Generation and In-planta expression of a recombinant single chain antibody with broad neutralization activity on Bothrops pauloensis snake venom.

The main systemic alterations present in bothropic envenomation are hemostasis disorders, for which the conventional treatment is based on animal-produced antiophidic sera. We have developed a neutralizing antibody against Bothrops pauloensis (B. pauloensis) venom, which is member of the genus most predominant in snakebite accidents in Brazil. Subsequently, we expressed this antibody in plants to evaluate its enzymatic and biological activities. The ability of single-chain variable fragment (scFv) molecules to inhibit fibrinogenolytic, azocaseinolytic, coagulant and hemorrhagic actions of snake venom metalloproteinases (SVMPs) contained in B. pauloensis venom was verified through proteolytic assays. The antibody neutralized the toxic effects of envenomation, particularly those related to systemic processes, by interacting with one of the predominant classes of metalloproteinases. This novel molecule is a potential tool with great antivenom potential and provides a biotechnological antidote to snake venom due to its broad neutralizing activity.

Causas de condenações post-mortem de perus abatidos em estabelecimento com Serviço de Inspeção Federal (SIF) no estado de Minas Gerais, Brasil

Este estudo objetivou identificar as principais causas de condenações de perus abatidos sob Serviço de Inspeção Federal, além de verificar as estações do ano em que as condenações são mais frequentes. Foram utilizados dados do Serviço de Inspeção Federal de um matadouro-frigorífico exportador de aves localizado na mesorregião do Triângulo Mineiro e Alto Paranaíba, Minas Gerais, no período compreendido entre agosto de 2008 a julho de 2009. Foram inspecionadas 8.153.866 aves, procedentes de 19 municípios localizados nesta mesorregião. Destas, 1.767.503 (21,68%), tiveram algum tipo de condenação post-mortem. As principais causas de condenação (parcial e total) foram agrupadas segundo o mês e estação do ano, a fim de se verificar quais foram as condenações de maior e menor frequência. Foram condenados parcialmente 1.750.863 perus (21,47%) e 16.640 (0,21%) foram condenados totalmente. Dentre o número total de condenações post-mortem, 99,06% foi devido a condenações parciais e 0,94% devido a condenações totais. As causas de condenação parcial mais frequentes foram devido à aerossaculite (41,24%), seguida de contaminação biliar (23,73%). As causas de condenação total mais frequentes foram em função de aspecto repugnante (51,62%) e dermatose (22,71%). A estação do ano que apresentou maior frequência de condenações tanto totais quanto parciais foi a primavera. Verificou-se que na mesor

Cutaneous hypersensitivity test to evaluate phage display anti-tick borne vaccine antigen candidates.

Early experiments performed by our group with the phage display technique revealed the potential for using epitope-displaying phages (mimotopes) as a tool for tick antigen discovery. Thus, as a preliminary study, inflammatory reactions induced by phage display tick-borne candidates were investigated by using the cutaneous hypersensitivity test. The profile of selected Rhipicephalus microplus mimotopes was assessed on tick field-exposed cattle and our data indicated a pattern similar to immediate hypersensitivity reaction and not a delayed immune response as expected. However, the wild-type phage inoculation surprisingly induced a strong immediate response on its own. Such reactions indicate that the wild-type phage may have hidden many of the potential reactions raised by the mimotopes. The study of the inflammatory reactions to these phage mimotopes in tick-infested hosts may provide basic information about the immune reaction. Finally, this work is of relevance for when considering research alternatives for finding and characterization of antigens by the phage display technique.

Antigen fingerprinting of polyclonal antibodies raised in immunized chickens with tick total proteins: a reservoir for the discovery of novel antigens.

Identification of tick-protective antigens remains the limiting step in vaccine development. The authors have generated several B cell epitope candidates by fingerprinting Rhipicephalus (Boophilus) microplus proteins that were characterized through bioselection of random peptide phage display libraries against polyclonal antibodies antitick proteins. From 280 clones selected and sequenced, 107 distinct reactive clones were validated by dot-blot assays. Eight consensus motifs were generated, and the most frequent ones were PXXKXH, NXXKXXL, and HTS (68.2%, 65%, and 42%, respectively). The consensus sequences identified potential vaccine targets by alignment with the protein database of R. microplus, which may have putative roles in the host response. Sequences that did not align with known proteins but shared extensive homology among each other were classified as conformational epitopes. Sequence alignments also recognized multiple targets, and the most predominant proteins were identified. Finally, immunized mice sera recognized tick proteins, demonstrating that functional epitope profiles can be identified through selection of phage-displayed peptide libraries with hyperimmune sera and revealing that the epitope-displaying phages can be used as potential vaccine immunogens.

Toxinas e perfil protéico de amostras de Actinobacillus suis provenientes de plantéis suínos norte-americanos / Toxins and proteic profile of Actinobacillus suis samples from North American hog herds

Actinobacillus suis (A.suis) surgiu como uma grande ameaça aos plantéis suínos norte-americanos. Os sinais clínicos e as lesões são particularmente variáveis e podem lembrar aquelas causadas por outros organismos, como o Actinobacillus pleuropneumoniae (App), podendo ter como causa a similaridade na produção das toxinas ApxI e ApxII. Os objetivos do estudo foram confirmar a produção das toxinas ApxI e ApxII, investigar a produção de toxina geneticamente semelhante à Apx III e analisar as proteínas totais, verificando se existe similaridade entre os isolados provenientes de diferentes plantéis de suínos norte-americanos. Neste estudo, todas as cepas de A. suis foram positivas para os genes codificadores das toxinas ApxI e ApxII, usando o método de reação em cadeia de polimerase - multiplex (PCR-multiplex); e as proteínas totais de 70 amostras de A. suis, oriundos de diferentes plantéis suínos norte-americanos, foram analisadas por meio de eletroforese em gel de poliacrilaminda desnaturante (SDS-PAGE) e foram idênticas. A similaridade eletroforética observada entre as proteínas totais das bactérias analisadas indica a possibilidade de haver uma proteção cruzada a partir de uma provável vacina universal desenvolvida com esses antígenos para A. suis.

Actinobacillus suis (A. suis) has arisen as a great threat to the North American hog herds. The clinical symptoms and lesions are particularly variable and may resemble the same caused by other pathogenic organisms, such as Actinobacillus pleuropneumoniae (App), which can similarly lead to the production of the toxins ApxI and ApxII. This study aimed to confirm the production of the toxins ApxI and ApxII, as well as, to investigate the production of toxins that are genetically similar to ApxIII, and analyze total protein to verify whether there is any similarity among the isolated samples obtained from different North American hog herds. In this study, all the strains of A. suis were positive for the genes that codify the toxins ApxI and ApxII using the multiplex polymerase chain reaction (PCR-multiplex) method; and total protein from 70 samples of A. suis, obtained from different North American hog herds, were analyzed through denaturing polyacrylamide gel electrophoresis (SDS-PAGE), and were identical. The electrophoretic similarity observed among total protein of the analyzed bacteria indicates that there is the possibility of existing a cross protection in case of developing a probable universal vaccine with the antigens of A. suis.

Recombinant peptides as new immunogens for the control of the bovine tick, Rhipicephalus (Boophilus) microplus.

A candidate vaccine that effectively controls Rhipicephalus microplus ticks in livestock is an attractive strategy in bovine management because of the expensive and labor-intensive treatment of animals with acaricides. However, the complex nature of ectoparasites has imposed restrictions on the development of such a vaccine, and its future efficacy is still under debate. Using Phage Display technology, we developed specific immunogens that mimic R. microplus tick antigens and were successfully validated by in vitro and in vivo assays. Nine peptides were used in the experimental vaccination of mice and cattle with different formulations. The peptides generated specific antibody response against R. microplus larval proteins in mouse immunization. A vaccine mixture of all nine selected phage clones was employed in cattle vaccination and the immune response induced specific changes in teleogine physiology, manifested as a hemorrhagic event in the gastrointestinal and reproductive tracts. A new means for vaccine development and the discovery of immunogens have been demonstrated, and our data may provide evidence that a putative vaccine for ectoparasite control is possible.

Selection of high affinity peptide ligands for detection of circulating antibodies in neurocysticercosis.

Neurocysticercosis (NC), caused by Taenia solium, is the most common infection caused by helminthes of the human central nervous system. In this study, a random peptide phage display library was used to isolate peptide ligands as potential markers for neurocysticercosis diagnosis, because occurrence of cross-reactions with other helminthes species in the current used markers. We selected different peptides using IgG purified from pooled sera of neurocysticercosis patients. To investigate the diagnostic potential of recombinant peptides, we have tested different panels of serum samples by Phage-ELISA, and 10 phage clones strongly bound to the anti-T. solium IgGs in NC sera, with an accuracy range from 84.2% to 95%. The phage clones, NC(4)1 and NC(2)8, presented the highest sensitivity and specificity (100%), respectively, and most important, some phage clones did not react with patients' sera from Echinococcus granulosus infected patients. The validation with a competitive ELISA assay demonstrated that the selected phages could mimic T. solium epitopes and bind specifically to the pool of NC sera. Finally, the two recombinant antigens may become potential biomarkers for serodiagnosis of NC, and the Phage-ELISA demonstrated to be a very good assay, being reproducible, simple, fast, and low-cost due to its production through Escherichia coli culture, allowing a high throughput screening of NC.

In silico analysis for identification of tick phagotopes selected by phage-displayed libraries.

Phage display techniques have been widely employed to map epitope structures which have served as the basis for developing molecular vaccines. We have applied this technique to map specific epitopes of Rhipicephalus (Boophilus) microplus. In the present study, we have identified the potential immunogens using a process in which the selected phage clones were analyzed through bioinformatics, prior to final field tests. The present study demonstrates the feasibility of identifying important R. (B.) microplus phagotopes for vaccine development through screening of phage-displayed random peptide libraries and bioinformatics tools.

In silico analysis for identification of tick phagotopes selected by phage-displayed libraries / Análises in sílico na identificação de fagotopos de carrapatos selecionados por bibliotecas de phage display

Phage display techniques have been widely employed to map epitope structures which have served as the basis for developing molecular vaccines. We have applied this technique to map specific epitopes of Rhipicephalus (Boophilus) microplus. In the present study, we have identified the potential immunogens using a process in which the selected phage clones were analyzed through bioinformatics, prior to final field tests. The present study demonstrates the feasibility of identifying important R. (B.) microplus phagotopes for vaccine development through screening of phage-displayed random peptide libraries and bioinformatics tools.

Técnicas de phage display têm sido amplamente empregadas para o mapeamento de epítopos os quais tem servido como base para o desenvolvimento de vacinas moleculares. Esta técnica foi aplicada no mapeamento de epítopos do Rhipicephalus (Boophilus) microplus. Neste estudo, potenciais imunógenos foram identificados pela adoção de um processo em que os clones de fagos foram analisados por bioinformática, previamente à realização dos testes. Os resultados demonstraram a possibilidade da identificação de importantes mimetopos do R. (B.) microplus para o desenvolvimento de vacinas através da seleção de bibliotecas de phage display associada à análise de bioinformática.

In silico analysis for identification of tick phagotopes selected by phage-displayed libraries

Phage display techniques have been widely employed to map epitope structures which have served as the basis for developing molecular vaccines. We have applied this technique to map specific epitopes of Rhipicephalus (Boophilus) microplus. In the present study, we have identified the potential immunogens using a process in which the selected phage clones were analyzed through bioinformatics, prior to final field tests. The present study demonstrates the feasibility of identifying important R. (B.) microplus phagotopes for vaccine development through screening of phage-displayed random peptide libraries and bioinformatics tools.

Técnicas de phage display têm sido amplamente empregadas para o mapeamento de epítopos os quais tem servido como base para o desenvolvimento de vacinas moleculares. Esta técnica foi aplicada no mapeamento de epítopos do Rhipicephalus (Boophilus) microplus. Neste estudo, potenciais imunógenos foram identificados pela adoção de um processo em que os clones de fagos foram analisados por bioinformática, previamente à realização dos testes. Os resultados demonstraram a possibilidade da identificação de importantes mimetopos do R. (B.) microplus para o desenvolvimento de vacinas através da seleção de bibliotecas de phage display associada à análise de bioinformática.