Nanotecnología : diagnóstico para la brucelosis porcina

Porcine brucellosis is an important zoonosis to public health and to agricultural economy since it causes significant losses in production, and the establishment of sanitary restrictions on local and international trade. Therefore, it is important to have a rapid and reliable diagnosis. To date, confirmatory diagnosis of brucellosis mainly relies on the isolation of the bacterium, but it is difficult to implement. Currently, most of the diagnostic tests are based on serological assays useful for diagnosing herds; however there is still not an accurate diagnosis to test individual animals. Thus, in the present work we developed and evaluated a novel indirect immunoassay (iELISA) using a recombinant glycoprotein antigen formed by the LPS O: 9-O polysaccharide of Yersinia enterocolitica (identical to the O polysaccharide of B. suis) covalently coupled to the carrier protein AcrA, derived from Campylobacter jejuni. This antigen was previously characterized and validated for the diagnosis of brucellosis in humans and cattle. From the above mentioned antigen we have developed and validated the glyco-iELISA using a panel of positive and negative sera previously characterized by agglutination buffered plate antigen techniques (BPA), and by fluorescent polarization assay (FPA). The results indicate that the glyco-iELISA differentiates positive animals from clearly negative ones. A cut-off value of 0.56 resulted in a diagnostic test sensitivity and specificity of 100 %, respectively. In addition, as a proof of concept we set to develop, with a small panel of sera, an indirect fluorescence immunoassay with OAg-AcrA coupled to magnetic beads. Due to the advantage of using magnetic racks instead of centrifugation, reduced incubation time and more efficient washes, this platform has shown to be a valuable practical diagnostic tool. Both developed assays discriminate positive from negative animals, even in infected herds specifically identifying the infected animal and differentiating it from negative animals exposed.\nFil: Balzano Parodi, Rodrigo E. Universidad de Buenos Aires. Facultad de Ciencias Veterinarias; ArgentinaLa brucelosis porcina es una zoonosis de importancia para la salud pública y la economía agropecuaria, ya que provoca importantes pérdidas en la producción y restricciones sanitarias en el comercio interno y en las exportaciones. Por ello, es importante tener un diagnóstico rápido y confiable. Hasta la fecha, la prueba confirmatoria de brucelosis es el aislamiento de la bacteria, lo cual es difícil de implementar. Los test diagnósticos vigentes se basan en pruebas serológicas que son útiles para el diagnóstico de piaras pero no permiten obtener un diagnóstico fiable de los animales a nivel individual. Por esta razón, en este trabajo se desarrolló y evaluó un nuevo test diagnóstico. El mismo consiste en un inmunoensayo (iELISA) utilizando como antígeno una glicoproteína recombinante, la cual está formada por el polisacárido O del LPS de Yersinia enterocolitica O:9 (idéntico al polisacárido O de B. suis) unido covalentemente a la proteína transportadora AcrA derivada de Campylobacter jejuni. Este antígeno fue previamente caracterizado y validado para el diagnóstico de brucelosis en humanos y bovinos. A partir del mismo, se desarrolló y validó el glico-iELISA utilizando un panel de sueros positivos y negativos previamente caracterizados por las técnicas de aglutinación con antígeno bufferado en placa (BPA) y Ensayo de Polarización Fluorescente (FPA). Los resultados indican que el glico-iELISA permite discriminar claramente animales positivos de negativos, con un valor de cut-off de 0,56, resultando en una sensibilidad y especificidad diagnóstica de 100%. Además, se desarrolló como prueba de concepto, un inmunoensayo de fluorescencia basado en la unión covalente del mencionado antígeno a micropartículas magnéticas con un panel reducido de sueros. Esta plataforma presenta las ventajas de reducir los tiempos de incubación y los lavados son más eficientes utilizando racks magnéticos sin necesidad de centrifugar, entre otras. Ambos ensayos desarrollados permitieron discriminar animales positivos de negativos, incluso en piaras infectadas identificando puntualmente al animal infectado y diferenciándolo de los animales negativos expuestos

A bacterial glycoengineered antigen for improved serodiagnosis of porcine brucellosis

Brucellosis is a highly zoonotic disease that affects animals and human beings. Brucella suis is the etiological agent of porcine brucellosis and one of the major human brucellosis pathogens. Laboratory diagnosis of porcine brucellosis mainly relies on serological tests, and it has been widely demonstrated that serological assays based on the detection of anti O-polysaccharide antibodies are the most sensitive tests. Here, we validate a recombinant glycoprotein antigen, an N-formylperosamine O-polysaccharide protein conjugate (OAg-AcrA), for diagnosis of porcine brucellosis. An indirect immunoassay based on the detection of anti-O-polysaccharide IgG antibodies was developed coupling OAg-AcrA to enzyme-linked immunosorbent assay plates (glycoiELISA). To validate the assay, 563 serum samples obtained from experimentally infected and immunized pigs, as well as animals naturally infected with B. suis biovar 1 or 2, were tested. A receiver operating characteristic (ROC) analysis was performed, and based on this analysis, the optimum cutoff value was 0.56 (relative reactivity), which resulted in a diagnostic sensitivity and specificity of 100% and 99.7%, respectively. A cutoff value of 0.78 resulted in a test sensitivity of 98.4% and a test specificity of 100%. Overall, our results demonstrate that the glyco-iELISA is highly accurate for diagnosis of porcine brucellosis, improving the diagnostic performance of current serological tests. The recombinant glycoprotein OAg-AcrA can be produced in large homogeneous batches in a standardized way, making it an ideal candidate for further validation as a universal antigen for diagnosis of "smooth" brucellosis in animals and humans.Fil: Cortina, María Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; ArgentinaFil: Balzano, Rodrigo E.. Ministerio de Agricultura, Ganadería, Pesca y Alimento. Servicio Nacional de Sanidad y Calidad Agroalimentaria; ArgentinaFil: Rey Serantes, Diego A. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; ArgentinaFil: Caillava, Ana Josefina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; ArgentinaFil: Elena, Sebastian. Ministerio de Agricultura, Ganadería, Pesca y Alimento. Servicio Nacional de Sanidad y Calidad Agroalimentaria; ArgentinaFil: Ferreira, A. C.. Instituto Nacional de Investigação Agrária e Veterinária; PortugalFil: Nicola, Ana M.. Ministerio de Agricultura, Ganadería, Pesca y Alimento. Servicio Nacional de Sanidad y Calidad Agroalimentaria; ArgentinaFil: Ugalde, Juan Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; ArgentinaFil: Comerci, Diego José. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; Argentina. Comisión Nacional de Energía Atómica; ArgentinaFil: Ciocchini, Andres Eduardo. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - La Plata. Instituto de Investigaciones Biotecnológicas. Instituto de Investigaciones Biotecnológicas ; Argentin

Nível plasmático de uréia sobre parâmetros reprodutivos de machos ovinos deslanados (Ovis aries, LINNAEUS, 1758)

The effect of increased plasma urea levels on reproductive parameters of Santa Ines Brazilian breed rams (Ovis aries) was determined during two phases, 60 days in length each. During the first phase, 18 animals divided into three blocks according to scrotal circumference were submitted to the following treatments: diet A1- with nitrogen levels according to ARC (1980); diet B1 - nitrogen levels 133% higher than A1; and diet C1 - nitrogen levels 166% higher than A1. During the second phase, 12 animals divided into two groups received diet A2- with nitrogen levels according to ARC (1980), and diet B2 - nitrogen levels 200% higher than diet A2, respectively. The increase in protein equivalents was obtained by adding 20, 40 and 60 g of feed grade urea to treatments B1, C1 and B2, respectively. Semen was collected daily using an artificial vagina and analyzed twice a week for progressive motility; vigor, whirling, density, pH, concentration, sperm morphology, ejaculated volume and total sperm per ejaculate. Urea concentration was determined weekly in semen and plasma. Urea level increased in plasma and semen (p < 0.0001) during both phases but did not affect the parameters studied.Estudou-se o efeito de níveis aumentados de uréia plasmática sobre parâmetros reprodutivos de machos ovinos (Ovis aries) deslanados da raça Santa Inês, em duas fases de 60 dias cada. Durante a primeira fase, 18 animais divididos em três blocos de acordo com perímetro escrotal foram submetidos aos seguintes tratamentos: dieta A1 - com níveis de nitrogênio de acordo com ARC(1980); dieta B1 - com níveis de nitrogênio 133% maiores que dieta A1; e dieta C1 - níveis de nitrogênio 166% maiores que dieta A1. Durante a segunda fase, 12 animais divididos em dois grupos receberam as dietas: A2 - com níveis de nitrogênio de acordo com ARC (1980) e dieta B2 - níveis de nitrogênio 200% maiores que dieta A2. O aumento no equivalente protéico foi obtido com adição de 20, 40 e 60 g de uréia aos tratamentos B1, C1 e B2, respectivamente. Foi coletado sêmen diariamente com vagina artificial e duas vezes por semana foram analisados: motilidade retilínea e progressiva, vigor, turbilhonamento, densidade, pH, concentração, morfologia espermática, volume ejaculado e total de espermatozóides por ejaculado. A concentração de uréia no plasma e no sêmen foi analisada semanalmente. O aumento (p < 0,0001) de uréia no sêmen e no plasma durante ambas as fases não afetou os parâmetros estudados

Influência do nível de uréia plasmático sobre parâmetros citogenéticos de machos ovinos deslanados

The experiment was planned to verify the effects of increasing blood urea levels resulting from increasing urea in the diet on cytogenetic parameters of Santa Ines Brazilian breed rams (Ovis aries), aged 13 to 18 months. Eighteen animals were divided into three experimental treatments consisted of: diet A ration with nitrogen levels, according to NRC (1985); diet B - ration with a 133% higher level of nitrogen than diet A; and C- ration with a 166% higher level of nitrogen than diet A. The increase in nitrogen level was obtained by adding 20, 40 g of feed grade urea in treatments B and C, respectively. Blood was collected on a weekly basis to determine urea concentration. Blood was also collected from each animal, 60 days after desired diet urea levels were reached, to obtain a lymphocyte culture in order to count chromosome number and determine mitotic index. Increases in plasma urea level were observed (p < 0.0001), but did not affect the observed traits.O experimento foi planejado para verificar o efeito do aumento dos níveis plasmáticos de uréia, resultantes do acréscimo de uréia à dieta, sobre parâmetros citogenéticos de ovinos (Ovis aries) deslanados brasileiros, da raça Santa Inês, com idade entre 13 e 18 meses. Dezoito animais foram distribuídos a três dietas que consistiam de: dieta A - ração contendo níveis de nitrogênio de acordo com NRC (1985); dieta B - ração contendo nível de nitrogênio 133% superior ao da ração A; e dieta C - ração contendo nível de nitrogênio 166% superior ao da ração A. O aumento no nível de nitrogênio foi obtido com acréscimo de 20 e 40 g de uréia grau alimento, para os tratamentos B e C, respectivamente. A colheita de sangue para determinação do nível de uréia no plasma foi semanal. Para a realização da cultura de linfócitos, para a verificação do número de cromossomos e índice mitótico, 60 dias após terem sido alcançados os níveis de uréia desejados na dieta, foi coletado sangue de cada animal. O aumento dos níveis de uréia no plasma (p < 0,0001) não afetou os parâmetros observados

Pandemic (H1N1) 2009 outbreak on pig farm, Argentina

In June-July 2009, an outbreak of pandemic (H1N1) 2009 infection occurred on a pig farm in Argentina. Molecular analysis indicated that the virus was genetically related to the pandemic (H1N1) 2009 influenza virus strain. The outbreak presumably resulted from direct human-to-pig transmission.Facultad de Ciencias Veterinaria

Pandemic (H1N1) 2009 Outbreak on Pig Farm, Argentina

In June–July 2009, an outbreak of pandemic (H1N1) 2009 infection occurred on a pig farm in Argentina. Molecular analysis indicated that the virus was genetically related to the pandemic (H1N1) 2009 influenza virus strain. The outbreak presumably resulted from direct human-to-pig transmission

Pandemic (H1N1) 2009 outbreak on pig farm, Argentina

In June-July 2009, an outbreak of pandemic (H1N1) 2009 infection occurred on a pig farm in Argentina. Molecular analysis indicated that the virus was genetically related to the pandemic (H1N1) 2009 influenza virus strain. The outbreak presumably resulted from direct human-to-pig transmission.Facultad de Ciencias Veterinaria