Results of a sample-to-cutoff ratio using Abbott Architect rHTLV-I/II assay allow to predict detection of HTLV-1 and HTLV-2 proviral DNA by real-time PCR / Resultados da razão sinal da amostra sobre o cutoff da reação no ensaio Abbott Architect rHTLV-I/II permite prever a detecção de DNA proviral por PCR em tempo real

The present study aims to correlate the sample-to-cutoff ratios (S/CO) distributions of reactive results for HTLV-1/2 antibodies with the detection of proviral DNA in a population of blood donor candidates. It was carried out a retrospective data search of 632 HTLV-1/2 reactive samples, submitted to confirmatory testing from January 2015 to December 2019. Serological screening was performed by chemiluminescent microparticle immunoassay Architect rHTLV-I/II, whereas confirmatory testing was performed by in-house real-time polymerase chain reaction method. 496 out of 632 samples (78%) had undetectable HTLV-1/2 proviral DNA and 136 (22%) had detectable proviral DNA. HTLV infection was not confirmed in any individual for whom the S/CO ratio value was <4, and proviral DNA detection rates gradually escalated as S/CO ratio values increased. The sensitivity and predictive positive value found for the Architect rHTLV-I/II was 100% and 22%, respectively. The receiver operating characteristic (ROC) curve analysis showed that the optimal S/CO ratio value for predicting the presence of HTLV-1/2 was 18.11. High S/CO ratios were more associated with the detection of proviral DNA. The S/CO ratio value <4 suggests excluding true HTLV infection and the risk of blood transmission (AU).

O estudo tem como objetivo correlacionar às distribuições das razões sample-to-cutoff (S/CO) de resultados reagentes para anticorpos HTLV-1/2 com a detecção de DNA proviral em uma população de candidatos à doação de sangue. Realizou-se uma busca retrospectiva de dados de 632 amostras reagentes para HTLV-1/2 submetidas à testagem confirmatória entre janeiro de 2015 a dezembro de 2019. A triagem sorológica foi realizada pelo imunoensaio quimioluminescente de micropartículas Architect rHTLV-I/II, enquanto o teste confirmatório foi realizado pelo método de PCR em tempo real in-house. 496 de 632 amostras (78%) apresentaram DNA proviral indetectável e 136 (22%) apresentaram DNA proviral detectável. A infecção por HTLV não foi confirmada em nenhum indivíduo com valor de S/CO <4 e as taxas de detecção de DNA proviral escalonaram gradualmente à medida que as razões S/CO aumentaram. A sensibilidade e valor preditivo positivo encontrados para o Architect rHTLV-I/II foram 100% e 22%, respectivamente. Utilizando análise de curva ROC, o valor de razão S/CO ideal para predizer a presença de DNA proviral foi de 18,11. Razões S/CO elevadas foram mais associadas à detecção de DNA proviral. Em suma, o valor de S/CO <4 sugere a exclusão de infecção por HTLV e o risco de transmissão pelo sangue (AU).

Anatomomopathological and immunohistochemical analyses of the spleen and lymph node of dogs seropositives for leishmaniasis in serological tests / Análises anatomopatológica e imuno-histoquímica do baço e linfonodo de cães soropositivos para leishmaniose em testes sorológicos

Canine leishmaniasis (CanL) is a zoonosis caused by the protozoan of the species Leishmania infantum. The spleen and lymph nodes undergo morphological changes during CanL. This research aimed to perform an anatomopathological and immunohistochemical study of these organs in dogs reactive to leishmaniasis in the Dual-path Platform chromatographic immunoassay (DPP®) and Enzyme Immunoabsorption Assay (ELISA). Twenty-seven dogs were evaluated for anatomopathological examination with 92.6% showing changes at gross evaluation, specially splenomegaly and lymphadenomegaly. All dogs showed changes in the spleen unrelated to the parasitic load, with granulomatous splenitis being the most severe change. Diffuse cortical and paracortical hyperplasia, and hyperplasia and hypertrophy of the medullary cords were observed in the lymph node. Amastigote forms of Leishmania spp. were found in the spleen and lymph node at histopathological and immunohistochemical evaluations, with good agreement between these evaluations (k = 0.55, p = 0.00124), but no difference was observed in the parasitic intensity of these organs at immunohistochemistry (p = 0.23). It was concluded that spleen and lymph node from dogs reactive to leishmaniasis on the DPP® and ELISA tests show histomorphological changes resulting from the disease, independent to the parasitic load, as well as these organs show similar parasitic load at immunohistochemical test.

A leishmaniose canina (CanL) é uma zoonose causada pelo protozoário da espécie Leishmania infantum. O baço e os linfonodos sofrem alterações morfológicas durante o CanL. Esta pesquisa teve como objetivo realizar um estudo anatomopatológico e imuno-histoquímico desses órgãos em cães reativos para leishmaniose aos testes de Imunoensaio Cromatográfico "Dual Path Platform" (DPP®) e Ensaio de Imunoabsorção Enzimática (ELISA). Vinte e sete cães foram avaliados ao exame anatomopatológico, com 92,6% exibindo alterações à avaliação macroscópica, especialmente esplenomegalia e linfadenomegalia. Todos os cães apresentaram alterações no baço não relacionadas à carga parasitária, sendo a esplenite granulomatosa a alteração mais grave. Hiperplasia cortical e paracortical difusa e hiperplasia e hipertrofia dos cordões medulares foram observadas nos linfonodos. Formas amastigotas de Leishmania spp. foram encontradas no baço e linfonodo às avaliações histopatológica e imuno-histoquímica, com boa concordância entre os métodos (k = 0,55, p = 0,00124), mas não foi observada diferença na intensidade parasitária entre esses órgãos à imuno-histoquímica (p = 0,23). Conclui-se que baço e linfonodo de cães reativos para leishmaniose aos testes DPP® e ELISA apresentam alterações histomorfológicas decorrentes da doença, independente da carga parasitária, assim como esses órgãos apresentam carga parasitária semelhante ao método imuno-histoquímico.

Epidemiological and clinical aspects of snakebites in the upper Juruá River region, western Brazilian Amazonia / Aspectos clínicos e epidemiológicos de acidentes ofídicos na região do Alto Juruá, oeste da Amazônia brasileira

This study addresses the clinical and epidemiological aspects of envenoming cases resulting from snakebites treated at a hospital in Cruzeiro do Sul, in the upper Juruá River region, western Brazilian Amazonia. The specific identity of snakes that caused the envenomings was inferred (a) from the diagnosis of patient symptoms and signs upon hospital admission, (b) by enzyme immunoassay for detection of Bothrops atrox and Lachesis muta venom from serum samples taken from patients before antivenom therapy, or (c) by direct identification of the snake, when it was brought along to the hospital or photographed. There were 133 snakebites (76.2 cases per 100,000 inhabitants) registered during one year (July 2017 to June 2018). Most snakebites (88.7%) were caused by Bothrops spp., and the rest by non-venomous snakes or dry bites. Snakebites tended to occur more often during the rainy season, coinciding with the period of greater reproductive activity of the snakes and greater availability of their prey. In addition, the increase in the water level of rivers and lakes during the rainy season tends to concentrate snakes in dry places and, thus, to increase encounters with humans. Information campaigns on prevention and first aid, specially among the most vulnerable groups (indigenous people, farmers, and children and teenagers in rural areas), and the importance of using protective equipment (boots, leggings, leather gloves) in certain high risk activities (e.g. agriculture and extractivism in forests) are fundamental for the reduction of snakebite morbidity.(AU)

Este estudo aborda os aspectos clínicos e epidemiológicos dos envenenamentos ofídicos tratados em um hospital em Cruzeiro do Sul, Acre, resultantes de acidentes que ocorreram na região do Alto Juruá, no oeste da Amazônia brasileira. A identidade específica das serpentes que causaram os envenenamentos foi inferida (a) pelos sinais e sintomas apresentados pelo paciente na admissão hospitalar, (b) por imunoensaio enzimático para detecção de veneno de Bothrops atrox e Lachesis muta em amostras de soro retiradas de pacientes antes da soroterapia, e (c) pela identificação direta da serpente, quando esta foi levada para o hospital ou fotografada. Houve 133 casos (76,2 casos por 100.000 habitantes) de acidentes ofídicos registrados durante um ano (julho 2017 a junho 2018). A maioria das picadas de serpentes (88,7%) foi causada por Bothrops spp., e o restante por espécies não peçonhentas ou picadas secas. Os acidentes ofídicos tenderam a ocorrer com maior frequência durante a estação chuvosa, coincidindo com o período de maior atividade reprodutiva das serpentes e maior disponibilidade de suas presas. Além disso, o aumento dos níveis de rios e lagos pode fazer com que esses animais procurem locais mais secos, aumentando a frequência de encontro com seres humanos. Campanhas educativas sobre prevenção e primeiros socorros, principalmente entre os grupos mais vulneráveis (indígenas, agricultores, crianças e adolescentes em áreas rurais), e sobre a importância da utilização de equipamentos de proteção (botas, perneiras, luvas de couro) em determinadas atividades de maior risco (e.g., agricultura e extrativismo em florestas), são fundamentais para reduzir a morbidade de picadas de serpentes.(AU)

Anticorpos policlonais para o imunodiagnóstico de vírus bovino / Polyclonal antibodies for the immunodiagnostic of bovine viruses

O objetivo do estudo foi produzir anticorpos policlonais para vírus bovino e testar a reatividade em imunoensaios como imunofluorescência, imunoperoxidase e slot blot. Como imunógeno utilizou-se cepas e/ou isolados dos herpesvírus bovino tipo 1, 2 e 5 (BoHV-1, BoHV-2 e BoHV-5), vírus da diarreia viral bovina (BVDV), vírus respiratório sincicial bovino (BRSV), vírus da língua azul (BTV) e vírus vaccina (VACV) amplificados em cultivo celular. Os coelhos foram imunizados em intervalos regulares e o soro hiperimune produzido foi utilizado como anticorpo primário nos imunoensaios. A diluição de trabalho para cada antissoro foi determinada por diluição seriada limitante e variaram entre 1:800 e 1:51.200. As maiores diluições foram observadas para imunoperoxidase, quando comparadas com a imunofluorescência e slot blot. Diluições menores que 1:800 foram associadas com aumento de reações inespecíficas. Os antissoros anti-BoHV-1, -BoHV-5, -BVDV e -BRSV reagiram frente a isolados heterólogos em ensaios de imunofluorescência e imunoperoxidase. Conclui-se que os antissoros produzidos possuem elevadas concentrações de anticorpos específicos para os vírus e é uma alternativa para a utilização em imunoensaios.

The objective of the study was to produce polyclonal antibodies to bovine viruses and to test the reactivity in immunoassays such as immunofluorescence, immunoperoxidase and slot blot. Bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV), bovine herpesvirus type 1, 2 and 5 (BoHV-1, BoHV-2 and BoHV-5) blue tongue virus (BTV) and vaccinia virus (VACV) were amplified in cell culture and used to immunize rabbits. Animals were immunized at regular intervals and the hyper immune serum produced was used as primary antibody in the immunoassays. The working dilution for each antiserum was determined by limiting serial dilution and varied from 1: 800 to 1: 51,200. The highest dilutions were observed for immunoperoxidase, when compared with immunofluorescence and slot blot. Dilutions lower than 1:800 were associated with the presence of nonspecific reactions. Anti-BoHV-1, -BoHV-5, -BVDV and -BRSV antisera reacted against heterologous isolates in immunofluorescence and immunoperoxidase assays. It is concluded that the produced polyclonal antibodies have high concentrations of virus-specific antibodies and are an alternative for use in immunoassays.

Anticorpos policlonais para o imunodiagnóstico de vírus bovino / Polyclonal antibodies for the immunodiagnostic of bovine viruses

O objetivo do estudo foi produzir anticorpos policlonais para vírus bovino e testar a reatividade em imunoensaios como imunofluorescência, imunoperoxidase e slot blot. Como imunógeno utilizou-se cepas e/ou isolados dos herpesvírus bovino tipo 1, 2 e 5 (BoHV-1, BoHV-2 e BoHV-5), vírus da diarreia viral bovina (BVDV), vírus respiratório sincicial bovino (BRSV), vírus da língua azul (BTV) e vírus vaccina (VACV) amplificados em cultivo celular. Os coelhos foram imunizados em intervalos regulares e o soro hiperimune produzido foi utilizado como anticorpo primário nos imunoensaios. A diluição de trabalho para cada antissoro foi determinada por diluição seriada limitante e variaram entre 1:800 e 1:51.200. As maiores diluições foram observadas para imunoperoxidase, quando comparadas com a imunofluorescência e slot blot. Diluições menores que 1:800 foram associadas com aumento de reações inespecíficas. Os antissoros anti-BoHV-1, -BoHV-5, -BVDV e -BRSV reagiram frente a isolados heterólogos em ensaios de imunofluorescência e imunoperoxidase. Conclui-se que os antissoros produzidos possuem elevadas concentrações de anticorpos específicos para os vírus e é uma alternativa para a utilização em imunoensaios.(AU)

The objective of the study was to produce polyclonal antibodies to bovine viruses and to test the reactivity in immunoassays such as immunofluorescence, immunoperoxidase and slot blot. Bovine viral diarrhea virus (BVDV), bovine respiratory syncytial virus (BRSV), bovine herpesvirus type 1, 2 and 5 (BoHV-1, BoHV-2 and BoHV-5) blue tongue virus (BTV) and vaccinia virus (VACV) were amplified in cell culture and used to immunize rabbits. Animals were immunized at regular intervals and the hyper immune serum produced was used as primary antibody in the immunoassays. The working dilution for each antiserum was determined by limiting serial dilution and varied from 1: 800 to 1: 51,200. The highest dilutions were observed for immunoperoxidase, when compared with immunofluorescence and slot blot. Dilutions lower than 1:800 were associated with the presence of nonspecific reactions. Anti-BoHV-1, -BoHV-5, -BVDV and -BRSV antisera reacted against heterologous isolates in immunofluorescence and immunoperoxidase assays. It is concluded that the produced polyclonal antibodies have high concentrations of virus-specific antibodies and are an alternative for use in immunoassays.(AU)

Sensitivity, specificity and comparison of three commercially available immunological tests in the diagnosis of Cryptosporidium species in animals

The study was conducted to compare the specificity of immunological diagnostic methods used for the diagnosis of Cryptosporidium species capable of causing life-threatening infection in both immunosuppressed and immunocompetent patients. For the detection of Cryptosporidium species in 79 animals with diarrhoea, we used three Copro-antigen tests: RIDASCREEN® Cryptosporidium test, Cryptosporidium 2nd Generation (ELISA) and RIDA®QUICK Cryptosporidium. For immunoassays we used positive and negative samples detected by means of polymerase chain reaction and validated by sequencing and nested polymerase chain reaction to confirm the presence six different species of Cryptosporidium species. Prevalence of cryptosporidiosis in the entire group determined by enzyme immunoassay, enzyme linked immunosorbent assay, immuno-chromatographic test and polymerase chain reaction was 34.17%, 27.84%, 6.33% and 27.84%, respectively. Sensitivity of animal samples with enzyme immunoassay, enzyme linked immunosorbent assay, and immuno-chromatographic test was 63.6%, 40.9% and 22.7%, resp., when questionable samples were considered positive, whereas specificity of enzyme immunoassay, enzyme linked immunosorbent assay and immuno-chromatographic test was 75.9%, 78.9% and 100%, respectively. Positive predictive values and negative predictive values were different for all the tests. These differences results are controversial and therefore reliability and reproducibility of immunoassays as the only diagnostic method is questionable. The use of various Cryptosporidium species in diagnosis based on immunological testing and different results obtained by individual tests indicate potential differences in Copro-antigens produced by individual Cryptosporidium species.(AU)

Survey of mycotoxins in Southern Brazilian wheat and evaluation of immunoassay methods

One hundred commercial wheat grain samples were collected during the 2015 sea-son across 78 municipalities in the states of Paraná (PR), Rio Grande do Sul (RS), and São Paulo (SP), Brazil. Separate subsamples were analyzed for the concentration of deoxynivalenol (DON), zearalenona (ZEA) and ochratoxin A (OTA) mycotoxins using two methods: UHPLC-MS/MS (reference method) and a commercial enzyme-linked immunosorbent assay (ELISA) (AgraQuant®). The OTA mycotoxin was not found in the samples by both methods. DON and ZEA were detected in 55 % and 39 % of the samples by the reference method, with overall mean levels of 795.2 g kg1 and 79.78 g kg1, respectively. There was a significant and positive correlation (Spearman rank) between DON and ZEA estimates by the reference method (r = 0.77, p 0.001). The DON levels estimated by the immunoassay agreed poorly with the reference, being largely overestimated. Based on a cut-off level of 1000 g kg1, the immunoassay correctly classified 57 samples as true negatives and 15 as true positives. Only 28 were classified as false positives. For ZEA, the levels estimated by the two methods were in better agreement than for DON. Using the cut-off level of 200 g kg1, 96 % of the samples were classified correctly as true positives and only one sample was classified as false positive. The levels for both mycotoxins were mostly acceptable for human consumption. Further studies should focus on multi-toxin methods compared with immunoassays to understand the reasons of overestimation and the role of immunoassays as a cost-effective solution for fast screening of mycotoxins in the food chain.

Can anti-bothropstoxin-I antibodies discriminate between Bothrops jararaca and Bothrops jararacussu venoms?

Background Snakes of the genus Bothrops, popularly known as pit vipers, are responsible for most cases of snakebite in Brazil. Within this genus, Bothrops jararacussu and B. jararaca deserve special attention due to the severity of their bites and for inhabiting densely populated areas. Regarding the treatment of snakebites by Bothrops jararacussu, questions have been raised about the effectiveness of the specific bothropic antivenom in neutralizing myotoxic effects; however, there are no accurate data for humans. Thus, the development of a differential diagnostic kit for this species would be of great interest because it provides, for healthcare professionals, a tool that would allow us to determine whether the accident was caused by B. jararacussu or other species of the genus. It would also make it possible to evaluate the specificity of the treatment and to provide data for epidemiological studies. Methods First, we produced a species-specific polyclonal antibody - a potential biomarker of Bothrops jararacussu venom - against bothropstoxin-I (BthTx-I), which is also found in smaller quantities in the venoms of B. jararaca from southern Brazil. Results Polyclonal antibodies against bothropstoxin-I could be separated into several species-specific immunoglobulins. Then, aiming to develop a system of safe and standardized immunoassay, we produced monoclonal antibodies. Seven hybridomas were obtained. Five of them were specific to the venom of B. jararacussu and two recognized the venom of B. jararaca from the southeastern population. The use of monoclonal antibodies also made it possible to differentiate B. jararacussu from B. jararaca venom obtained from the southern population. Analyzing the reactivity of monoclonal antibodies against other bothropic venoms, we found mAb Bt-3 to be more specific than others for B. jararacussu venom. Conclusions These results show the potential of BthTx-I for producing monoclonal antibodies that differentiate between B. jararacussu and other Bothrops species venoms.(AU)

Survey of mycotoxins in Southern Brazilian wheat and evaluation of immunoassay methods

One hundred commercial wheat grain samples were collected during the 2015 sea-son across 78 municipalities in the states of Paraná (PR), Rio Grande do Sul (RS), and São Paulo (SP), Brazil. Separate subsamples were analyzed for the concentration of deoxynivalenol (DON), zearalenona (ZEA) and ochratoxin A (OTA) mycotoxins using two methods: UHPLC-MS/MS (reference method) and a commercial enzyme-linked immunosorbent assay (ELISA) (AgraQuant®). The OTA mycotoxin was not found in the samples by both methods. DON and ZEA were detected in 55 % and 39 % of the samples by the reference method, with overall mean levels of 795.2 g kg1 and 79.78 g kg1, respectively. There was a significant and positive correlation (Spearman rank) between DON and ZEA estimates by the reference method (r = 0.77, p 0.001). The DON levels estimated by the immunoassay agreed poorly with the reference, being largely overestimated. Based on a cut-off level of 1000 g kg1, the immunoassay correctly classified 57 samples as true negatives and 15 as true positives. Only 28 were classified as false positives. For ZEA, the levels estimated by the two methods were in better agreement than for DON. Using the cut-off level of 200 g kg1, 96 % of the samples were classified correctly as true positives and only one sample was classified as false positive. The levels for both mycotoxins were mostly acceptable for human consumption. Further studies should focus on multi-toxin methods compared with immunoassays to understand the reasons of overestimation and the role of immunoassays as a cost-effective solution for fast screening of mycotoxins in the food chain.(AU)

Can anti-bothropstoxin-I antibodies discriminate between Bothrops jararaca and Bothrops jararacussu venoms?

Snakes of the genus Bothrops, popularly known as pit vipers, are responsible for most cases of snakebite in Brazil. Within this genus, Bothrops jararacussu and B. jararaca deserve special attention due to the severity of their bites and for inhabiting densely populated areas. Regarding the treatment of snakebites by Bothrops jararacussu, questions have been raised about the effectiveness of the specific bothropic antivenom in neutralizing myotoxic effects; however, there are no accurate data for humans. Thus, the development of a differential diagnostic kit for this species would be of great interest because it provides, for healthcare professionals, a tool that would allow us to determine whether the accident was caused by B. jararacussu or other species of the genus. It would also make it possible to evaluate the specificity of the treatment and to provide data for epidemiological studies. Methods First, we produced a species-specific polyclonal antibody - a potential biomarker of Bothrops jararacussu venom - against bothropstoxin-I (BthTx-I), which is also found in smaller quantities in the venoms of B. jararaca from southern Brazil. Results Polyclonal antibodies against bothropstoxin-I could be separated into several species-specific immunoglobulins. Then, aiming to develop a system of safe and standardized immunoassay, we produced monoclonal antibodies. Seven hybridomas were obtained. Five of them were specific to the venom of B. jararacussu and two recognized the venom of B. jararaca from the southeastern population. The use of monoclonal antibodies also made it possible to differentiate B. jararacussu from B. jararaca venom obtained from the southern population. Analyzing the reactivity of monoclonal antibodies against other bothropic venoms, we found mAb Bt-3 to be more specific than others for B. jararacussu venom. Conclusions These results show the potential of BthTx-I for producing monoclonal antibodies that differentiate between B. jararacussu and other Bothrops species venoms.(AU)

Development of an ELISA for detection of serum antibodies against bovine leukemia virus / Desenvolvimento de um ELISA para a detecção de anticorpos séricos contra o vírus da leucose bovina

The Enzootic bovine leucosis is a chronic disease that causes economic losses to cattle. The detection of infected animals is an important step to control the disease. Therefore, this study aimed to investigate the prevalence of the virus in the Alto Uruguai Catarinense (Brazil) region and in parallel develop an enzyme linked immunosorbent assay (ELISA) able to discriminate serologically positive and negative animals for bovine leukemia virus (BLV). For ELISA standardization, antigen and serum controls block titrations were performed. Then, 289 bovine serum samples collected from 42 dairy herds were analyzed. The results obtained were compared with Agar Gel Immunodiffusion (AGID). The comparative analysis concluded that the developed ELISA showed a satisfactory performance and was able to discriminate between infected and uninfected animals. Although the sensitivity (80.2%) and specificity (71.2%) of the test showed lower values than those found in previous studies using commercial kits or in-house assays, the ELISA developed can be associated with IDGA as a screening test, in order to reduce the number of false negatives. The prevalence of antibodies against BLV found was 31% for AGID and 45% for ELISA. From 42 properties, 47.6% had at least one positive animal in the ELISA. These results indicate that bovine leukosis is widespread in dairy herds of the region analyzed. Therefore, the test developed could be used as an initial screening tool, thus contributing to the serological monitoring of the herd.

A leucose enzoótica bovina é uma enfermidade crônica que acarreta prejuízos econômicos ao produtor. Detectar a presença de animais infectados é uma etapa importante no controle desta doença. Portanto, neste trabalho procurou-se investigar a prevalência do vírus na região do Alto Uruguai Catarinense e, paralelamente, desenvolver um ensaio imunoenzimático (ELISA) capaz de discriminar animais sorologicamente positivos e negativos para o vírus da leucose bovina (BLV). Para a padronização do ELISA foram realizadas titulações em bloco do antígeno e de amostras de soros controles. Em seguida, foram analisadas 289 amostras de soro bovino, colhidas em 42 rebanhos leiteiros. Os resultados obtidos foram comparados aos da imunodifusão em gel de ágar (IDGA). A análise dos critérios de comparação permitiu concluir que o ELISA desenvolvido apresentou um desempenho satisfatório e foi capaz de discriminar animais infectados e não infectados. Embora os valores de sensibilidade (80,2%) e especificidade (71,2%) do teste tenham sido menores aos relatados em estudos utilizando kits comerciais ou testes in house, o ELISA desenvolvido pode ser associado à IDGA como teste de triagem, visando diminuir o número de falso-negativos. A prevalência de anticorpos contra o BLV na população amostrada foi de 31% para a IDGA e 45% para o ELISA. Das propriedades, 20 (47,6%) possuíam ao menos um animal positivo no ELISA. Estes dados indicam que a leucose enzoótica bovina (LEB) está amplamente disseminada nos rebanhos leiteiros da região. Portanto, o teste desenvolvido pode servir como uma ferramenta de triagem inicial, contribuindo desta maneira para o monitoramento sorológico do rebanho.

Development of an ELISA for detection of serum antibodies against bovine leukemia virus / Desenvolvimento de um ELISA para a detecção de anticorpos séricos contra o vírus da leucose bovina

The Enzootic bovine leucosis is a chronic disease that causes economic losses to cattle. The detection of infected animals is an important step to control the disease. Therefore, this study aimed to investigate the prevalence of the virus in the Alto Uruguai Catarinense (Brazil) region and in parallel develop an enzyme linked immunosorbent assay (ELISA) able to discriminate serologically positive and negative animals for bovine leukemia virus (BLV). For ELISA standardization, antigen and serum controls block titrations were performed. Then, 289 bovine serum samples collected from 42 dairy herds were analyzed. The results obtained were compared with Agar Gel Immunodiffusion (AGID). The comparative analysis concluded that the developed ELISA showed a satisfactory performance and was able to discriminate between infected and uninfected animals. Although the sensitivity (80.2%) and specificity (71.2%) of the test showed lower values than those found in previous studies using commercial kits or in-house assays, the ELISA developed can be associated with IDGA as a screening test, in order to reduce the number of false negatives. The prevalence of antibodies against BLV found was 31% for AGID and 45% for ELISA. From 42 properties, 47.6% had at least one positive animal in the ELISA. These results indicate that bovine leukosis is widespread in dairy herds of the region analyzed. Therefore, the test developed could be used as an initial screening tool, thus contributing to the serological monitoring of the herd.(AU)

A leucose enzoótica bovina é uma enfermidade crônica que acarreta prejuízos econômicos ao produtor. Detectar a presença de animais infectados é uma etapa importante no controle desta doença. Portanto, neste trabalho procurou-se investigar a prevalência do vírus na região do Alto Uruguai Catarinense e, paralelamente, desenvolver um ensaio imunoenzimático (ELISA) capaz de discriminar animais sorologicamente positivos e negativos para o vírus da leucose bovina (BLV). Para a padronização do ELISA foram realizadas titulações em bloco do antígeno e de amostras de soros controles. Em seguida, foram analisadas 289 amostras de soro bovino, colhidas em 42 rebanhos leiteiros. Os resultados obtidos foram comparados aos da imunodifusão em gel de ágar (IDGA). A análise dos critérios de comparação permitiu concluir que o ELISA desenvolvido apresentou um desempenho satisfatório e foi capaz de discriminar animais infectados e não infectados. Embora os valores de sensibilidade (80,2%) e especificidade (71,2%) do teste tenham sido menores aos relatados em estudos utilizando kits comerciais ou testes in house, o ELISA desenvolvido pode ser associado à IDGA como teste de triagem, visando diminuir o número de falso-negativos. A prevalência de anticorpos contra o BLV na população amostrada foi de 31% para a IDGA e 45% para o ELISA. Das propriedades, 20 (47,6%) possuíam ao menos um animal positivo no ELISA. Estes dados indicam que a leucose enzoótica bovina (LEB) está amplamente disseminada nos rebanhos leiteiros da região. Portanto, o teste desenvolvido pode servir como uma ferramenta de triagem inicial, contribuindo desta maneira para o monitoramento sorológico do rebanho.(AU)

Standardization of dot-ELISA methodology for detection of IgG antibodies anti-Toxoplasma gondii in saliva / Padronização da metodologia dot-ELISA para detecção de anticorpos IgG anti-Toxoplasma gondii em saliva

The diagnosis of T. gondii infection is usually performed by serological tests, but the blood collection could be restricted in some groups as children. Antibodies are also found in other biological materials, such as saliva, whose sampling has been done by means of non-invasive procedure. Commercially available assays for performing antibody detection are standardized for analyzing serum samples. There are alternative techniques for detecting antibody in saliva, however they demand the use of equipment which is not easy to be used in field. Dot-ELISA is highly sensitivity and the results are of visual reading without any equipments, being available to be used in field studies, by means a rapid and efficient screening technique. Thus, a dot-ELISA with high sensitivity was standardized for detecting anti-T. gondii antibodies in saliva and serum by using samples from 20 adult volunteers. Sensitivity and specificity of the standardized dot-ELISA were similar in both saliva and serum samples, and precisely distinguishing the positive and negative samples, even in low antibody concentration-containing sample as saliva. Saliva showed to be as a potential biological material for detecting anti-T. gondii antibody in epidemiological studies on toxoplasmosis in children or other protected groups, where the blood collection is restricted.(AU)

O diagnóstico da infecção pelo T. gondii é usualmente feito pelas técnicas sorológicas, mas a amostra (soro ou plasma) pode ser restrita em determinados grupos protegidos, em que a coleta de sangue é considerada agressiva e invasiva. Os anticorpos são encontrados em outros materiais biológicos, de coleta não invasiva, como a saliva. Os métodos de detecção de anticorpos no mercado estão padronizados para utilizar amostras de soro, e há metodologias alternativas de maior sensibilidade utilizando-se saliva, mas estas requerem equipamentos de difícil uso no campo. Dot-ELISA tem alta sensibilidade e leitura visual sem equipamentos, que facilita a execução de ensaio em campo utilizando-se técnica de triagem rápida e eficiente. Neste contexto, foi padronizado o dot-ELISA de alta sensibilidade para detecção de anticorpos anti-T. gondii em saliva e soro, utilizando-se amostras de 20 voluntários adultos. A sensibilidade e a especificidade do dot-ELISA padronizado foram semelhantes em soro e saliva, com exata distinção de amostras positivas e negativas, mesmo na ocorrência de baixas concentrações de anticorpos como na saliva. A saliva mostra ser material biológico adequado para detecção de anticorpos anti-T. gondii em estudos epidemiológicos da toxoplasmose em crianças ou outros grupos protegidos, em que a coleta de sangue é restrita.(AU)

Importância e metodologias para mensuração dos esteroides sexuais em ruminantes / Importance and methodologies to measure sexual steroids progesterone and estradiol in ruminants

As dosagens dos esteroides sexuais, como progesterona e estradiol, pela sua importância, foram econtinuam sendo largamente utilizadas nas pesquisas em reprodução animal, nos estudos de fisiologia, patologiae biotecnologia reprodutiva. Vários métodos para quantificação desses esteroides foram desenvolvidos, porémcada metodologia analítica possui vantagens e limitações. O radioimunoensaio é o mais amplamente utilizado,pela sua eficiência e aplicabilidade em diversas espécies, mas tem como principal desvantagem o envolvimentode material radioativo. A preocupação nesse setor, aliada às exigências das legislações nacionais e internacionaisem questões ambientais, tem aumentado a necessidade do desenvolvimento de novas técnicas sem a utilização dematerial radioativo. Uma possibilidade que se apresenta é a tecnologia xMAP-Multiplex®. Esta tecnologiaapresenta vantagens sobre o radioimunoensaio, como ausência de radioatividade e detecção simultânea dehormônios, promovendo a diminuição do tempo gasto para dosagem e menor custo por amostra dosada. Porém,ainda sem estudos na endocrinologia reprodutiva de animais domésticos, torna-se necessária avaliá-la e validá-lapara cada espécie.(AU)

Quantifications of sexual steroids, such as progesterone and estradiol, because of their importance,have been widely used in many researches of animal´s reproduction to study reproductive physiology, pathologyand biotechnology. Several methods to quantify these steroids have been developed, but each analyticalmethodology has advantages and limitations. Radioimmunoassay is the most widely used method because of itsefficiency and applicability in several species, but it has as major disadvantage the use of radioactive material.These concerns added to the requirements of national and international´s legislations about environmentaltopics have increased the need to develop new techniques without the use of radioactive material. One newpossibility is the xMAP-Multiplex® technology. This technology offers advantages over radioimmunoassay, suchas absence of radioactivity and simultaneous detection of hormones, which decreases the time taken to quantifythem and lower price for each quantified sample. Although, there are no studies at reproductive endocrinologyat domestic animals and it is necessary to evaluate and validate it for each specie.(AU)

Importância e metodologias para mensuração dos esteroides sexuais em ruminantes / Importance and methodologies to measure sexual steroids progesterone and estradiol in ruminants

As dosagens dos esteroides sexuais, como progesterona e estradiol, pela sua importância, foram econtinuam sendo largamente utilizadas nas pesquisas em reprodução animal, nos estudos de fisiologia, patologiae biotecnologia reprodutiva. Vários métodos para quantificação desses esteroides foram desenvolvidos, porémcada metodologia analítica possui vantagens e limitações. O radioimunoensaio é o mais amplamente utilizado,pela sua eficiência e aplicabilidade em diversas espécies, mas tem como principal desvantagem o envolvimentode material radioativo. A preocupação nesse setor, aliada às exigências das legislações nacionais e internacionaisem questões ambientais, tem aumentado a necessidade do desenvolvimento de novas técnicas sem a utilização dematerial radioativo. Uma possibilidade que se apresenta é a tecnologia xMAP-Multiplex®. Esta tecnologiaapresenta vantagens sobre o radioimunoensaio, como ausência de radioatividade e detecção simultânea dehormônios, promovendo a diminuição do tempo gasto para dosagem e menor custo por amostra dosada. Porém,ainda sem estudos na endocrinologia reprodutiva de animais domésticos, torna-se necessária avaliá-la e validá-lapara cada espécie.

Quantifications of sexual steroids, such as progesterone and estradiol, because of their importance,have been widely used in many researches of animal´s reproduction to study reproductive physiology, pathologyand biotechnology. Several methods to quantify these steroids have been developed, but each analyticalmethodology has advantages and limitations. Radioimmunoassay is the most widely used method because of itsefficiency and applicability in several species, but it has as major disadvantage the use of radioactive material.These concerns added to the requirements of national and international´s legislations about environmentaltopics have increased the need to develop new techniques without the use of radioactive material. One newpossibility is the xMAP-Multiplex® technology. This technology offers advantages over radioimmunoassay, suchas absence of radioactivity and simultaneous detection of hormones, which decreases the time taken to quantifythem and lower price for each quantified sample. Although, there are no studies at reproductive endocrinologyat domestic animals and it is necessary to evaluate and validate it for each specie.

ELISA and modified toxin-binding inhibition test for quality control of the clostridial vaccine processes / ELISA direto e ToBI-test modificado para o controle de qualidade de processos de vacinas clostridiais

This study aimed to assess and standardize the ELISA and modified ToBI test in vitro methods in order to verify the potency of epsilon toxicoid in comparison with the in vivo TCP method. The following epsilon toxoids were used: NIBSC standard from batches 375/07, 532/08, 551/08, 373/07 and 378/07. These were evaluated using a TCP test, ELISA and ToBI tests. The results indicate that the correlation ratio between the dilutions of standard NIBSC toxicoid and absorbance values of 89.44% obtained with the ELISA method support the use of the curve to evaluate epsilon toxoids. However, it was observed that the absorbance values were similar for all toxoids, thus presenting no significant difference between higher and lower concentration toxoids. For the ToBI test, the correlation ratio of 96.76, obtained in the curve pattern, demonstrates the effectiveness of the curve to be used in the epsilon toxoid evaluation. The correlation ratio between the titration degrees of toxoids obtained through TCP and ToBI tests was higher than 90%. It is concluded that the type of ELISA test used does present discriminative power for toxoids with different concentrations, which does not support the use of this technique for such a purpose. The ToBI test can be used as a screening method for it is sensitive and effective to detect epsilon toxicoid produced by C. perfringens type D.(AU)

Teve-se por objetivo avaliar e padronizar as metodologias in vitro, ELISA e ToBI-test modificado, para a análise de toxoide épsilon, em comparação com a metodologia in vivo TCP. Foram utilizados os seguintes toxoides épsilon: padrão NIBSC e os lotes 375/07, 532/08, 551/08, 373/07 e 378/07, os quais foram avaliados por métodos in vivo, TCP, e in vitro, ELISA e ToBI-test. A análise do título de toxoide épsilon por meio dos métodos in vitro foi realizada a partir de uma curva-padrão, estabelecida previamente. Os principais resultados mostram que os valores de absorbância foram semelhantes para todos os toxoides, não apresentando diferença significativa entre os toxoides mais concentrados e menos concentrados. No ToBI-test, o coeficiente de correlação de 96,76%, obtido na curva-padrão, demonstra a eficiência da curva para avaliação do toxoide épsilon. O coeficiente de correlação entre os títulos de toxoide obtidos pelo TCP e ToBI-test foi superior a 90%. Conclui-se que o tipo de ELISA utilizado não apresenta poder discriminativo para toxoides com diferentes concentrações, inviabilizando a técnica para esse fim. O ToBI-test pode ser utilizado como um método de triagem sensível e eficaz para a detecção de toxoide épsilon de C. perfringens tipo D.(AU)

Dosagem de metabólitos de glucocorticoides e progesterona em fezes de papagaio-verdadeiro (Amazona aestiva) / Measurement of glucocorticoid and progesterone metabolites in feces of blue fronted parrot (Amazona aestiva)

The objectives of the present study were to evaluate fecal concentrations of metabolites of glucocorticoids, measured by enzyme immunoassay with a cortisol antibody and by radioimmunoassay with a corticosterone antibody, and progesterone by radioimmunoassay with a progesterone antibody in blue-fronted parrot (Amazona aestiva) after ACTH challenge. The adrenal stimulation with ACTH (25 UI/animal) resulted in an increase of fecal glucocorticoids metabolites concentration, but it did not affect the concentrations of fecal progesterone metabolites. Although there were no synchronized peaks of glucocorticoid metabolites excretion measured by enzyme immunoassay and radioimmunoassay, there were two peaks of excretion, one at 2-4 hours and other at 8-10 hours. Despite the occurrence of peaks, the analysis of fecal glucocorticoids metabolites and progesterone metabolites showed no effect of group (control and treatment), moment (hours of sampling) and sex.

Os objetivos do presente trabalho foram avaliar as concentrações fecais de metabólitos de glicocorticoides, mensurados por enzimaimunoensaio, empregando-se anticorpos contra cortisol, e por radioimunoensaio, empregando-se anticorpo contra corticosterona, e dos metabólitos da progesterona, mensurados por raioimunoensaio empregando-se anticorpo contra progesterona, em papagaios-verdadeiros (Amazona aestiva) após desafio com ACTH. A estimulação da adrenal com 25 UI/animal de ACTH resultou na elevação das concentrações de metabólitos de glicocorticoides, mas não modificou a dos metabólitos da progesterona fecal em papagaio-verdadeiro. Embora não tenha sido observada a sincronização dos picos de excreção fecal dos metabólitos de glicocorticoides mensurados por enzimaimunoensaio e radioimunoensaio, houve dois picos de excreção, um entre 2 e 4 horas e outro entre 8 e 10 horas. Apesar dos picos, não foram detectados efeitos de Grupos (Tratamento x Controle), momento (horas de coleta) ou sexo (macho x fêmea) nos resultados observados nas concentrações fecais de metabólitos de glicocorticoides e de progesterona, com os métodos empregados.

Immunological assays employed for the elucidation of an histoplasmosis outbreak in São Paulo, SP

Several reports showed outbreaks of histoplasmosis acquired while bat-inhabited caves were visited by tourists, miners or researchers. We evaluated the performance of double immunodifusion (DI) and immunoblotting (IB) assays, employed for the histoplasmosis outbreak elucidation occurred in Vale do Paraíba, São Paulo. The existence of epidemiologic link, four patients with clinical signs suggestive of histoplasmosis and mycological confirmation has made that all 35 individuals involved to the cave visit were subjected to serological evaluation. By DI, we observed reactivity against H. capsulatum antigen in a single serum examined nearly 20 days after exposure to fungal propagules. On the other hand, IB showed reactivity against H and M fractions in 50% of samples evaluated. The analysis of the second sample batch, collected two months after the exposure showed that 96.7% were reactive by DI with antibodies titers ranging from 1 to 16 and 100% of reactivity against H and M fractions, by IB, suggesting an acute infection. The analysis of the overall agreement between the methods showed to be reasonable (κ = 0.37). This study confirms the importance and efficacy of more sensitive methodologies, such as IB assay, to early elucidation of disease, especially in cases of patients without mycological information.

Dosagem de metabólitos de glucocorticoides e progesterona em fezes de papagaio-verdadeiro (Amazona aestiva) / Measurement of glucocorticoid and progesterone metabolites in feces of blue fronted parrot (Amazona aestiva)

The objectives of the present study were to evaluate fecal concentrations of metabolites of glucocorticoids, measured by enzyme immunoassay with a cortisol antibody and by radioimmunoassay with a corticosterone antibody, and progesterone by radioimmunoassay with a progesterone antibody in blue-fronted parrot (Amazona aestiva) after ACTH challenge. The adrenal stimulation with ACTH (25 UI/animal) resulted in an increase of fecal glucocorticoids metabolites concentration, but it did not affect the concentrations of fecal progesterone metabolites. Although there were no synchronized peaks of glucocorticoid metabolites excretion measured by enzyme immunoassay and radioimmunoassay, there were two peaks of excretion, one at 2-4 hours and other at 8-10 hours. Despite the occurrence of peaks, the analysis of fecal glucocorticoids metabolites and progesterone metabolites showed no effect of group (control and treatment), moment (hours of sampling) and sex. (AU)

Os objetivos do presente trabalho foram avaliar as concentrações fecais de metabólitos de glicocorticoides, mensurados por enzimaimunoensaio, empregando-se anticorpos contra cortisol, e por radioimunoensaio, empregando-se anticorpo contra corticosterona, e dos metabólitos da progesterona, mensurados por raioimunoensaio empregando-se anticorpo contra progesterona, em papagaios-verdadeiros (Amazona aestiva) após desafio com ACTH. A estimulação da adrenal com 25 UI/animal de ACTH resultou na elevação das concentrações de metabólitos de glicocorticoides, mas não modificou a dos metabólitos da progesterona fecal em papagaio-verdadeiro. Embora não tenha sido observada a sincronização dos picos de excreção fecal dos metabólitos de glicocorticoides mensurados por enzimaimunoensaio e radioimunoensaio, houve dois picos de excreção, um entre 2 e 4 horas e outro entre 8 e 10 horas. Apesar dos picos, não foram detectados efeitos de Grupos (Tratamento x Controle), momento (horas de coleta) ou sexo (macho x fêmea) nos resultados observados nas concentrações fecais de metabólitos de glicocorticoides e de progesterona, com os métodos empregados.(AU)

Validação laboratorial e fisiológica de conjunto comercial para a quantificação de corticóides fecais em chimpanzé (Pan troglodytes) e orangotango (Pongo pygmaeus), cativos e submetidos a enriquecimentos ambientais / Laboratorial and physiological validation of comercial kits for quantification of fecal corticoids of captive chimpanzees (Pan troglodytes) and orangutangs (Pongo pygmaeus) under environmental enriched conditions

Neste trabalho foi realizado estudo comparativo dos níveis decorticóides fecais (CF) de chimpanzé (Pan troglodytes) e orangotango(Pongo pygmaeus). Foram analisadas amostras coletadas em duas fasesdistintas, relacionadas com a introdução de técnicas de enriquecimentoambiental, a saber: Base (antes da introdução) e Habituação(imediatamente após). Realizamos as validações do conjunto comercialpara radioimunoensaio ImmunuChem™ Double AntibodyCorticosterone da MP Biomedicals, para mensuração de CF. A validaçãolaboratorial dos conjuntos diagnósticos para uso em extrato fecal deprimatas foi realizada pelo método de paralelismo, no qual, para cadaespécie, concentrações conhecidas de corticosterona foram adicionadasa um pool de extratos fecais, sendo estas amostras analisadas emseguida. As inclinações das curvas obtidas nestes ensaios e da curvapadrão do ensaio foram então comparadas. Os resultados obtidospara chimpanzé e orangotango, foram respectivamente, Y=17,23+1,31*X;R

A comparative study of fecal corticoids (FC) concentrations was carriedout with chimpanzees (Pan troglodytes) e orangutans (Pongo pygmaeus).Fecal samples were collected before (Basal) and just after (Habituation)enrichment introduction and analyzed. We performed biochemical andphysiological validations of the ImmunuChem™ Double AntibodyCorticosterone kit for radioimmunoassay from MP Biomedicals forquantifying FC concentrations. To establish the biochemical validity ofour assay we performed parallelism assays in which pooled fecal extractsfrom both species were spiked with known quantities of corticosteronestandard and the slopes of the curves obtained with these samples andthe standard curves of the kits were compared. The correlation coefficientswere R