticos com menor custo de produção e em escala industrial, pois estes são necessários e importantes para a implantação de medidas de controle adequadas para esta parasitose nos rebanhos bovinos.

Made available in DSpace on 2014-08-20T13:32:49Z (GMT). No. of bitstreams: 1 dissertacao_andressa_fisch.pdf: 708004 bytes, checksum: 48d7e7a2a394dafd06e3209289b60dde (MD5) Previous issue date: 2014-02-19 / Enzootic Pneumoniae (EP), caused by Mycoplasma hyopneumoniae, generates significant economic losses to the pig industry worldwide. The currently available vaccines confer partial protection against the EP and the development of new vaccine strategies have proven necessary. In this paper, humoral immune response in mice of six comercial bacterins against sexteen recombinant antigens previously characterized of M. hyopneumoniae was evaluated by indirect ELISA. Seroconversion Seroconversion was used to determine biologically significant reactivity. The antigen MHP_0067 was recognized by sera from one inoculated group. The antigens MHP_0513 and MHP_0580 were recognized by sera from four inoculated groups each. For other antigens, not seroconversion was detected. Identify potentially protective antigens underexpressed in these vaccines and associate them with conventional vaccination may promote increased levels of protection. These antigens are potential targets for the composition of a recombinant subunit vaccine associated with the commercial vaccine. / A Pneumonia Enzoótica Suína (PES), causada pela bactéria Mycoplasma hyopneumoniae, gera importantes perdas econômicas para a indústria suinícola em todo o mundo. As vacinas atualmente disponíveis diminuem as perdas produtivas, mas não impedem a infecção dos rebanhos. O desenvolvimento de novas estratégias vacinais têm se mostrado necessário. Neste trabalho, avalIou-se a resposta imune humoral induzida pela inoculação, em camundongos, de seis bacterinas comerciais contra quinze fatores de patogenicidade de M. hyopneumoniae previamente caracterizados por nosso grupo. A detecção de anticorpos foi realizada através de ELISA indireto. Soroconversão foi utilizada para determinar reatividade biologicamente significativa. O antígeno MHP_0067 foi reconhecido pelo soro de um único grupo inoculado. Os antígenos MHP_0513 e MHP_0580 foram reconhecidos pelos soros de quatro grupos inoculados cada. Para os demais antígenos não houve soroconversão pelos grupos inoculados com as bacterinas comerciais. Identificar antígenos potencialmente protetores ausentes nestas vacinas e associá-los à vacinação convencional pode promover o aumento dos níveis de proteção. Estes antígenos são potenciais alvos para a composição de uma vacina de subunidade recombinante associada à vacina comercial.

En France, quelques cas de toxoplasmose sévère ont été liés à la consommation de viande de cheval qui avait été importée du continent américain où les souches atypiques de Toxoplasma gondii sont plus fréquentes qu’en Europe. De nombreuses études de séroprévalence existent dans la littérature mais l’estimation du risque d’infection par T. gondii après consommation de viande de cheval est impossible à cause de l’absence de validation des tests sérologiques et la corrélation inconnue entre la détection des anticorps anti T. gondii et la présence de kystes dans les tissus. Nous avons utilisé la technique de capture magnétique-réaction de polymérisation en chaine (CM-PCR) pour détecter l’ADN de T. gondii dans 231 échantillons de viande de cheval achetés dans des supermarchés en France et évalué la performance et le niveau d’agrément du test d’agglutination modifié (MAT) et de la méthode immuno-enzymatique ELISA dans les jus de viande. Nous avons également utilisé 196 sérums de chevaux provenant de l'institut français du cheval et de l'équitation à Chamberet, en France, pour évaluer la précision des tests sérologiques ELISA, MAT et le test d’immunofluorescence (IFAT). Les tests sérologiques manquaient de sensibilité, de spécificité, d’agrément entre eux et il n’y avait pas de corrélation avec la présence d’ADN de T. gondii dans la viande de cheval, ce qui suscite des doutes sur la fiabilité des données de séroprévalence de T. gondii chez les chevaux dans la littérature. L’ADN de T. gondii a été détecté dans 43% des échantillons de viande de cheval mais l’absence d’isolement de souche chez la souris suggère une faible répartition des kystes dans les muscles squelettiques et un faible risque d’infection par T. gondii avec la consommation de viande de cheval. Cependant, pour éviter tout risque de toxoplasmose, il est recommandé de bien cuire la viande. / In France, some cases of severe toxoplasmosis have been linked to the consumption of horse meat that had been imported from the American continent where atypical strains of Toxoplasma gondii are more common than in Europe. Many seroprevalence studies exist in the literature but the risk assessment of T. gondii infection after horse meat consumption is impossible because of the absence of validation of serological tests and the unknown correlation between detection of antibodies against T. gondii and presence of tissue cysts. We performed magnetic capture-polymerase chain reaction (MC-PCR) to detect T. gondii DNA in 231 horse meat samples purchased in supermarkets in France and evaluated the performance and level of agreement of the modified agglutination test (MAT) and Enzyme-linked immunosorbent assay (ELISA) in the meat juices. We also tested 196 horse sera from - institut français du cheval et de l'équitation, Chamberet, France - to assess the accuracy of ELISA, MAT and immunofluorescence antibody test (IFAT). The serological tests lacked sensitivity, specificity, agreement between them, and there was no correlation with the presence of T. gondii DNA in horse meat, raising concerns about the reliability of T. gondii seroprevalence data in horses from the literature. T. gondii DNA was detected in 43% of horse meat samples but the absence of strain isolation in mice from more than 100 horse meat samples suggest a low distribution of cysts in skeletal muscles and a low risk of T. gondii infection associated with horse meat consumption. However, to avoid any risk of toxoplasmosis, a thorough cooking of horse meat is recommended.

Parmi les phlébovirus (famille des Bunyaviridae, genre Phlebovirus), ceux qui sont transmis par les phlébotomes de l'Ancien Monde sont largement distribués dans le bassin méditerranéen. Les infections humaines causées certains de ces phlébovirus sont connues depuis longtemps, mais elles restent tout de même négligées en médecine en raison de l'absence de données épidémiologiques solides (problème des réactions croisées) et d'outils de diagnostic rapides et fiables.La première partie de cette thèse a été consacrée à l'optimisation d'un test de neutralisation du virus pour étudier la séroprévalence de 5 virus, et leur capacité respective à infecter les humains et les animaux.La deuxième partie visait à mesurer la séroprévalence de phlébovirus appartenant aux 3 complexes antigéniques transmis par les phlébotomes dans le bassin méditerranéen (Sandfly fever Naples, Sandfly fever Sicilian et Salehabad). Ces études ont été menées sur des sérums de chiens et de chats en Tunisie, Portugal, Grèce/Chypre.La troisième partie a montré la capacité de virus récemment découverts dans le serocomplexe Salehabad (Adana et Medjerda valley virus) à infecter l'homme et les animaux traduisant un potentiel pathogène à explorer par des études spécifiques.La dernière partie a démontré la présence du virus Toscana en Kabylie (Algérie du Nord), et l'exposition extrêmement élevée des populations humaines vivant dans la région, avec des prévalence 10 fois plus élevées que dans les régions les plus à risque du sud-est de la France. / Sandfly-borne phleboviruses, transmitted by phlebotomine sandflies and belonging to the genus Phlebovirus within the Bunyaviridae family are widely distributed in Mediterranean basin. Human diseases caused by infection with phleboviruses are known for a long time, but they are still neglected due to the lack of epidemiological knowledge and of diagnostic tools.The first part of this thesis was dedicated to optimize a comparative virus neutralisation test to study the seroprevalence of selected phleboviruses and to assess the capacity of each virus to infect humans and animals. The second part aimed to estimate the epidemiology of phlebovirus serocomplexes (Naples, Sicilian and Salehabad) in Mediterranean basin. In order to update the presence of these viruses and their capacity to infect animals, several serologic studies were carried out on animal blood samples in Tunisia, Portugal, Greece and Cyprus. The results demonstrated that the phleboviruses belonging to 3 distinct groups are widely circulating and capable to infect non human vertebrate at different rates in studied countries.The third part showed the capacity of newly discovered viruses (Adana and Medjerda valley viruses) belonging to Salehabad serocomplex to infect human and animal at low and high rates, respectively. These findings suggest the medical and veterinary importance of these viruses. The last part of this thesis, confirm the circulation of Toscana virus by seroprevelance study which was carried out in local population in north Algeria where Toscana virus was isolated recently. The high rate of circulate suggests that Toscana virus is heavily affecting sandfly-exposed people in Algeria.

Les maladies allergiques constituent une part importante des préoccupations de santé publique. Parmi elles, la maladie du poumon d’éleveur d’oiseaux (PEO) est une pathologie respiratoire liée à l’inhalation répétée de protéines antigéniques, localisées dans les fientes, les plumes et les squames des oiseaux. Actuellement, on connait peu de chose sur la nature des antigènes impliqués dans la maladie.La mise en évidence d’immunoglobulines G (IgG) spécifiques des agents étiologiques est un élément important dans la prise en charge diagnostique et thérapeutique. L’objectif de la thèse est d’identifier les protéines antigéniques de pigeon et de les produire par génie génétique afin de développer un test sérologique efficace et standardisé de type ELISA. L’approche immuno-protéomique développée combine l’utilisation d’analyses sérologiques (western blot, ELISA) et l’identification par spectrométrie de masse des protéines antigéniques et des protéines totales (shotgun) des fientes, squames et sérum de pigeon. Ainsi, 14 protéines antigéniques principalement localisées dans les fientes et les squames de pigeon ont été identifiées et 2 protéines recombinantes ont été produites, puis testées en ELISA. Les protéines recombinantes Immunoglobulin-lambda-like polypeptide-1 et Proprotéinase E sont très performantes pour le diagnostic sérologique du PEO avec une spécificité et une sensibilité respectives de 100% et 84%. Des protéines orthologues, potentiellement impliquées dans les réactions antigéniques croisées ont été identifiées à partir des fientes de perruche et de poule. Ce travail a permis d’identifier les protéines antigéniques des fientes de pigeon, d’apporter des précisions sur leur localisation, et de développer des antigènes recombinants standardisés et performants pour améliorer le diagnostic sérologique du PEO. Des études complémentaires, sur des modèles animaux et cellulaires, devront être menées pour explorer l'implication de ces protéines dans l'induction de la maladie. / Allergic diseases represent one of the most important public health concerns. Among them, Bird Fancier’s Lung disease (BFL) is a respiratory illness associated with repeated inhalation of antigenic proteins, present in bird droppings, feathers and blooms. Currently, little is known about the nature of the antigens involved in the disease. The detection of immunoglobulin G (IgG) specific etiologic agents is an important factor in diagnostic and therapeutic management.The objective of this thesis is to identify the antigenic proteins of pigeons and to produce them by genetic engineering in order to develop an effective and standardized ELISA-type serological test. The immuno-proteomic approach created combines the use of serological analyses (western blot, ELISA) and the identification by mass spectrometry of antigenic proteins and total proteins (shotgun) of pigeon droppings, blooms and serum. Thus, 14 antigenic proteins mainly located in droppings and blooms were identified, and 2 recombinant proteins were produced and then tested with ELISA.The recombinant proteins Immunoglobulin-lambda-like polypeptide-1 and Proproteinase E are highly effective for the serological diagnosis of BFL,with specificity and sensitivity rates of 100% and 84%, respectively. Orthologous proteins potentially involved in cross-antigen reactions were identified from budgerigar and hen droppings. This work made it possible to identify the antigenic proteins of pigeon droppings, to provide further details on their location, and to develop standardized and efficient recombinant antigens to improve the serological diagnosis of BFL.Additional studies on animals and cell models will be needed to explore the role of these proteins in the induction of the disease.

A Leishmaniose visceral é um problema que cresce no Estado de São Paulo afetando o homem e o cão. Os exoantígenos da membrana das leishmanias são liberados no meio de cultura. Os exoantígenos são importantes na indução da imunidade mediada pelas células T e B estimulando a produção elevada de anticorpos. Realizamos uma avaliação comparativa por ELISA e Immunoblotting de exoantígenos e antígenos totais de L. (L.) amazonensis, L. (V.) braziliensis e L. (L.) chagasi, no diagnóstico da leishmaniose visceral humana e canina. Obteve-se por ELISA sensibilidade de 100% para ambos os preparados antigênicos independente da espécie de Leishmania. A melhor especificidade em humanos e cães foi com os exoantígenos. O exoantígeno da L. (L.) chagasi teve a melhor especificidade e média de absorbâncias comparadas aos das outras espécies (p<0.005). Para o hospedeiro humano o ELISA com exoantígenos, não discriminou pacientes com leishmaniose cutânea e ou mucocutânea. O Immublotting dos exoantígenos de L. (L.) chagasi (IBleish) apresentou 100% de sensibilidade e especificidade para os cães. Os dados do IBleish-L. (L.) chagasi demonstraram a possibilidade de sua utilização como método confiável para a confirmação do diagnóstico da leishmaniose canina. / The visceral leishmaniasis is a new problem that grows in the State of São Paulo, affecting men and dogs. The exoantigens from the membrane of the Leishmania, are released out of culture medium. The exoantigens are important in the induction of immunity mediated by T and B cells, stimulating the production of antibodies. This work was carried out an evaluation of ELISA and Immunoblotting using comparatively exoantigens and total antigens of L. (L.) amazonensis, L. (V.) braziliensis and L. (L.) chagasi for the diagnosis of canine and human visceral leishmaniasis. ELISA sensitivity was 100% for all different antigens, independent of the species of Leishmania employed. The best specificity for both human and dogs were obtained with exoantigens. Exoantigen from L. (L.) chagasi was what showed the best specificity and mean absorbance compared to those of other species (p<0.05). The ELISA with exoantigens for the human host not discriminate individuals with leishmaniasis cutaneous or mucocutaneous. The Immunoblotting that used the exoantigens of L. (L.) chagasi (IBleish) showed 100% sensitivity and specificity for the dogs. The IBleish-L. (L.) chagasi showed the possibility of its use as a reliable method to confirm the diagnosis of canine leishmaniasis.