Diagnóstico e fatores de risco do complexo teníase-cisticercose bovina no município de Salinas, Minas Gerais / Diagnosis and risk factors of bovine taeniasis-cysticercosis complex in Salinas, Minas Gerais, Brazil

Com o objetivo de diagnosticar a situação do complexo teníase-cisticercose bovina no município de Salinas, Minas Gerais, foram coletadas amostras de sangue de 355 bovinos distribuídos em 18 propriedades rurais, sorteadas aleatoriamente. Em cada propriedade, foi aplicado um questionário socioeconômico para a análise de fatores que favorecem a manutenção do complexo teníase-cisticercose bovina. Foi realizado também um levantamento epidemiológico dos casos de teníase diagnosticados nos laboratórios credenciados pela Secretaria Municipal de Saúde de Salinas, no período de 2007 a 2010. A prevalência de cisticercose bovina foi de 4,70% enquanto as prevalências de teníase, encontradas durante os quatro períodos avaliados, foram de 0,29%, 0,36%, 0,24% e 0,24%. Entre os fatores de risco para a manutenção do complexo teníase-cisticercose analisados, foi observada uma relação estatisticamente significativa entre a ocorrência de cisticercose bovina e a ingestão de carne malpassada pelos entrevistados. Foi concluído que a cisticercose bovina está presente no município de Salinas, Minas Gerais, sendo o tratamento térmico ineficiente da carne bovina o principal fator de risco para a manutenção do complexo teníase-cisticercose, o que reforça a necessidade da adoção de medidas de controle com contínua vigilância epidemiológica e sanitária.(AU)

In order to diagnose the situation of bovine taeniasis-cysticercosis complex in the municipality of Salinas, Minas Gerais, Brazil, blood samples were collected from 355 cattle in 18 randomly selected farms. A socioeconomic questionnaire was filled in each farm for the analysis of factors which favor the maintenance of the taeniasis-cysticercosis complex. An epidemiological survey of human taeniasis was performed through analyses of the Municipal Health Department in the 2007-2010 period. A prevalence of 4.7% for bovine cysticercosis and the frequency of 0.29, 0.36, 0.24 and 0.24% for human taeniasis, during the evaluated period, was found. Among the risk factors, a statistically significant correlation was found between the occurrence of bovine cysticercosis and the ingestion of undercooked meat. It was concluded that bovine cysticercosis is present in the municipality of Salinas, due to inefficient heat treatment of the meat as the main risk factor for maintenance of the taeniasis-cysticercosis complex, reinforcing the need to adopt control measures with continuous epidemiological and health surveillance.(AU)

Intra-uterine exposure of horses to Sarcocystis spp. antigens / Exposição intrauterina ao Sarcocystis spp. em equinos

The aim of this study was to examine the intra-uterine exposure to Sarcocystis spp. antigens, determining the number of foals with detectable concentrations of antibodies against these agents in the serum, before colostrum ingestion and collect data about exposure of horses to the parasite. Serum samples were collected from 195 thoroughbred mares and their newborns in two farms from southern Brazil. Parasite specific antibody responses to Sarcocystis antigens were detected using the indirect immunofluorescent antibody test (IFAT) and immunoblot analysis. In 84.1% (159/189) of the pregnant mares and in 7.4% (14/189) of foals we detected antibodies anti-Sarcocystis spp. by IFAT. All samples seropositive from foals were also positive in their respective mares. Serum samples of seropositive foals by IFAT, showed no reactivity on the immunoblot, having as antigens S. neurona merozoites. In conclusion, the intra-uterine exposure to Sarcocystis spp. antigens in horses was demonstrated, with occurrence not only in mares, but also in their foals, before colostrum ingestion these occurrences were reduced.

O objetivo deste estudo foi avaliar a exposição intrauterina ao Sarcocystis spp., para determinar o número de potros que possuem concentrações detectáveis de anticorpos contra esses agentes no soro, antes da ingestão do colostro, por meio da coleta de dados sobre a exposição a esses protozoários nos equinos. Amostras de soro foram coletadas de 195 éguas puro-sangue e seus respectivos potros recém-nascidos, em duas fazendas localizadas na região Sul do Brasil. Os testes utilizados na detecção de anticorpos específicos para o Sarcocystis foram a reação de imunofluorescência indireta (RIFI) e análise por meio de immunoblot. Pela RIFI, em 84,1% (159/189) das éguas e em 7,4% (14/189) dos potros foram detectados anticorpos anti-Sarcocystis spp. Todas as amostras soropositivas dos potros também foram positivas para suas respectivas mães. As amostras de soro dos potros soropositivos na RIFI, não apresentaram reatividade no immunoblot, tendo como antígenos merozoítos de S. neurona. Em conclusão, foi demonstrada a exposição intrauterina de Sarcocystis spp. em equinos, com ocorrência em éguas, porém, em seus respectivos potros, antes da ingestão de colostro a ocorrência foi reduzida.

Relevant peptides of Taenia crassiceps for the diagnosis of bovine cysticercosis by immunoblot / Peptídeos relevantes de Taenia crassiceps para o diagnóstico da cisticercose bovina pelo immunoblot

Given the limited knowledge about the diagnosis of bovine cysticercosis by immunoblot, the aim of this study was to assess the applicability of this test, identifying key peptides with diagnostic value. Immunoblot assays were performed using total larval antigen of Taenia crassiceps and 60 sera of positive bovines for cysticercosis (30 naturally and 30 experimentally infected with T. saginata eggs), 30 sera of negative bovines for cysticercosis and 30 sera of bovines with other diseases (fascioliasis, hydatidosis and tuberculosis). The peptides of greater diagnostic importance, in descending order of accuracy (%), were as follows: 6-8kDa (90.8%), 129-143kDa (74.2%), 99-105kDa (71.7%) and 14-19kDa (71.1%). Cross-reactions, due to fascioliasis and hydatidosis, were observed in the four intervals of peptides highlighted. The results demonstrate that the total antigen of T. crassiceps has peptides with a high diagnostic potential; therefore, the immunoblot is useful in the diagnosis of bovine cysticercosis.

Tendo em vista o conhecimento limitado sobre o diagnóstico da cisticercose bovina pelo immunoblot, o objetivo deste trabalho foi avaliar a aplicabilidade desse teste, identificando os principais peptídeos com valor diagnóstico. Foram realizados ensaios de immunoblot, utilizando antígeno total de larva de Taenia crassiceps e 60 soros de bovinos positivos para a cisticercose (30 naturalmente e 30 experimentalmente infectados com ovos de T. saginata), 30 soros de bovinos negativos para a cisticercose e 30 soros de bovinos com outras patologias (fasciolose, hidatidose e tuberculose). Os peptídeos de maior importância diagnóstica, em ordem decrescente de acurácia (%), foram os seguintes: 6-8 kDa (90,8%), 129-143 kDa (74,2%), 99-105 kDa (71,7%) e 14-19 kDa (71,1%). Reações cruzadas, em decorrência da fasciolose e hidatidose, foram observadas nos quatro intervalos de peptídeos ressaltados. Os resultados demonstram que o antígeno total de T. crassiceps possui peptídeos com alto potencial diagnóstico, sendo, portanto, o immunoblot útil no diagnóstico da cisticercose bovina.

Proteínas imunorreativas de Conidiobolus lamprauges isoladas de ovinos infectados naturalmente / Immunoreactive proteins of Conidiobolus lamprauges isolated from naturally infected sheep

O estudo de conidiobolomicose ovina tem sido realizado nos seus aspectos clínicos, epidemiológicos, patológicos e moleculares. Informações, entretanto, sobre a resposta imune do hospedeiro na infecção por Conidiobolus lamprauges são inexistentes. Este estudo teve por objetivo a identificação de proteínas imunorreativas que possam desempenhar papel importante na resposta imune de ovinos naturalmente infectados por C. lamprauges. Para a caracterização protéica e imunológica foi utilizada a cepa de C. lamprauges (FIOCRUZ-INCQS 40316) isolada de ovino com sinais clínicos de conidiobolomicose no Estado do MT e cinco amostras de soro de ovinos infectados naturalmente pelo fungo. A presença de anticorpos IgG foi observada em todos os animais doentes com títulos reagentes em diluições de até 1:1.600. Na técnica do immunoblot, o perfil antigênico frente aos soros ovinos com a doença apresentou doze bandas reativas, com massas moleculares variando de 35 a 198 kDa. Dentre estas, a proteína de 198 kDa foi reativa em 3 soros de ovinos e a de 53 kDa apresentou a maior intensidade comparativamente com outras bandas, sendo provavelmente imunodominante. Amostras de soro de animais sadios não apresentaram reatividade demostrando a especificidade da técnica. A presença de proteínas antigênicas de C. lamprauges e IgG específicos em soros de ovinos observados no presente trabalho poderá auxiliar no desenvolvimento de métodos de diagnóstico precoces e na utilização de proteínas candidatas a vacinas para o controle e prevenção da infecção em animais e humanos.(AU)

The study of sheep conidiobolomycosis has been carried out in its clinical, epidemiological, pathological and molecular aspects. Information, however, about the host immune response in infection Conidiobolus lamprauges is absent. This study aimed to identify immunoreactive proteins that may play an important role in the immune response of sheep naturally infected by C. lamprauges. For protein and immunological characterization, C. lamprauges (strain FIOCRUZ-INCQS 40316) isolated from a sheep with clinical signs of conidiobolomycosis in the MT state and five sera samples of naturally infected sheep were used. The presence of IgG antibody was observed in all patients with reagent titers in dilutions up to 1:1600. In immunoblot technique, the antigenic profile against infected sheep sera showed twelve reactive bands with molecular weights ranging from 35 to 198 kDa. Among them, the 198 kDa protein was reactive against sera from three sheep and the 53 kDa showed increased intensity compared to other bands probably being immunodominant. Healthy animal serum samples showed no reactivity demonstrating the specificity of the technique. The presence of antigenic proteins of C. lamprauges and specific IgG in sheep sera observed in this study may assist in the development of early diagnostic methods and the use of protein as candidate vaccines for the control and prevention of infection in animals and human.(AU)

Evaluation of glycoproteins purified from adult and larval camel ticks (Hyalomma dromedarii) as a candidate vaccine

In order to identify antigens that can help prevent camel tick infestations, three major glycoproteins (GLPs) about 97, 66 and 40 kDa in size were purified from adult and larval Egyptian ticks, Hyalomma (H.) dromedarii, using a single-step purification method with Con-A sepharose. The purified GLPs were evaluated as vaccines against camel tick infestation in rabbits. The rabbits received three intramuscular inoculations of GLPs (20 microg/animal) on days 0, 14, and 28. In the immunoblot analysis, Sera from the immunized rabbits recognized the native GLPs and other proteins from larval and adult H. dromedarii ticks along with those from other tick species such as Rhipicephalus sanguineus but not Ornithodoros moubata. The effects of immunity induced by these GLPs were determined by exposing rabbits to adult H. dromedarii ticks. These results demonstrated that GLP immunization led to a slightly decreased reproductive index and significantly reduced rates of egg hatchability. These results demonstrated that immunization with the purified GLPs can provide protection against infestation by H. dromedarii and some other tick species. Further studies are needed to confirm the effectiveness of immunization with GLPs against other tick species.

Detecção de peptídeos importantes para o diagnóstico da cisticercose bovina no immunoblot / Detection of important peptides for bovine cysticercosis diagnosis in immunoblot

A dificuldade de se localizar cisticercos em bovinos com infecção discreta torna o diagnóstico tradicional da cisticercose bovina em matadouros um recurso de baixa sensibilidade, o que gera uma demanda de diagnóstico por métodos alternativos como o immunoblot. Para tanto, neste trabalho, foram realizados ensaios de immunoblot a fim de analisar os peptídeos responsáveis pela reação do antígeno vesicular de Taenia crassiceps. Foram utilizadas 28 amostras de soro de bovinos comprovadamente negativos e 28 amostras coletadas de bovinos infectados experimentalmente com ovos de Taenia saginata. Os resultados dos ensaios de immunoblot com as amostras de soros-controle mencionadas indicaram que os peptídeos de 4-6, 14 e 18kDa destacaram-se entre os demais, mostrando altas taxas de desempenho no diagnóstico da cisticercose bovina e uma aparência diferenciada, com área e largura maiores, ao contrário dos peptídeos de média e alta massa molecular, que se apresentaram sob a forma de linha e com reações inespecíficas. O immunoblot mostrou ter um potencial como uma alternativa de diagnóstico da cisticercose bovina, incluindo sua aplicação em animais vivos, como método auxiliar de diagnóstico em estudos epidemiológicos da doença.

The diagnosis of cysticercosis in bovines that presents discreet infection is committed by the conventional methodology of detection employed in slaughterhouses. This deficiency leads to the development of alternative methods, such as immunoblot, in order to improve the detection of this disease. An immunoblot assay was developed to detect peptides from Taenia crassiceps antigenand it was used to test 56 samples of bovine serum (28 confirmed as negative and 28 from experimentally infected animals with Taenia saginata eggs). The peptides 4-6, 14 and 18 kDa showed better performance in bovine cysticercosis diagnosis due to an enhanced appearance, once they showed higher area and wide, contrasting with medium and high molecular weight peptides, that showed unspecific reactions and discreet appearance. The obtained results indicated that immunoblot can represent a potential alternative in bovine cysticercosis diagnosis, including its use in live animals, improving the detection of this disease even in epidemiological studies.

Caracterização de anticorpos monoclonais contra rotavírus bovino e suas aplicações como ferramenta de diagnóstico / Characterization of monoclonal antibodies against bovine rotavirus and their diagnostic applications

Anticorpos monoclonais (AcM) para rotavírus bovino foram caracterizados para sua aplicação como ferramenta de diagnóstico, utilizando-se as técnicas de isotipificação, dot-blot, western-blot, imunofluorescência indireta (IFI) e ELISA de captura. A caracterização imunoquímica demonstrou que os cinco AcM 1G5, 4F7, 1E12, 4F3 e 3C12 foram do isótipo IgG2a. Pela técnica de dot-blot, os AcM 1G5, 4F7, 1E12, 4F3 detectaram antígenos do rotavírus, em diferentes concentrações, e dois AcM (1E12 e 4F3) reconheceram proteínas virais pela técnica de western-blot. Todos os AcM reagiram positivamente na técnica de IFI em cultivo celular e foram capazes de detectar antígeno viral em amostras fecais bovinas e humanas, pela técnica de ELISA de captura. Identificaram-se dois grupos de AcM, um deles formado pelos AcM 4F7, 1E12 e 1G5, para seu possível uso na detecção de antígeno viral em fezes por meio do ELISA de captura ou dot-blot e outro pelos 4F3 e 3C12, que podem ser usados para detectar antígeno viral em culturas de células por meio de IFI.

This work was carried out to characterize and evaluate five bovine rotavirus, monoclonal antibodies (MAbs), as a diagnosis tool, by isotyping, dot-blot, western-blot, indirect immunofluorescence (IFI) and ELISA techniques. The immunochemistry characterization showed that all five MAbs (4F7, 4F3, 1G5, 1E12 and 3C12) were IgG2a isotype. The dot-blot immunoassay showed that 1G5, 4F7, 1E12 and 4F3 detected viral antigen in different concentrations and two MAbs (1E12 and 4F3) recognized viral proteins by western-blot. All MAbs detected viral antigen in bovine and human fecal samples by capture ELISA technique and viral antigen in infected MA-104 cell culture by IFI. In conclusion, two groups of Mabs were indetified: one with Mabs 4F7, 1E12 and 1G5 showed the best results to detect rotavirus antigen in fecal samples by capture ELISA or dot-blot techniques assay and other with 4F3 and 3C12 which may be used to detect rotavirus antigens in cell culture by IFI. The results showed the potential use of these MAbs as diagnosis tools in diarrheas by rotavirus in bovines.

Development of a monoclonal antibody-based co-agglutination test to detect enterotoxigenic Escherichia coli isolated from diarrheic neonatal calves

Escherichia coli (E. coli) strains were collected from young diarrheic calves in farms and field. Strains that expressed the K99 (F5) antigen were identified by agglutination tests using reference antibodies to K99 antigen and electron microscopy. The K99 antigen from a selected field strain (SAR-14) was heat-extracted and fractionated on a Sepharose CL-4B column. Further purification was carried out by sodium deoxycholate treatment and/or ion-exchange chromatography. Monoclonal antibodies to purified K99 antigen were produced by the hybridoma technique, and a specific clone, NEK99-5.6.12, was selected for propagation in tissue culture. The antibodies, thus obtained, were affinity-purified, characterized and coated onto Giemsastained Cowan-I strain of Staphylococcus aureus (S. aureus). The antibody-coated S. aureus were used in a coagglutination test to detect K99+ E. coli isolated from feces of diarrheic calves. The specificity of the test was validated against reference monoclonal antibodies used in co-agglutination tests, as well as in ELISA. Specificity of the monoclonal antibodies was also tested against various Gram negative bacteria. The developed antibodies specifically detected purified K99 antigen in immunoblots, as well as K99+ E. coli in ELISA and co-agglutination tests. The co-agglutination test was specific and convenient for large-scale screening of K99+ E. coli isolates.

Production of monoclonal antibodies against serum immunoglobulins of black rockfish (Sebastes schlegeli Higendorf)

The present study was undertaken to produce monoclonal antibodies (MAbs) against immunoglobulin (Ig) purified from black rockfish (Sebastes schlegeli Higendorf) serum using protein A, mannan binding protein, and goat IgG affinity columns. These three different ligands were found to possess high affinity for black rockfish serum Ig. All of the Igs purified eluted at only 0.46 M NaCl concentration in anion exchange column chromatography and consisted of two bands at 70 kDa and 25 kDa in SDS-PAGE; they also had similar antigenicity for MAbs to Ig heavy chain in immunoblot assays. Therefore, black rockfish Ig is believed to exist as a single isotype within serum. The MAbs produced against Ig heavy chain reacted specifically with spots distributed over the pI range from 4.8 to 5.6 with a molecular weight of 70 kDa on two dimensional gel electrophoresis immunoblot profiles.

Characterization and localization of the unique Marek's disease virus type 2 ORF873 gene product

Studies on Marek's disease virus (MDV)-unique genes are important for understanding the biological nature of the virus. Based on complete DNA sequence analyses of the MDV genomes, the MDV genomes contain presumably at least five MDV-unique genes, which are commonly conserved among the three MDV serotypes. A recombinant baculovirus that contains the MDV serotype 2 (MDV2)-unique gene, ORF873, under the polyhedrin promoter was constructed and designated rAcORF873. Polyclonal and monoclonal antibodies, which recognize the recombinant MDV2 ORF873 protein in Spodoptera frugiperda clone 9 (Sf9) cells infected with rAcORF873, were prepared by immunizing mice with a recombinant fusion protein expressed in Escherichia coli. Immunoblot analyses with the antibodies revealed a major protein band with a molecular mass of 108-kDa in both MDV2-infected chick embryo fibroblasts (CEF) and rAcORF873-infected Sf9 cells. By indirect immunofluorescence analyses using monoclonal antibody, the authentic ORF873 protein was localized in the cytoplasm of MDV2-infected CEF cells. The monoclonal and polyclonal sera, which were generated in the present study and reacted effectively to MDV2 ORF873 protein, are considered to be useful reagents for further studying the role(s) of the ORF873 protein in MDV2 infection.

Specific and common antigens of Clonorchis sinensis and Opisthorchis viverrini (Opisthorchidae, Trematoda)

The antigenic characterizations and serological reactions of human liver flukes, Clonorchis sinensis and Opisthorchis viverrini, were analyzed by immunoblot. The antigenic profiles of the crude extract of Clonorchis contained major proteins of 8, 26-28, 34-37, 43, and 70 kDa, and those of Opisthorchis 34-37, 43, 70, and 100 kDa. Of these, the 8, 26-28 and 34-37 kDa bands of Clonorchis and the 100 kDa of Opisthorchis were major components of each excretory-secretory antigen. The 8 and 26-28 kDa bands were specific to Clonorchis but the 100 kDa of Opisthorchis cross-reacted with the sera of clonorchiasis, and the 34-37, 70 and 100 kDa bands cross-reacted with sera of other helminthiases. The frequency and intensity of the immunoblot reactions were positively correlated with the intensity of the liver fluke infection.