O gênero Candida acomete cerca de 80% das infecções fúngicas no ambiente hospitalar e constitui causa relevante de infecções na corrente sangüínea. Espécies de Candida não-albicans respondem atualmente por pelo menos 50% das infecções invasivas por Candida spp, apresentando peculiaridades em termos clínicos e suscetibilidade a drogas antifúngicas. A mortalidade geral de fugemias por Candida spp é da ordem de 40-60%, tornando esta complicação infecciosa um grande desafio aos clínicos. O objetivo deste estudo foi a padronização de um ensaio imuno-enzimático (ELISA) para o diagnóstico de infecções hematogênicas devido a Candida spp. Vinte e cinco soros de pacientes com candidemia obtidos do Hospital de Clínicas de Porto Alegre e trinta e dois soros de indivíduos hígidos obtidos do Centro de Biotecnologia da Universidade Federal do Rio Grande do Sul foram analisados. Foram utilizados os extratos protéicos totais de Candida albicans, Candida tropicalis, Candida parapsilosis, Candida guilliermondii, Candida glabrata, Candida dubliniensis e Candida krusei como antígenos. Nossos resultados demonstraram reatividade cruzada entre as espécies bem como alguns resultados falsonegativos. Estes resultados falso-negativos podem ser devido a resposta imune do paciente estar atrasada, diminuída ou ausente. Isto pode ser melhorado com amostras seriadas do mesmo paciente. Os resultados falso-positivos podem ser minimizados selecionando-se antígenos específicos apropriados, moléculas purificadas ou antígenos recombinantes. / Candida spp is associated to almost 80% of all nosocomial fungal infections and is considered a major cause of blood stream infections. This yeast is the fourth most common cause of blood stream infections in the United States, where this agent is responsible for 8% of all invasive infections documented in this site and the crude mortality rate is between 40 and 60%. At the present, non-albicans species are related to at least 50% of all invasive infections due to Candida spp and they present differences in terms of clinical outcome as well as susceptibility to antifungal drugs. Nevertheless, the hematogenous infections due to Candida spp diagnosis remain an important challenge for all clinicians. The objective of this study was the standardization of an enzyme-linked immunosorbent assay (ELISA) to diagnose hematogenous infections due to Candida spp. Twenty five sera of patients with candidaemia from Hospital de Clínicas de Porto Alegre, a general tertiary care hospital in Southern Brazil and thirty two sera from healthy people from Centro de Biotecnologia da Universidade Federal do Rio Grande do Sul were analyzed. As antigens for ELISA, protein extracts from Candida albicans, Candida tropicalis, Candida parapsilosis, Candida guilliermondii, Candida glabrata, Candida dubliniensis and Candida krusei were used. Our results had demonstrated cross-reactivity between species as well as some false-negatives. These false-negatives may be due to a delayed, reduced or absent antibody response, which might be improved increasing the number of samples tested. The false-positives can be improved by selecting more appropriate specific antigens, as purified molecules or recombinant antigens.

Pasteurella multocida é um importante patógeno para suínos, causando rinite atrófica progressiva, pneumonia, pleurite, e septicemia. Para implantação de estratégias de controle e prevenção desta infecção, torna-se necessário o conhecimento a respeito do perfil de disseminação do agente em condições naturais e em diferentes tipos de sistema de produção. Tendo em vista a inexistência de técnicas sorológicas padronizadas para esta finalidade na espécie suína, o presente estudo teve por objetivos avaliar em um grupo de 90 animais a influência de diferentes sítios de coleta de amostra na detecção de animais portadores de P. multocida através da PCR e comparar estes dados a detecção de anticorpos na espécie suína através de um kit de ELISA comercial registrado para detecção de anticorpos contra P. multocida em aves. Foram coletados suabes de cavidade nasal, suabes de tonsila e sangue de 90 animais em idade de abate provenientes de dois sistemas de produção de suínos. Dentre os 90 animais, 14 (15,55%) foram positivos para detecção de P. multocida em suabes nasais e nenhum foi positivo em suabe de tonsila. Através do ELISA, 25 (27,77%) animais apresentaram anticorpos contra o agente. Através da análise de comparação de proporção, houve diferença significativa em relação à metodologia de diagnóstico nos diferentes testes realizados considerando-se valor de p < 0,0001 e intervalo de confiança de 95%. Ou seja, a freqüência de positivos foi significativamente maior no teste de ELISA, seguido pela reação em cadeia pela polimerase em suabes nasais, sugerindo que a cavidade nasal é o sitio primário de colonização dos animais por este agente e que o ELISA testado pode ser facilmente adaptado para avaliação do perfil sorológico do agente na espécie suína. / Pasteurella multocida is an important pathogen for pigs, causing progressive atrophic rhinitis, pneumonia, pleurisy, and septicemia. For implementation of strategies to control and prevent infections, it is necessary to know about the profile of agent spread in natural conditions and in different types of production system. Given the lack of standardized serological techniques for this purpose, this study aims to evaluate the influence of different methods and sites of sample collection in the detection of animals with P. multocida by polymerase chain reaction (PCR) and ELISA. We evaluated different pairs of primers specific for the agent and the reaction that have lower detection threshold will be used in the evaluation of the study sites. Swabs were collected from the nasal cavity, tonsil and also blood of 90 animals. Among these, 14 (15.55%) were positive for Pasteurella multocida by polymerase chain reaction (PCR), all of which were nasal swabs. There are no positive animals among the tonsil swabs. In the ELISA, 25 (27.77%) were positive, and of these, three (3.33%) were positive in both the polymerase chain reaction and the ELISA and the remaining 22 (24.44%) was positive by ELISA and negative by polymerase chain reaction. Using comparison of proportion analysis, was observed a significant differences in the methodology of diagnosis in different tests considering p-value <0.0001 and a confidence interval of 95%. Concluding, the frequency of positives was significantly higher in ELISA than by the polymerase chain reaction.

CAVALHEIRO, N.P. Análise dos sorotipos do VHC identificados em pacientes da cidade de São Paulo, através de método imunoenzimático. São Paulo, 1999. 97p. Dissertação de mestrado - Faculdade de Medicina, Universidade de São Paulo. Com o objetivo de analisar a prevalência dos diferentes tipos do vírus da Hepatite C (VHC) em uma população de pacientes portadores crônicos do VHC, através de um método sorológico (MUREX HCV Serotyping Assay), foram estudados 219 pacientes, que apresentaram positiva a Reação em Cadeia da Polimerase (PCR), nested-PCR. Estes soros foram submetidos ao teste imunoenzimático para detecção dos anticorpos contra os tipos 1,2,3,4,5,6 do VHC. As amostras foram diluídas e incubadas na presença de peptídeos heterólogos de competição, com antígenos sorotipo-específicos do VHC. Dos 219 pacientes, foi possível detectar o sorotipo do VHC em 166, revelando uma sensibilidade de 75,8%. Os resultados apresentaram a predominância do tipo 1 (70,0%) em nosso meio, seguido pelo tipo 3 (22,3%) e tipo 2 (4,2%). Os sorotipos 4 e 5 estiveram presentes para 1,8% dos pacientes, sempre associados com o sorotipo 1. Estas amostras, apesar de cumprirem os quesitos de validade do teste, apresentaram leituras de Densidade Ótica muito altas para todos os tipos virais testados, inclusive controles positivo e negativo e a possibilidade de reações cruzadas, nestes casos, deve ser considerada. A confirmação por genotipagem e uma investigação mais detalhada sobre a procedência e formas de aquisição do VHC destes pacientes devem ser pesquisados. O tipo 6 não foi confirmado em nenhuma das amostras testadas e provavelmente não estava presente nesta coleção de amostras avaliadas. Os parâmetros epidemiológicos avaliados foram: idade, sexo e vias de transmissão. Dos 166 pacientes com diagnóstico para o tipo do VHC, 108 (65,1%) eram homens e 58 (34,9%) mulheres. A idade dos pacientes variou de 12 a 73 anos (média 41,1). As formas de transmissão mencionadas foram 52 (31,3%) transfusão de sangue, 18 (10,8%) uso de drogas injetáveis, 8 (4,8%) tatuagens, 6 (3,6%) provável via sexual, 3 (1,8%) acidente com agulha, 2 (1,2%) trabalho em área de saúde, 1 (0,6%) acupuntura, 1 (0,6%) hemofílico. Sessenta e um pacientes (36,7%) não apresentaram fatores de risco que justificassem a aquisição da infecção pelo VHC. Não foi verificada diferença significativa entre os tipos do VHC encontrados e os parâmetros epidemiológicos estudados. A predominância dos tipos 1, 3 e 2 é compatível com outros estudos, de genotipagem, que envolveram amostras brasileiras, particularmente da cidade de São Paulo. As amostras que apresentaram leituras de Densidade Ótica altas ou baixas, para todos os poços de uma mesma amostra testada, inclusive controles positivo e negativo, sugerem a possibilidade de reações inespecíficas ou cruzadas e devem ser confirmadas por outras técnicas de genotipagem ou seqüenciamento. A praticidade oferecida pelo teste de sorotipagem do VHC, apesar de não identificar os subtipos, pode ser útil na prática clínica e auxiliar no prognóstico da doença, não necessitando da tecnologia exigida pelos testes que envolvem biologia molecular. / CAVALHEIRO, N.P. Analysis of serotypes of HCV in patients from the city of São Paulo, by means of a enzyme-immunoassay method. São Paulo, 1999. 97p. Dissertação de Mestrado - Faculdade de Medicina, Universidade de São Paulo. With the objetive of analysing the prevalence of the different types of Hepatitis C Virus (HCV) in a population of chronic carriers of HCV, through a sorologic method (MUREX HCV Serotyping Assay), 219 patients were studied who showed a positive polymerase chain reaction. This sera were submitted to immunoenzimatic tests for the detection of antibodies in relation to HCV types 1, 2,3,4,5 and 6. The samples were diluted and incubated in the presence of heterologous competing peptides, with microwells coated with serotype-specific antigens of HCV. Of the 219 patients, it was possible to detect the HCV serotype in 166, revealing a sensitivity of 75.8%. The results showed a predominance of type 1 (70.0%) in our medium, followed by type 3 (22.3%) and type 2 (4.2%). Serotypes 4 and 5 were present in 1.8% of the patients, but always associated with serotype 1. These samples, in spite of fulfilling the prerequisites of validity for testing, showed a very high optical density reading for all types of viruses tested, including positive and negative controls. The possibility of cross reactions in these cases should be considered. Confirmation by genotyping and a more detailed investigation on the origin and mode of acquisition of the HCV of these patients should be researched. Type 6 was not confirmed in any of the samples tested and probably was not present in this particular collection. The epidemiological parameters evaluated were: age, sex and means of transmission. Of the 166 patients diagnosed with the HCV, 108 (65.1%) were men and 58 (34.9%) were women. The age of the patients varied from 12 to 73 years, the average being 41.1 years. The means of transmission mentioned were blood transfusion in 52 (31.3%) cases, intravenous drug use in 18 (10.8%) cases, by tatoos in 8 (4.8%) cases, 6 (3.6%) cases were sexually transmitted, 3 (1.8%) were by accident with a needle, 2 (1.2%) through work in the health field, one (0.6%) through acupunture and one by being hemophiliac. Sixty one (36.7%) patients were not able to offer any risk factor which justified the acquisition of the HCV infection. No significant difference was verified among the different types of HCV found and the different epidemiological parameters studied. The predominance of types 1, 3 and 2 is compatible with other genotyping studies which involved Brazilian samples, particularly in the city of São Paulo. The samples which showed high or low dense optical reading for all the wells of the same samples tested even the positive or negative controls, suggested confirmation by sequecing or genotyping. The praticality obtained by the HCV serotyping test, in spite of the fact that it does not identify the sub type, can be useful in clinical pratice and helpful in the prognostication of the disease, not needing the tecnology demanded by the tests which involve molecular biology.

A lipoproteína de baixa densidade eletronegativa (LDL-) é um importante antígeno envolvido na patogênese da aterosclerose. A LDL(-) provoca resposta inflamatória e imunológica, levando à produção de autoanticorpos anti-LDL(-) e a formação subsequente de imunocomplexos (IC-LDL-), os quais também contribuem com o processo aterosclerótico. Diante disso, este trabalho teve como objetivo o desenvolvimento e validação de imunoensaios para quantificação plasmática de LDL(-), anti-LDL(-) e IC-LDL(-), assim como o desenvolvimento de ferramentas aplicáveis em um biossensor para LDL(-). Após a padronização de cada ELISA, foram avaliadas as características de desempenho dos métodos: limites de detecção (LD) e quantificação (LQ), precisão intra e inter-ensaios, exatidão, linearidade de diluição e interferentes. Os LD e LQ do ELISA para LDL(-) foram 0,423 mg/L e 0,517 mg/L de LDL(-), respectivamente. As concentrações plasmáticas de LDL(-) apresentaram linearidade quando os plasmas foram diluídos 1:1000, 1:2000, 1:4000 e 1:8000. Os LD e LQ do ELISA para anti-LDL(-) foram 0,0028 mg/L e 0,0032 mg/L de anti-LDL(-), respectivamente. Os plasmas apresentaram linearidade na diluição quando diluídos 1:100, 1:200, 1:400 e 1:800. Os LD e LQ do ELISA para IC-LDL(-) foram 0,023 g/L e 0,034 g/L de IC-LDL(-), respectivamente. Os plasmas apresentaram linearidade quando diluídos 1:12,5, 1:25, 1:50 e 1:100. Os três ELISAs apresentaram precisão intra e inter-ensaios e recuperação dentro dos limites requeridos para imunoensaios. Para o desenvolvimento de um biossensor para LDL(-), uma proteína recombinante, denominada GFP5-scFv, foi expressa em bactérias Escherichia coli da linhagem BL21(DE3). Para obtenção dessa proteína foi realizada a inserção da sequência de DNA de um fragmento variável de cadeia única (scFv) anti-LDL(-) em um vetor bacteriano com a sequência de DNA da proteína verde fluorescente (GFP5). Dessa forma, a GFP5-scFv é fluorescente e tem afinidade pela LDL(-). Também foram sintetizadas nanopartículas de ouro, as quais podem ser eficientemente utilizadas na supressão da emissão da fluorescência de GFP5-scFv. Portanto, os imunoensaios validados e os aplicativos desenvolvidos para o biossensor são ferramentas que tem potencial para serem utilizadas na avaliação da patogênese da aterosclerose. / The electronegative low-density lipoprotein (LDL) is an important antigen involved in the pathogenesis of atherosclerosis. The LDL(-) causes inflammatory and immune response, leading to production of autoantibodies anti-LDL(-) and the subsequent formation of immune complexes (IC-LDL), which also contribute to the atherosclerotic process. Thus, this study aimed to develop and validate immunoassays for quantification of plasma LDL(-), anti-LDL(-) and LDL-IC(-) as well as developing tools applicable to a biosensor for LDL(-). After the standardization of each ELISA, were evaluated the performance characteristics of methods: limits of detection (LD) and quantification (LQ), intra- and inter-assays precision, accuracy, linearity of dilution and interferences. The LD and LQ of LDL(-) ELISA were 0.423 mg/L and 0.517 mg/L of LDL(-), respectively. Plasmas showed linearity when diluted 1:1000, 1:2000, 1:4000 and 1:8000. The LD and LQ of anti-LDL(-) ELISA were 0.0028 mg/L and 0.0032 mg/L of anti-LDL(-), respectively. Plasmas showed linearity when diluted 1:100, 1:200, 1:400 and 1:800. The LD and LQ of IC-LDL(-) ELISA were 0.023 g/L and 0.034 g/L of LDL-IC(-), respectively. Plasma showed linearity when diluted 1:12.5, 1:25, 1:50 and 1:100. The three ELISAs showed good intra- and inter-assays precision and recovery within the limits required for immunoassays. To develop a biosensor for LDL(-), a recombinant protein, called GFP5-scFv was expressed in Escherichia coli BL21(DE3) strain. The obtainment of this protein was performed inserting the DNA sequence of an anti-LDL(-) single chain variable fragment (scFv) in a bacterial vector with a DNA sequence of green fluorescent protein (GFP5). Thus, the scFv-GFP5 is fluorescent and has affinity for LDL(-). Were also synthesized gold nanoparticles, which can be efficiently used for quenching the fluorescence emission of GFP5-scFv. Therefore, immunoassays validated and developed applications for the biosensor are tools that have potential to be used in evaluating the pathogenesis of atherosclerosis.

A dengue é uma doença infecciosa viral transmitida pela picada de mosquitos do gênero Aedes e é um importante problema de saúde pública mundial. A infecção por qualquer um dos quatro sorotipos (DENV 1-4) pode apresentar diferentes quadros clínicos: pode ser assintomática, causar uma síndrome febril indiferenciada, ou a febre da dengue (DF), a evolução do quadro clínico pode levar a dengue hemorrágica com ou sem choque (DHF/DSS). Um número crescente de casos de infecção por dengue em crianças têm sido observados nos últimos anos. Estudos de prevalência da dengue em regiões endêmicas são importantes para avaliar a incidência da infecção por dengue em crianças. Assim, o objetivo deste estudo é investigar a prevalência de anticorpos anti-dengue IgM e IgG em crianças atendidas no Centro de Saúde Escola Dr. Edgard Aché, localizado na região oeste de Ribeirão Preto-SP. Crianças (n = 271) de 1-15 anos de idade foram recrutadas de março de 2010 até maio de 2011. Depois de um termo de consentimento ter sido assinado pelo responsável, uma amostra de sangue das crianças, participantes deste estudo, foi coletada. As crianças foram classificadas em assintomáticas (n= 174) ou sintomáticas (n = 97), quando estas apresentavam um ou mais de um sintoma sugestivo de dengue, de acordo com critérios da Organização Mundial de Saúde. Anticorpos IgM e IgG anti-dengue foram detectado nas amostras de soro por um ELISA de captura padronizado em nosso laboratório. O ELISA de captura para IgG mostrou uma positividade de 9,23% (25/271) e o ELISA de captura para IgM de 8,49% (23/271). O ELISA de captura para IgG foi positivo em 10,31% (10/97) das crianças sintomáticas e 8,62% (15/174) das crianças assintomáticas, enquanto que o ELISA de captura para IgM foi positivo em 15,46% (15/97) das crianças sintomáticas e 4,6% (8/174) em crianças assintomáticas. Este estudo mostrou a alta prevalência de anticorpos anti-dengue em crianças da região oeste do município de Ribeirão Preto; mostrou também que a infecção pode ser causada de forma assintomática, e que a infecção por este vírus pode ser um grave problema de saúde nesta população, servindo como um alerta às autoridades de saúde. / Dengue is an infectious viral disease transmitted by the biting of mosquitoes of Aedes genus and it is an important public health problem worldwide. Infection with any of the four serotypes (DENV 1-4) may be asymptomatic or causes illness ranging from mild viral syndrome, dengue fever (DF) to dengue hemorrhagic fever (DHF). An increasing number of dengue infection cases in children have been noted in the last years. A dengue surveillance study might be an important tool in endemic region to evaluate the incidence of dengue infection in children. Thus, the aim of this study was to investigate the prevalence of anti-dengue IgM and IgG antibodies in children in the Primary Health Care Center, Dr. Edgard Aché, located in the west region of Ribeirão Preto-SP. Children (n=271) from 1 to 15 years old were recruited during March 2010 until May 2011. After a signed consent by the person responsible for the children to participate in this study, a blood sample was collected. The children were classified in asymptomatic (n=174) or symptomatic (n=97) when they had more than one symptom suggestive of dengue according to the World Health Organization criterions. Anti-dengue IgM and IgG were detected in serum samples by a capture ELISA standardized in our laboratory. IgG capture ELISA was positive in 9,23% (25/271) and IgM capture ELISA in 8,49% (23/271) of the children. IgG capture ELISA was positive in 10,31% (10/97) of the symptomatic children and 8,62% (15/174) in asymptomatic children; while IgM capture ELISA was positive in 15,46% (15/97) of symptomatic children and 4,6% (8/174) in asymptomatic children. This study showed the high prevalence of anti-dengue antibodies in children in the west region of Ribeirão Preto. In addition, a high prevalence of dengue-infected children without symptoms was observed. The present survey demonstrated that dengue virus infection might be a problem in children\'s from Ribeirão Preto city and serve as an alert to the health authorities.

A decreased concentration of pepsinogen A in serum was found to be a marker of gastric cancer, similarly as a low ratio of pepsinogen A to pepsinogen C. In the present study we have compared properties of immunoglobulin fraction isolated from the egg yolks after immunization of laying hens with pepsinogen isolated from porcine gastric mucosa with those of present in rabbit antiserum obtained after the animal immunization with the same antigen. The characteristics of chicken antibodies against porcine pepsinogen and the comparison with rabbit antibodies raised against the same antigen was carried out using the following methods: ELISA, affinity chromatography on immobilzed antigen and bovine serum albumin, SDS and native electrophoresis and MALDI-TOF MS/MS. While the rabbit specific antibodies interacted with the used antigen and only slightly with bovine serum albumin and there was a diference between pre-immune IgG and specific IgG, in the case of chicken antibodies IgY it did not work. No diference was observed between ELISA tests performed with pre-immune serum and the serum after immunization with porcine pepsinogen and a high interaction of IgY with bovine serum albumin in pre-immune serum and specific IgY after the immunization were detected. Similar results were obtained in experiments with...

No description available.

Las pruebas serológicas de Inhibición de la Hemaglutinación (HI) y la Prueba de Inmunoensayo con enzimas asociadas (ELISA) son usadas rutinariamente para la detección de anticuerpos contra el virus de la Enfermedad de Newcastle (vENC). El antígeno de la prueba de HI puede ser una cepa de tropismo respiratorio o de tropismo entérico. En el presente estudio fue analizada la concordancia entre las dos pruebas de HI y la prueba de ELISA. Con tal fin fueron analizados los resultados serológicos obtenidos en 196 sueros colectados en pollos de engorde de 26 y 42 días, antes y después de ser desafiados con una cepa velogénica viscerotrópica del virus de la enfermedad de Newcastle. Los anticuerpos detectados por ambas pruebas fueron agrupados en cuatro escalas según su nivel de detección (negativo, bajo, medio y alto) para posteriormente ser analizados por la prueba estadística de Kappa de Cohen. Se analizó la concordancia entre las dos pruebas de HI con antígeno respiratorio o entérico y, entre cada una de estos últimas (HI respiratoria y HI entérica) con la prueba de ELISA. Se determinó que la concordancia entre las prueba de HI y ELISA, tanto usando antígeno entérico como antígeno respiratorio fue muy pobre, mientras que la concordancia entre las dos pruebas de HI (con antígeno respiratorio o entérico) fue moderada. Palabras claves: virus de la enfermedad de Newcastle (ENC), ELISA, HI, índice de kappa, concordancia. / --- Hemagglutination Inhibition (IH) and Enzyme-linked Immunosorbent Assay (ELISA) Serological tests are routinely used for antibodies detection against the virus of Newcastle disease (NDV). Antigen for IH test may be a strain of respiratory tropism or enteric tropism. This study analyzed the concordance between the HI and ELISA tests. For this purpose, the serological results obtained on 196 sera collected in broilers of 26 and 42 days old were analyzed, before and after being challenged with a velogenic viscerotropic strain of newcastle disease virus. The antibodies detected by both tests were grouped into four scales according to their detection level (negative, low, medium and high) in order to later be analyzed by the Kappa Cohen statistic test. Concordance between the two tests of IH with respiratory or enteric antigen was analyzed, and concordance between each these (IH respiratory and IH enteric) with the ELISA test was analyzed. It was found that the concordance between IH test and ELISA, using both enteric antigen and respiratory antigen was very poor, while the concordance between the two IH tests (respiratory or enteric antigen) was moderate. Keywords: virus Newcastle disease (NDV), ELISA, HI, kappa index, concordance.

Ruminant alphaherpesviruses related to bovine herpesvirus 1 (BoHV-1) are a cluster of viruses antigenically and genetically closely related. The prototype of this cluster, BoHV-1, is a major pathogen of cattle associated with various clinical manifestations including infectious bovine rhinotracheitis (IBR) and infectious pustular vulvovaginitis (IPV). IBR is a disease of major economic concern in many parts of the world and especially in Europe, both in countries where this infection has been eradicated and in those where the control of IBR is currently or will be undertaken. The massive use of vaccination allowed a significant reduction of the number of IBR clinical cases. However, the existence of closely related viruses to BoHV-1 is a threat for IBR eradication programmes. Consequently, the main objective of the present work is dedicated to afford a better knowledge of the interaction between alphaherpesviruses and their ruminant hosts in order to contribute to improve the control of IBR. To meet the objective, two approaches have been developed: the study of the viral diversity aiming to extend both epidemiological and virological data about ruminant alphaherpesviruses related to BoHV-1 and the study of the heterologous protection aiming to protect minor ruminant species by the concept of the cascade vaccination. Illustrating the problematic of the cluster of ruminant alphaherpesviruses related to BoHV-1, an original situation has been described recently in Belgium. During 2001 and 2002 hunting seasons, 28.9% of red deer were detected seropositive to BoHV-1. Due to an apparent lack of contact between cattle and red deer, it was suggested that a BoHV-1 related virus was spreading in the Belgian red deer population. Thus, the first isolation of cervid herpesvirus 1 (CvHV-1) in wild fauna is reported, which brings the opportunity to deeper analyse the antigenic, genomic and genetic relationship between BoHV-1 and its related ruminant alphaherpesviruses. This isolation demonstrates that a ruminant can be strongly identified as BoHV-1 positive while in actual fact it is infected with a related but distinct alphaherpesvirus and this ruminant will be declared as false positive. The problem is even greater when these viruses become latent allowing their possible reactivation and persistence for a very long time in their ecological niches. It is necessary to have tests which can differentiate related alphaherpesviruses that infect different ruminant species. The control of IBR relies on the use of BoHV-1 gB and gE blocking enzyme linked immunosorbent assays (ELISA) in order to differentiate infected and gE-negative vaccinated animals. Knowing that CpHV-1 is the most distant virus from BoHV-1, it can be hypothesised that a BoHV-1 gB blocking ELISA detects CpHV-1 but that CpHV-1 infection could be discriminated by a BoHV-1 gE blocking ELISA. CpHV-1 being mainly distributed in the Mediterranean part of Europe as Greece, Spain and Italy, the analysis was performed with field serums collected in France with the aim to update the epidemiological situation of the infection in Europe. Besides BoHV-1, CpHV-1 is the most relevant infection in Europe but is sadly neglected. The first reason is that economic losses are restricted to a herd level in contrast with IBR that brings an economical impact at a country level. The second reason is that goat is considered as a minor species. In this context, the problem is still not big enough for commercial interest towards vaccine development. The European Union has recently pointed out the problem of minor uses and minor species and allowed off label use of veterinary medicinal products or the use of a product licensed for a major species when an authorised veterinary medicinal product is not available (cascade principle). Goat being a minor species and CpHV-1 sharing close antigenic and genetic properties with BoHV-1, a live attenuated gE-negative BoHV-1 vaccine has been assessed in goats to protect against either a nasal or a genital CpHV-1 infection.

Listeria monocytogenes is a bacterial pathogen that is typically transmitted to humans through consumption of contaminated foods. Infection with this organism can lead to a severe and life-threatening illness referred to as listeriosis. The goal of this study was to develop monoclonal antibodies (MAbs) with high specificity and affinity to proteins found on the surface of all strains of L. monocytogenes while not cross-reacting with non-pathogenic Listeria spp. or other major bacterial pathogens commonly found in foods. A literature search was conducted to identify ten candidate surface proteins involved or putatively involved in the virulence of L. monocytogenes. Bioinformatics analyses using BLAST on the NCBI website showed that five of the ten candidate proteins were potentially present in L. monocytogenes strains but absent from strains of other Listeria spp. Genes encoding for these five proteins, ActA, InlA, InlC2, InlJ and LapB, were cloned and expressed in Escherichia coli. MAbs were raised against recombinant LapB, InlJ and InlC2 proteins using hybridoma technology. A total of 48 anti-LapB, 33 anti-InlJ and 37 anti-InlC2 MAbs were developed. Based on the comparison of IFM signal of each MAb against L. monocytogenes cells, seven anti-LapB MAbs and six anti-InlC2 MAbs were selected for further characterization. All of the anti-InlJ MAbs showed weak IFM signals and negative reactivity in ELISA against L. monocytogenes cells. The selected anti-LapB and anti-InlC2 MAbs were further characterized by assessing their ability to bind to cells of 51 strains representing 11 L. monocytogenes serotypes using ELISA. Six anti-LapB MAbs (M3484, M3495, M3500, M3509, M3517, M3519) reacted strongly with 44 of 51 strains representing 9 of the 11 L. monocytogenes serotypes tested. Five anti-InlC2 MAbs (M3607, M3618, M3630, M3633, M3636) reacted strongly with 47 strains representing 10 of the 11 L. monocytogenes serotypes tested. These results indicate that anti-LapB and anti-InlC2 MAbs could potentially be used as diagnostic reagents for isolation and detection of almost all L. monocytogenes strains in contaminated foods.