Risk factors and detection of brucella ovis in naturally infected rams and ewes / Fatores de risco e detecção de brucella ovis em carneiros e ovelhas naturalmente infectados

The objective of this study was to verify the occurrence of ovine brucellosis using Agar Gel Immunodiffusion (AGID) and Polymerase Chain Reaction (PCR) techniques, as well as to identify the main risk factors associated with infection in sheep flocks belonging to municipalities in the microregion from Teresina, PI, Brazil. A total of 100 urine and blood samples were collected from sheep aged 6 months or older. The urine samples were submitted to conventional PCR and the blood samples were examined by the AGID technique. Of the 100 blood samples, 17 (17%) were reactive to the AGID test. In conventional PCR of 100 urine samples, six (6%) were positive. Risk factors associated to infection by B. ovis included the rearing system (OR=0.19), feed management (OR=0.05), presence of dystotic births (OR=4.50), miscarriages (OR=3.75) and source of water offered to the animals (OR=0.19). Thus, it was concluded that it is possible to detect the occurrence of animals with ovine brucellosis since PCR is a reliable method to confirm infection. Furthermore, there are risk factors associated to infection by B. ovis in the municipalities studied.

Objetivou-se verificar a ocorrência da brucelose ovina através das técnicas de Imunodifusão em Gel de Ágar (IDGA) e Reação em Cadeia da Polimerase (PCR), bem como identificar os principais fatores de risco associados à infecção nos rebanhos ovinos pertencentes a municípios da microrregião de Teresina, PI, Brasil. Foram colhidas 100 amostras de urina e de sangue de ovinos com idade superior ou igual a seis meses. As amostras de urina foram submetidas a PCR convencional e as amostras de sangue à técnica de IDGA. Das 100 amostras de sangue 17 (17%) foram reagentes ao teste de IDGA. Já na PCR convencional das 100 amostras de urina, seis (6%) foram positivas. Ressalta-se que três animais foram positivos em ambos os testes. Como fatores associados à infecção por B. ovis, observou-se o tipo de sistema de criação (OR=0,19), o manejo alimentar (OR=0,05), presença de partos distócicos (OR=4,50), abortamentos (OR=3,75) e a fonte de água fornecida aos animais (OR=0,19). Assim, conclui-se que foi possível detectar a ocorrência de animais com brucelose ovina, uma vez que a PCR é um método confirmatório. Além disso, há fatores de risco associados à infecção por B. ovis nos municípios estudados.

Síntesis intratecal de IgG anti-herpes virus como evidencia neuroinmunológica en pacientes pediátricos / Intrathecal synthesis of IgG against herpes virus as neuroimmunological evidence in pediatric patients

En los procesos neuroinflamatorios se produce a nivel de líquido cefalorraquídeo una activación policlonal y poliespecífica. Esta activación se produce desde los primeros días y puede permanecer por períodos prolongados. Luego por mecanismos de apoptosis los clones que no responden directamente contra los agentes biológicos involucrados no proliferan. El Reibergrama permite saber si las inmunoglobulinas presentes en el líquido cefalorraquídeo se sintetizaron o no en el sistema nervioso central (SNC) y el Índice de Anticuerpo (IA) determina la especificidad de las mismas en caso de que exista síntesis intratecal. Con estas herramientas nos propusimos identificar la respuesta neuroinmunológica frente a agentes de la familia herpesvirus en pacientes pediátricos con proceso inflamatorio del SNC a partir de sus respectivos IA. Para lograr esto se cuantificaron los niveles de IgG y albúmina en suero y líquido cefalorraquídeo (LCR) mediante inmunodifusión radial simple y por ensayo inmunoenzimático, con lo cual se construyó el Reibergrama que permitió la selección de 85 pacientes pediátricos con síntesis intratecal de inmunoglobulinas, que se diferenciaron en cuatro grupos según sus edades. Mediante ensayo inmunoenzimático se cuantificaron los niveles de IgG específica contra citomegalovirus, virus varicela zoster y virus herpes simple, tanto en suero como en LCR y se determinó el IA específico. La respuesta contra los virus estudiados fue similar para los distintos grupos de edades, lo cual nos permite afirmar la exposición temprana a los mismos(AU)

In a neuroinflammatory process a polyclonal and poly-specific activation is produced in cerebrospinal fluid. This activation starts from the first days and may persist for a long time. The clones not related directly against the biological agent do not proliferate by apoptosis. Reibergram determine if part of the immunoglobulins content in cerebrospinal fluid belongs from the blood or it is synthesized in the central nervous system. Antibody index determines if the specific antibodies was synthesized intrathecally. By these tools it can be possible to identify the humoral immune response against some herpes virus in pediatric patients suffering from a central nervous system inflammatory process. Quantification of specific IgG against citomegalovirus, varicella zoster and herpes simplex virus in serum and cerebrospinal fluid was done by ELISA. Specific Antibody index against these viruses were similar for the different age groups, which confirm the early exposure of the population(AU)

Performance assessment of imported ELISA in the serodiagnosis of the enzootic bovine leukosis in herds of Pernambuco state, Brazil / Avaliação do desempenho de ELISA importado no sorodiagnóstico da leucose enzoótica bovina em rebanhos do estado de Pernambuco, Brasil

Enzootic bovine leukosis (EBL) is an infectious disease of cosmopolitan distribution and chronic character caused by a virus of the Retroviridae family, bovine leukemia virus (BLV). The epidemiological situation of EBL in Brazil has motivated studies to improve its diagnosis, based on the recommended serological techniques: agar gel immunodiffusion (AGID) and enzyme-linked immunosorbent assay (ELISA). This study was designed to evaluate the use of imported ELISA for the detection of BLV in dairy herds raised in Pernambuco, Brazil, comparing it to AGID. Blood serum samples from 327 dairy cattle from the state of Pernambuco were tested to AGID and the imported commercial ELISA CHEKIT-Leucose-serum, produced by the IDEXX® laboratory for the diagnosis of EBL. Discarding 25 inconclusive samples from one or both tests, 302 samples were analyzed, being 24.1% positive (73/302) in the AGID and 45% (136/302) in the ELISA, which compared to the AGID, a technique considered standard, presented sensitivity of 98.6%, specificity of 72% and Kappa coefficient of 0.55. The lack of agreement in the diagnostic methods was probably due to the high sensitivity of the ELISA, which makes it possible to detect antibodies even in situations with low serum levels. Although AGID has been shown to be an efficient test so far, in more advanced stages of an EBL control and eradication program, with low prevalence rates, ELISA will present better performance, due to its higher sensitivity, avoiding the permanence of animals that spread the disease in the herds.(AU)

A leucose enzoótica bovina (LEB) é uma doença infecciosa de distribuição cosmopolita e caráter crônico causada por um vírus da família Retroviridae, o vírus da leucemia bovina (VLB). A situação epidemiológica da LEB no Brasil vem motivando estudos para o aprimoramento do seu diagnóstico, tendo como base as técnicas sorológicas recomendadas: imunodifusão em gel de ágar (IDGA) e Enzyme-Linked Immunoabsorbent Assay (ELISA). Este estudo teve como objetivo avaliar o uso de ELISA importado para a detecção do VLB em rebanhos leiteiros criados em Pernambuco, Brasil, comparando-o ao IDGA. Amostras de soro sanguíneo de 327 bovinos leiteiros do estado de Pernambuco foram testadas para IDGA e ELISA comercial importado CHEKIT-Leucose-serum, produzido pelo laboratório IDEXX® para o diagnóstico da LEB. Descartadas 25 amostras inconclusivas de um ou ambos os testes, foram analisadas 302 amostras, sendo 24,1% positivas (73/302) na IDGA e 45% (136/302) no ELISA, que em relação à IDGA, técnica considerada padrão, apresentou sensibilidade de 98,6%, especificidade de 72% e coeficiente Kappa de 0.55. A falta de concordância entre os métodos diagnósticos deveu-se, provavelmente, à elevada sensibilidade do ELISA, que possibilita detectar anticorpos mesmo em situações com baixos teores séricos. Apesar da IDGA se mostrar até o momento um teste eficiente, em etapas mais avançadas de um programa de controle e erradicação da LEB, com baixos índices de prevalência, o ELISA apresentará melhor desempenho, por possuir maior sensibilidade, evitando-se a permanência de animais disseminadores da doença nos rebanhos.​(AU)

Neurobrucelosis evaluada por reibergrama. Presentación de un caso / Neurobrucellosis evaluated by reibergram. A case report

Introducción: La brucelosis es una enfermedad zoonótica y endémica en muchas partes del mundo. La causa principal de la infección se produce por la ingestión de leche no pasteurizada o por el contacto con animales infectados. La neurobrucelosis incluye afecciones en el sistema nervioso central y periférico. Las principales manifestaciones clínicas son la meningitis, la encefalitis, la neuritis óptica y la periférica. Objetivo: Evaluar, mediante reibergrama, la dinámica intratecal de las clases mayores de inmunoglobulinas y el estado de la barrera sangre/LCR de un paciente con neurobrucelosis. Presentación del caso: Los niveles de IgA, IgM. IgG y albúmina en suero y líquido cefalorraquídeo fueron cuantificados por inmunodifusión. Los resultados fueron colocados en el reibergrama correspondiente. El paciente mostró síntesis intratecal de las tres clases mayores de inmunoglobulinas, sin disfunción de la barrera sangre/LCR. Conclusión: El estudio neuroinmunológico del líquido cefalorraquídeo puede indicar el curso activo de la respuesta inmune intratecal contra el patógeno, donde la síntesis intratecal de inmunoglobulinas y el funcionamiento de la barrera sangre/líquido cefalorraquídeo constituyeron los principales marcadores en el diagnóstico de la neuroinflamación(AU)

Introduction: Brucellosis is a zoonotic and an endemic disease in many areas around the world. The main cause of infection is the intake of unpasteurized milk or the contact with infected animals. Neurobrucellosis includes pathologic conditions in the central and peripheral nervous systems. The main clinical manifestations are meningitis, encephalitis, optical neuritis, and peripheral neuritis. Objective: To evaluate, through reibergram, the intrathecal dynamics of the major immunoglobulin classes and the blood-CSF barrier function in one patient with neurobrucellosis. Case report: IgA, IgM, IgG and albumin levels in serum and cerebrospinal fluid were quantified by using a radial immunodiffusion technique. Results were placed in the corresponding reibergram. The patient showed evidences of intrathecal synthesis of the three major immunoglobulins without blood-CSF barrier dysfunction. Conclusion: The neuroimmunological study of cerebrospinal fluid can indicate the active course of the intrathecal immune response against this pathogen, where the intrathecal synthesis of immunoglobulins and blood-cerebrospinal fluid barrier function constitute the main markers in the diagnosis of neuroinflammation(AU)

Optimización del uso de las Placas de Inmunodifusión Radial Simple de la firma Siemens / Optimization of the usage of single radial immunodiffusion plates from Siemens

Introducción: La Inmunodifusión radial simple es una técnica con fundamento inmunológico confiable por su especificidad para la cuantificación de inmunoglobulinas principales y se emplea también para otras proteínas. Las placas de Inmunodifusión comerciales se ofertan con un número determinado de pocillos donde se coloca la muestra biológica que contiene la proteína a cuantificar. Objetivo: Evaluar la sensibilidad y la especificidad de la modificación introducida para optimizar el uso de las placas de inmunodifusión radial simple de la marca SIEMENS por aumento del número de muestras por placas. Material y Métodos: Se presenta una innovación que permite optimizar el área biológicamente activa de la placa no utilizada para emplearla para la cuantificación de otras muestras. Se realizan montajes paralelos de muestras de controles en los pocillos tradicionales y en los realizados en los espacios disponibles para cuantificar IgG y albúmina para suero y líquido cefalorraquídeo. Resultados: La sensibilidad del empleo por el método tradicional y por el nuevo no presenta diferencias significativas. En cuanto a la especificidad tampoco existen diferencias significativas menos en las placas para cuantificar albúmina en suero por lo que se recomienda diluir la muestra de suero antes de ser utilizada en el área disponible. En el caso de las placas NOR y LC Partigen® el número de muestras a ser beneficiadas con la cuantificación se duplica, pero de igual manera puede ser aplicada en otras placas de otras firmas comerciales. Conclusiones: Esta innovación permite hacer un uso óptimo de las placas de inmunodifusión con el consiguiente ahorro de material de importación y se puede aplicar fácilmente en todos los laboratorios del país(AU)

Introduction: Single radial immunodiffusion assay is a technique with immunological base, which is reliable because of its specificity in the quantification of main immunoglobulins, although it is also used for other proteins. Commercial immunodiffusion plates are offered with a determined number of holes where the biological samples containing protein to be quantified are placed. Objective: To evaluate the sensitivity and specificity of the modification implemented to optimize the usage of single radial immunodiffusion plates from Siemens by increasing the number of samples in the plates. Materials and Methods: An innovating procedure that allows to optimize the non-used biologically active area and use it in the quantification of other samples is presented. A parallel quantification of control samples from traditional holes and the other ones opened in available spaces was performed in order to quantify IgG and albumin in serum and in cerebrospinal fluid. Results: Sensitivity was not affected significantly between the normal plates and the usage of the new procedure. Regarding specificity, there are also no significant differences except in the plates used to quantify serum albumin; so, it is recommended to dilute serum samples before the application. In case of NOR and LC Partigens®, this proposed modification duplicates the number of samples to be quantified in each plate, but otherwise, it could be applied in other commercial immunoplates. Conclusions: This innovation allows to make an optimal usage of immunodiffusion plates with the consequent saving of import materials, which can be easily applied in all the laboratories of the country(AU)

Diagnóstico serológico de la esporotricosis mediante el empleo del antígeno de micelio de Sporothrix schenckii sensu stricto / Serological diagnosis of sporotrichosis using an antigen of Sporothrix schenckii sensu stricto mycelium

En este estudio se desarrolló y se evaluó el ensayo por inmunoabsorción ligado a enzimas (ELISA), para la detección de anticuerpos en sueros de pacientes con esporotricosis, para lo cual se empleó un antígeno crudo de Sporothrix schenckii sensu stricto obtenido a partir de la forma micelial. Los sueros positivos para esporotricosis fueron ensayados por otras técnicas serológicas: inmunodifusión doble (IDD) y contrainmunoelectroforesis (CIE). El ensayo fue validado utilizando sueros de otras patologías como histoplasmosis, paracoccidioidomicosis, tuberculosis, leishmaniasis, lupus y sueros de individuos sanos como controles negativos. Se encontró una especificidad de 100 % con las técnicas utilizadas y una sensibilidad del antígeno de S.schenckii sensu stricto, por encima del 98% para IDD, CIE y ELISA. Estos resultados demuestran la alta sensibilidad y especificidad del antígeno de S. schenckii sensu stricto, para el diagnóstico de la esporotricosis, empleando las técnicas de IDD, CIE y ELISA. Los resultados sugieren, que este antígeno podría ser usado en conjunto con otras pruebas convencionales para el diagnóstico diferencial y puede ser útil para monitorizar la evolución de la enfermedad y respuesta al tratamiento.

We developed and analyzed an Enzyme-Linked Immunosorbent Assay (ELISA) in order to detect antibodies in sera from sporotrichosis patients. We used a crude antigen of Sporothrix schenckii sensu stricto, obtained from the mycelial phase of the fungi. Positive sera were analyzed by other serological techniques such as double immunodiffusion (IGG) and counterimmunoelectrophoresis (CIE). The assay was validated by using sera from patients with other pathologies such as: histoplasmosis, paracoccidioidomycosis, tuberculosis, leishmaniasis, lupus and healthy individuals as negative controls. For the Sporothrix schenckii sensu stricto antigen, we found a 100% of specificity by every technique and sensitivity higher than 98% with IDD, CIE and ELISA. Our results show a high sensitivity and specificity for the Sporothrix schenckii sensu stricto antigen, so it can be used for IDD, CIE and ELISA. The results suggest that this antigen could be used in conjunction with other conventional tests for differential diagnosis and may be useful for monitoring the disease progression and response to treatment.

Ferramentas diagnósticas de Lentivirose de Pequenos Ruminantes: padronização da técnica de ELISA indireto / Diagnostic tools of small ruminant lentiviruses: standardization of indirect ELISA

As Lentiviroses de Pequenos Ruminantes (LVPR) incluem a Maedi-Visna (MV) em ovinos e a Artrite Encefalite Caprina (CAE). Essas enfermidades estão difundidas no mundo e são responsáveis por grandes perdas na produtividade destes animais. Os LVPR são vírus RNA da subfamília Lentivirinae que causam uma infecção persistente, sendo a detecção precoce uma das formas mais eficientes para limitar sua disseminação no rebanho. Visando contribuir com essas questões, este experimento foi realizado na Universidade Federal do Piauí (UFPI) em parceria com a Embrapa Caprinos e Ovinos, com o objetivo de padronizar a técnica de ensaio imunoenzimático indireto e compará-lo com a imunodifusão em gel de agarose no diagnóstico da CAE. Foram utilizadas 696 amostras de soros de caprinos machos e fêmeas oriundas do banco de soros da Unidade de Pesquisa de LVPR do Centro de Ciências Agrárias da UFPI. As amostras foram coletadas no período de janeiro de 2007 a março de 2010. Na padronização, verificou-se que 0,25 µg de proteína/poço, diluição de 1:200 do soro e concentração de 1:3.000 do conjugado anticorpo anti-IgG cabra apresentaram os melhores resultados. O ponto de corte obtido foi de 0,36. Na comparação, o Imunodifusão em Gel de Ágar (IDGA) detectou 128 (18,4%) amostras positivas, e o ELISA indireto (ELISA-i), 259 (37,2%). A sensibilidade e a especificidade do teste ELISA-i com relação ao IDGA foi de 94,5% e 75,7%, respectivamente. Verificou-se maior índice de positividade em caprinos acima de seis meses (p < 0,05), e nos machos obteve-se prevalência de 56,7% em comparação às fêmeas, 35,4%, (p < 0,01).(AU)

The Small Ruminant Lentiviruses (SRLVs) include Maedi-Visna (MV) of sheep and Caprine Arthritis-Encephalitis (CAE). These diseases are widespread and responsible for major production losses regarding sheep and goats. The SRLV is a RNA virus of the subfamily Lentivirus genus that causes persistent infections in goats. Early detection is one of the best ways to limit its spread in the herd. To contribute to these issues, this experiment was conducted at Universidade Federal do Piauí in partnership with Embrapa Goats and Sheep, with the objective of standardizing the technique of indirect ELISA (i-ELISA) and to compare it with Immunodiffusion in Agarose Gel to diagnose Caprine Lentiviruses (LC). Six hundred ninety six serum samples were used from the University Veterinary Hospital, Universidade Federal do Piauí, from January 2007 to March 2010. Standardization showed that 0.25 µg protein/well, a 1:200 dilution of the serum and concentration of 1:3,000 of the conjugated anti-goat IgG presented the best results. It was observed that the Agar Gel Immunodiffusion (AGID) detected 128 (18.4%) positive samples, and ELISA, 259 (37.2%). The sensitivity and specificity of i-ELISA regarding AGID were 94.5% and 75.7%, respectively. A higher prevalence was observed among animals older than six months (p < 0.05). The prevalence among males was of 56.7%, and among females, 35.4% (p < 0.01).(AU)

Técnicas electroforéticas en el HYDRASYS 2. Utilidad diagnóstica en diferentes enfermedades / Electrophoretic techniques in HYDRASYS 2. Diagnostic usability in different diseases

Se presentan los resultados de la estandarización de las técnicas de electroforesis de hemoglobina (Hb), isoenzimas de la deshidrogenasa láctica (LDH) y proteinuria en el equipo Hydrasys 2, así como el estudio de pacientes atendidos en el Instituto de Hematología e Inmunología y en otros centros hospitalarios del país. Se realizó el diagnóstico de 149 portadores de hemoglobinopatías (AS, AC, b talasemia heterocigótica, variante rápida), 60 enfermos (SS, SC, CC), 24 pacientes con a talasemia o deficiencia de hierro y se cuantificó la hemoglobina fetal a 93 casos con hemoglobinopatía S. Se determinaron los valores normales de actividad e isoenzimas de LDH en la población mediante el estudio de 50 donantes de sangre. En los pacientes con anemia drepanocítica se encontró un aumento significativo de la isoenzima 1 (p= 0,000) y disminución de isoenzimas 3 (p= 0,002). Se realizó el estudio de proteínas en orina a 8 pacientes con enfermedades hematológicas que presentaron microalbuminuria al menos en 2 ocasiones, con concentraciones ³ 0,04 g/L. En 2 pacientes el resultado fue normal; en 2 se encontraron proteínas de origen tubular; y en otros 2, proteínas de origen glomerular

We present the results of the standardization of the techniques of electrophoresis of hemoglobin (Hb), lactate dehydrogenase isoenzymes (LDH) and proteinuria in HYDRASYS 2 equipment, and the study of patients treated at the Institute of Hematology and Immunology and other hospitals in the country. 149 hemoglobinopathies carriers were diagnosed (AS, AC, b thalassemia heterozygous fast variant), 60 patients (SS, SC, CC), 24 patients with athalassemia or iron deficiency. Fetal hemoglobin was quantified in 93 cases with hemoglobinopaty S. Normal values of activity and LDH isoenzymes were determined in the population through the study of 50 blood donors. In patients with sickle cell anemia we found a significant increase in isoenzyme 1 (p=0.000) and isozyme 3 decreased (p=0.002). We performed the study of proteins in urine in 8 patients with hematologic malignancies who had microalbuminuria at least 2 times, with concentrations ³ 0.04 g / L. In 2 patients the results were normal, in 2 proteins were tubular origin, and in 2, proteins of glomerular origin

Development of agar diffusion method for dosage of gramicidin / Desenvolvimento do método de difusão em agar para dosagem de gramicidina

Gramicidin, an antimicrobial peptide active against Gram positive bacteria, is commonly used in pharmaceutical preparations for topical use. Considering that only the turbidimetric method has been described in the literature, the present study sought to develop and validate an agar diffusion method for the dosage of gramicidin. The method was developed and validated using the Kocuria rhizophila ATCC 9341 as a test microorganism. Two designs were used: a 3x3 parallel-line model, and a 5x1 standard curve. The validation demonstrated that the method follows the linear model (r²= 0.994), presenting a significant regression between the zone diameter of growth inhibition and the logarithm of the concentration within the range of 5 to 25.3 µg/mL. The results obtained for both designs were precise, having a relative standard deviation (R.S.D.) for intra-day precision of 0.81 for the 3x3 assay and 1.90 for the 5x1 assay. For the inter-day precision, the R.S.D. was 1.35 for the 3x3 and 2.64 for the 5x1. The accuracy was verified and results confirmed to be accurate, having a tolerance interval of 95 percent, which lay within permitted limits and appropriate trueness. In addition, the method was considered selective, with limit of detection and upper and lower limits of quantification of 2.00, 5.00 and 25.3 µg/mL, respectively. No difference in precision between the designs used in the agar diffusion method was evident (p>0.05). The method proved to be appropriate for the microbiological dosage of the raw material gramicidin.

A gramicidina, um peptídeo antimicrobiano ativo contra bactérias Gram positivo, é utilizada em preparações farmacêuticas de uso tópico. Neste trabalho procurou-se desenvolver e validar outro método para o doseamento de gramicidina tendo em vista que somente o método turbidimétrico é descrito. O método de difusão em ágar foi desenvolvido e validado utilizando como microrganismo teste Kocuria rhizophila ATCC 9341. Foram utilizados dois delineamentos: retas paralelas 3x3 e curva padrão 5x1. A validação demonstrou que o método segue o modelo linear (r²= 0,994) havendo regressão significativa entre o diâmetro dos halos de inibição e o logaritmo da concentração na faixa de 5,00 a 25,3 µg/mL. Os resultados obtidos por ambos os delineamentos foram precisos apresentando desvio padrão relativo (DPR) para precisão intra-dia de 0,81 para ensaio 3x3 e de 1,90 para ensaio 5x1. Para a precisão inter-dias o DPR foi de 1,35 para 3x3 e de 2,64 para 5x1. A exatidão foi verificada e os resultados foram exatos apresentando intervalo de tolerância a 95 por cento dentro dos limites permitidos e veracidade adequada. O método foi seletivo com limites de detecção e quantificação inferior e superior iguais a 2,00, 5,00 e 25,3 µg/mL, respectivamente. Não foi observada diferença entre a precisão dos delineamentos empregados no método de difusão em ágar (p>0.05). O método se mostrou adequado para a dosagem microbiológica de gramicidina matéria-prima.

Diagnosis of giardiasis in asymptomatic and symptomatic patients using G. lamblia antigen[s] and IgA antibody in saliva

Giardiasis may be asymptomatic or symptomatic with possible extra-intestinal complications such as maculopapular rashes, polyarthritis and urticaria. Diagnosis by stool examination may give false negative results. Accurate exclusion of giardiasis is important to differentiate it from other causes of these manifestations. To assess the employment of saliva for diagnosing G. lamblia infections. The study attempted to determine the presence of G. lamblia antigen in human saliva by immunodiffusion technique and the associated specific secretory IgA [sIgA] immune response by immunoblot technique. Samples of saliva were collected from 80 subjects; 40 Giardia-infected individuals [symptomatic and asymptomatic], 20 subjects infected with other intestinal parasites and 20 healthy individuals as control groups of similar age and sex. Giardia cysts were collected from stools of heavily infected individuals, excysted and the harvested forms were cultivated on TYI-S-33 medium to prepare trophozoite antigen. The latter was used to produce hyperimmune serum in rabbits that was employed to detect the presence of specific antigen [s] in saliva by double gel immunodiffusion technique. Cysts were also used for antigen preparation which was fractionated by SDS-PAGE and tested to detect sIgA by immunoblotting of saliva samples. G. lamblia antigen was not detected in the saliva of any of the individuals enrolled in the study. Secretory IgA was detected in 75% of all infected individuals with 75% sensitivity and 90% specificity and in 90% of symptomatic and 60% of asymptomatic infected individuals. Molecular weight [MW] bands of 170 and 133 kDa were recognized by specific salivary sIgA by immunoblot analysis. Sensitivity and specificity of the 170 kDa band recognition were 75% and 90%, respectively with 88.2% and 78.2% positive [PPV] and negative [NPV] predictive values, respectively. The 133 kDa band gave 42.5% sensitivity, 100% specificity with 100% PPV and 62.3% NPV. Absence of specific antigen in the saliva refutes the assumption that G. lamblia antigens may reach the blood and questions the possibility of tissue invasion. The 170 and 133 kDa of Giardia cysts antigens form together useful molecules for diagnosis of giardiasis by IgA immunoblotting

Diffusional restrictions in glyoxyl-agarose immobilized penicillin G acylase of different particle size and protein loading / Restricciones para la difusión de penicilina G acilasa inmovilizada en glicosil agarosa

Particle size and enzyme protein loading are design parameters of enzyme immobilization affecting biocatalyst performance that can be varied within broad margins. Their effect on mass transfer limitations at different bulk penicillin G concentrations has been studied with glyoxyl agarose immobilized penicillin G acylase biocatalysts of average particle size of 5·10-5m and 10·10-4m at protein loadings from 15 to 130 mg/g gel. Internal diffusional restrictions were evaluated for such biocatalysts: Thiele modulus varied from 1.17 for the small particles at the lower protein load to 5.84 for the large particles at the higher protein load. Effectiveness factors at different bulk substrate concentrations were determined for all biocatalysts, values ranging from 0.78 for small particle size at 25 mM penicillin G to 0.15 for large particle size at 2 mM penicillin G. Enzyme protein loading had a strong impact on the effectiveness factors of immobilized penicillin G acylase, being it more pronounced in the case of large particle size biocatalysts. At conditions in which 6-aminopenicillanic acid is industrially produced, all biocatalysts tested were mass-transfer limited, being this information valuable for reactor design and performance evaluation.

Detection of native and modified soybean in some meat production in Assiut city, Egypt

High meat prices prompted the meat industries in Egypt to produce various meat brands extended with soybean proteins. Genetically modified foods are often in the news. Much of the world has experienced strong and increasing resistance to the introduction of any genetically modified foods to the market place. Agar gel immunodiffusion [AGID] and polymerase chain reaction [PCR] were used to detect soybeans in some meat products [minced meat, raw kofta, sausage and beef burger]. PCR was applied due to stability of deoxyribonucleic acid [DNA] at high temperature and highly conserved structure of DNA within all tissues of an individual. Soybean was detected with AGID at 12%, 30% and 20% in raw kofta, sausage and beef burger, respectively, but not detected in minced meat. By using PCR native and modified soybeans were detected in 100% and 69%, respectively in beef burger and at lower rates in other products

Soroprevalência de Maedi-Visna em ovinos na região de Araçatuba, SP / Occurrence of Maedi-visna in sheep from Araçatuba region - SP - Brazil

The seroprevalence of Maedi-Visna in sheep from Araçatuba region - SP, was determined and correlated to age, gender, breed, or sheep production systems. Blood samples were collected from 444 sheep, aging from two to 12 year-old. Both sexes and different breeds were sampled in 20 farms of this region. Physical examination was performed in all animals. Agar gel immunodiffusion test kit was used to diagnose in serum samples. Twelve animals, from five different farms, were AGID positive, yielding a seroprevalence of 2.7%, with no correlation among breed, gender, or sheep production systems and the detection of the disease. No animal considered positive for Maedi-Visna showed clinical signs compatible with Maedi-Visna.

Verificação da atividade antibacteriana de sabonete líquido contendo extrato glicólico de Dimorphandra mollis Benth. / Verification of the antibacterial activity of liquid soap containing glycolic extract of Dimorphandra mollis Benth.

Dimorphandra mollis Benth., Composita e, falso barbatimão, é utilizada topicamente como cicatrizante, adstringente e antimicrobiano. No presente estudo, verificou-se a atividade antibacteriana de sabonete líquido contendo extrato glicólico de D. mollis (EGD) em diferentes concentrações (8, 15 e 20%) e em diferentes pHs (6 e 8). Foram preparadas cinco formulações (F) de sabonete: F1 - triclosan (0,1%), F2 - EGD (8%), F3- EGD (15%), F4 - EGD (20%) e F5 - sem conservante. Cascas de D. mollis foram secas em estufa de ar circulante e pulverizadas. Os extratos brutos foram preparados por turbo-extração utilizando-se etanol. Após filtração, os extratos foram concentrados em evaporador rotatório, liofilizados e ressuspendidos em propilenoglicol para a obtenção do extrato glicólico. A atividade antibacteriana foi verificada pelo método de difusão em ágar, empregando cilindros em placa. Placas contendo Staphylococcus aureus, Pseudomonas aeruginosa e Escherichia coli foram incubadas a 37ºC durante 24 horas. Após incubação, as leituras foram realizadas com paquímetro, observando-se o diâmetro do halo de inibição de crescimento bacteriano. Verificou-se que o sabonete líquido contendo triclosan provocou inibição do crescimento bacteriano em ambos os pHs; já os sabonetes sem conservante e contendo EGD, independente da concentração e do pH empregados, não apresentaram atividade antibacteriana.

Dimorphandra mollis Benth., Compositae, false barbatimão, has been used topically as a healing, astringent and antibacterial. In this study, antibacterial activity was verified on liquid soap containing glycolic extract of D. mollis (DGE) at different concentrations (8, 15 and 20%) and at different pH levels (6 and8). Five soap formulations (F) were prepared: F1 -tryclosan (0.1%), F2 - DGE (8%), F3 - DGE (15%),F4 - DGE (20%) and F5 - without preservatives. Bark of D. mollis were dried in a circulating air oven and ground. The rude extracts were prepared by turboextraction with ethanol. After screening, the extract were concentrated in rotating evaporator, lyophilized and resuspended in propileneglycol to obtain the glycolic extract. The antimicrobial activity was verified by diffusion in agar method, using cylinder in plate. Plates containing Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia coli were incubated at 37ºC for 24 hours. After incubation, the results were analysed with a pachy meter, observing the bacterial grouth inhibition halo diameter. It was verified that the liquid soap containing tryclosan caused on inhibition of bacterial growth at both pH levels; the soaps without preservatives and containing DGE, independently of the concentration and pH levels used, did not present antibacterial activity.

Agar gel immunodiffusion analysis using baculovirus-expressed recombinant bovine leukemia virus envelope glycoprotein (gp51/gp30T-)

Bovine leukemia virus (BLV) envelope glycoprotein (gp51/gp30T-), consisting of BLV gp51 and BLV gp30 that lacked its C-terminal transmembrane domain, was expressed in insect cells under the control of the baculovirus polyhedron promoter. Recombinant BLV gp51/gp30T- secreted from insect cells was determined by immunofluorescence, enzyme-linked immunosorbent and western blot assays using a BLV-specific monoclonal antibody and BLV-positive bovine antibodies. An agar gel immunodiffusion (AGID) test using gp51/gp30T- as the antigen for the detection of BLV antibodies in serum was developed and compared to traditional AGID, which uses wild type BLV antigen derived from fetal lamb kidney cells. AGID with the recombinant BLV gp51/gp30T- was relatively more sensitive than traditional AGID. When the two methods were tested with bovine sera from the field, the recombinant BLV gp51/gp30T- and traditional antigen had a relative sensitivity of 69.8% and 67.4%, respectively, and a relative specificity of 93.3% and 92.3%. These results indicated that the recombinant BLV gp51/gp30T- is an effective alternative antigen for the diagnosis of BLV infection in cattle.

Situação epidemiológica da leucose bovina enzoótica em Portugal entre os anos de 1995 e 2005 / Epidemiological survey of enzootic bovine leukosis in Portugal from 1995 to 2005

This paper evaluated the epidemiological situation of the enzootic bovine leucosis from 1995 to 2005, in Portugal. With exception of the South region, Algarve, the disease was distributed throughout the country, being more prevalent in the north, between Douro and Minho and Trás-os-Montes, than in the centre. A decrease in prevalence and incidence of the infection throughout the studied period was also observed.

Avaliação da atividade antimicrobiana de tinturas fitoterápicas sobre porphyromonas gingivales e prevotella melaninogênica / Evaluation of photherapy antimicrobial activity against porphyronomas gingivales and prevotella melaninogênica

O objetivo do presente trabalho foi verificar, in vitro, o efeito antimicrobiano dessas soluções sobre duas espécies bacterianas causadoras de doença periodontal. Método: As tinturas e as soluções de clorexidina a 0,12 por cento como grupo controle positivo, e álcool de cereais, utilizado no preparo das tinturas, como controle negativo, foram diluídas em solução salina de 1:2 até 1:128. Utilizando o método da difusão em agar,as cepas de referência Porphyromonas gingivales ATCC 49417 e Prevotella melaninogenica ATCC 25845, foram semeadas em meio BHI agar enriquecido com extrato de levedura (0,5 por cento) e incubadas em anaerobiose, à 37º C durante 3 dias.Resultados: Os resultados demonstraram que a Tanchagem e a Sálvia possuem ação antibacteriana sobre as duas cepas em teste, assim como a clorexidina. Porém a tintura de Ipê-roxonão interferiu no crescimento de P. gingivales, sendo P.melaninogenica única sensível a esta tintura. As cepas apresentaram-se resistentes ao álcool de cereais. Conclusão:Conclui-se que as tinturas de tanchagem e sálvia apresentam maior espectro de atuação antibacteriana, quando comparadas à tintura de Ipê-roxo, sendo seu efeito não influenciado pelo álcool utilizado na sua fabricação

Evaluación del desempeño diagnóstico del medio de difusión en agar Etest para el estudio de sensibilidad a los antifúngicos / Assessment of diagnostic performance of Agar Etest for antifungal sensitivity testing

El aumento de la incidencia de las micosis profundas ha generado la necesidad de desarrollar técnicas para el estudio de la susceptibilidad in vitro a los antifúngicos. El método de microdilución en caldo es el de referencia, sin embargo métodos alternativos surgen ante la necesidad de diagnóstico en el laboratorio clínico, siendo el Etest el método de difusión en agar m s utilizado en nuestro medio. En este trabajo se evaluó el desempeño diagnóstico del Etest en comparación con la técnica de referencia y se correlacionaron las concentraciones inhibitorias mínimas (CIMs) por ambos métodos. Se estudiaron 80 cepas de Candida spp., proces ndose por Etest y por microdilución en caldo (protocolo M27-A2 del National Committee for Clinical Laboratory Standards [NCCLS]). Se probaron: anfotericina B (AB), itraconazol (ITZ) y fluconazol (FLZ). Se evaluó el desempeño diagnóstico del Etest con respecto a la metodología de referencia (EPIDAT, versión 2.0 para Windows) y se calculó la correlación mediante el coeficiente de Pearson. El Etest presentó para el ITZ una especificidad y sensibilidad diagnóstica de 44 por ciento y 92 por ciento respectivamente, valor predictivo positivo (VPP) de 60por ciento y valor predictivo negativo (VPN) de 85 por ciento. Para el FLZ la especificidad y sensibilidad fue de 85 por ciento y 12,5 por ciento, respectivamente, VPP de 45 por ciento y VPN de 49 por ciento. Para AB el Etest no captó ninguna de las cepas resistentes. Los valores predictivos fueron calculados para detectar resistencia empleando los puntos de corte del National Committee for Clinical Laboratory Standards (NCCLS). Los coeficientes de correlación fueron los siguientes: r = - 0,02 (AB), r = - 0,03 (FLZ) y r = 0,4 (ITZ). No se validó el método desde el punto de vista analítico por no existir correlación entre las CIMs obtenidas por el Etest y la técnica de referencia. De acuerdo con los resultados obtenidos, el desempeño diagnóstico de la técnica es poco confiable. Analizando el desempeño global del Etest a la luz de los conocimientos actuales no parece un método confiable para su uso en los laboratorios de an lisis clínicos.

Prevalência de anticorpos contra o vírus da língua azul em bovinos e ovinos do Sudoeste e Sudeste do Rio Grande do Sul / Bluetongue virus antibodies in cattle and sheep in Southwest and Southeast regions of Rio Grande do Sul, Brazil

It was studied bluetongue virus antibodies prevalence for sheep and cattle in Southwest and Southeast regions of Rio Grande do Sul State. A total of 2613 serum samples (1272 bovine and 1341 ovine) were tested by agar gel immunodiffusion. Eight bovine and two ovine samples were positive meaning a prevalence of 0.63 percent and 0.15 percent, respectively. These results show that most of animals in these regions are negative to bluetongue.

Comparative study between Elisa immuno diffusion and cell bound immuno assay techniques for detection of anti bovine viral diarrhea antibodies in calves of some farms in Alexandria and Behira Governorates

Rapid, highly sensitive and specific technique is essential need for early diagnosis of Bovine Viral Diarrhea [BVD] infection in calves to prevent, control and eradicate the persistently infected animals; so, ELISA, Immuno-diffusion [ID] and Cell bound immuno assay [CBIA] techniques were compared for detection of anti-Bovine Viral Diarrhea [anti-BVD] antibodies in 240 calves blood samples of some farms in Alexandria and Behira Govcrnorates [120 each]. Out of total 240 tested blood samples; 143 [59.6%], 47 [19.6%], and 155 [64.6%] were positive for anti-BVD antibodies using ELISA, ID and CBIA respectively. The highest detection rate was found in diseased calves at Behira Governorate [83.5%> using CBIA technique, while the lowest detection rate was found in contact calves at Alexandria Governorate [3.2%] using ID technique. The percentage of agreement between CBIA and each of ELISA and ID was 95% and 55% respectively for detection of anti-BVD antibodies in Calve sera. The percentage of sensitivity between CBIA and each of ELISA and ID was 100%, while the percentage of specificity between CBIA and each of ELISA and ID was 87.6% and only 44% repectively for detection of anti-BVD antibodies in calves sera. This study showed that, the CBIA as well as ELISA techniques were highly sensitive and specific tests for detection of anti-BVD antibodies in calves sera when compared to ID technique. Moreover, CBIA has many advantages over ELISA technique; Firstly. CBIA is rapid, simple, cheap and not need to ELISA reader as in ELISA technique. Secondarily; the microtitre plate containing BVD infected tissue culture monolayer can be preserved for long time in comparison to prepared ELISA plates