Structure analysis of capsid protein of Porcine circovirus type 2 from pigs with systemic disease

Abstract Economic losses with high mortality rate associated with Porcine circovirus type 2 (PCV2) is reported worldwide. PCV2 commercial vaccine was introduced in 2006 in U.S. and in 2008 in Brazil. Although PCV2 vaccines have been widely used, cases of PCV2 systemic disease have been reported in the last years. Eleven nursery or fattening pigs suffering from PCV2 systemic disease were selected from eight PCV2-vaccinated farms with historical records of PCV2 systemic disease in Southern Brazil. PCV2 genomes were amplified and sequenced from lymph node samples of selected pigs. The comparison among the ORF2 amino acid sequences of PCV2 isolates revealed three amino acid substitutions in the positions F57I, N178S and A190T, respectively. Using molecular modeling, a structural model for the capsid protein of PCV2 was built. Afterwards, the mutated residues positions were identified in the model. The structural analysis of the mutated residues showed that the external residue 190 is close to an important predicted region for antibodies recognition. Therefore, changes in the viral protein conformation might lead to an inefficient antibody binding and this could be a relevant mechanism underlying the recent vaccine failures observed in swine farms in Brazil.

Structure analysis of capsid protein of Porcine circovirus type 2 from pigs with systemic disease

Economic losses with high mortality rate associated with Porcine circovirus type 2 (PCV2) is reported worldwide. PCV2 commercial vaccine was introduced in 2006 in U.S. and in 2008 in Brazil. Although PCV2 vaccines have been widely used, cases of PCV2 systemic disease have been reported in the last years. Eleven nursery or fattening pigs suffering from PCV2 systemic disease were selected from eight PCV2-vaccinated farms with historical records of PCV2 systemic disease in Southern Brazil. PCV2 genomes were amplified and sequenced from lymph node samples of selected pigs. The comparison among the ORF2 amino acid sequences of PCV2 isolates revealed three amino acid substitutions in the positions F57I, N178S and A190T, respectively. Using molecular modeling, a structural model for the capsid protein of PCV2 was built. Afterwards, the mutated residues positions were identified in the model. The structural analysis of the mutated residues showed that the external residue 190 is close to an important predicted region for antibodies recognition. Therefore, changes in the viral protein conformation might lead to an inefficient antibody binding and this could be a relevant mechanism underlying the recent vaccine failures observed in swine farms in Brazil.(AU)

Analysis of Bm86 conserved epitopes: is a global vaccine against Cattle Tick Rhipicephalus microplus possible? / Análise de epítopos conservados da Bm86: é possível uma vacina global contra Carrapato-do-Boi Rhipicephalus microplus?

The cattle tick Rhipicephalus microplus causes significant economic losses in agribusiness. Control of this tick is achieved mainly through the application of chemical acaricides, often resulting in contamination of animal food products and of the environment. Another major concern associated with acaricide use is the increasing reports of resistance of this tick vector against the active ingredients of many commercial products. An alternative control method is vaccination. However, the commercially available vaccine based on a protein homologous to Bm86 exhibits variations in efficacy relative to the different geographical locations. This study aimed to identify antigenic determinants of the sequences of proteins homologous to Bm86. Phylogenetic analyses were performed to determine the extent of divergence between different populations of R. microplus to identify the sequence that could be used as a universal vaccine against the multiple geographically distinct populations of R. microplus and related tick species. Considering the extensive sequence and functional polymorphism observed among strains of R. microplus from different geographical regions, we can conclude that it may be possible to achieve effective vaccination against these cattle ticks using a single universal Bm86-based antigen.(AU)

O carrapato Rhipicephalus microplus é responsável por perdas significativas no agronegócio. O controle deste carrapato é feito principalmente por meio da aplicação de acaricidas químicos, geralmente resultando na contaminação de produtos de origem animal e do meio ambiente. Outra preocupação importante associada ao uso de acaricidas é o crescente aumento de relatos sobre a resistência deste carrapato a princípios ativos de vários produtos comerciais. Uma alternativa de controle é por meio de vacinação. Porém, a vacina comercializada contendo proteína homóloga à Bm86, apresenta variações de eficácia em relação às diferentes localizações geográficas. Este estudo buscou identificar determinantes antigênicos das sequencias de proteínas homólogas a Bm86. As análises filogenéticas foram feitas para determinar a extensão da divergência entre diferentes populações de R. microplus com o objetivo de identificar a sequência que poderia ser usada como vacina universal contra as múltiplas populações geograficamente distintas de R. microplus e espécies de carrapatos relacionados. Considerando-se a extensa sequência e o polimorfismo observados entre linhagens de R. microplus de diferentes regiões geográficas, podemos concluir que pode ser possível obter uma vacinação efetiva contra esses carrapatos bovinos utilizando um único antígeno universal baseado em Bm86.(AU)

Mapping of the continuous epitopes displayed on the Clostridium perfringens type D epsilon-toxin

The epsilon toxin, produced by Clostridium perfringens, is responsible for enterotoxemia in ruminants and is a potential bioterrorism agent. In the present study, 15 regions of the toxin were recognized by antibodies present in the serum, with different immunodominance scales, and may be antigen determinants that can be used to formulate subunit vaccines.(AU)

Preparation of monoclonal antibodies against gamma-type phospholipase A2 inhibitors and immunodetection of these proteins in snake blood

Abstract Background: The gamma-type phospholipase A2 inhibitor (LI) is a natural protein commonly found in snake serum, which can neutralize pathophysiological effects of snake venom phospholipases A2. Therefore, this protein is a potential candidate to the development of a novel antivenom. To the best of our knowledge, there is no antibody currently available for PLI identification and characterization. Methods: Bioinformatics prediction of epitope using DNAStar software was performed based on the sequence of Sinonatrix annularis PLI (SaPLI). The best epitope 151CPVLRLSNRTHEANRNDLIKVA172 was chosen and synthesized, and then conjugated to keyhole limpet hemocyanin and bovine serum albumin for use as an immunogen and plate-coating antigen, respectively. Results: Eighteen IgG anti-PLI mAb hybridoma cell strains were obtained, and all the mAbs had positive interaction with recombinant His6-PLI and natural SaPLI. Moreover, the mAb from 10E9 strain was also successfully used for the immunodetection of other snake serum PLIs. cDNA sequence alignment of those PLIs from different snake species showed that their epitope segments were highly homologous. Conclusions: The successful preparation of anti-PLImAb is significant for further investigation on the relationship between the structure and function of PLIs, as well as the interaction between PLIs and PLA2s.

Preparation of monoclonal antibodies against gamma-type phospholipase A2 inhibitors and immunodetection of these proteins in snake blood

Background: Paracoccidioidomycosis (PCM) is a neglected systemic mycosis caused by a dimorphic fungus of the Paracoccidioides genus. The standard diagnosis is based on isolation of the fungi in culture, and by microscopic visualization of characteristic multiple budding yeast cells in biological samples. However, in some situations, access to the site of injury prevents the collection of biological material. A variety of immuno-serological techniques has proven useful for allowing inferring diagnosis with a certain degree of certainty, thus optimizing time. The aim of this study was to standardize and validate the Dot-ELISA (DE) assay, comparing it with the serological standard, double immunodiffusion (DI). Methods: In order to standardize the DE assay, 143 serum samples were used. Out of those, 23 were from apparently healthy patients, 77 were from patients with confirmed PCM and 43 were from patients with other lung infections (tuberculosis, aspergillosis and histoplasmosis). To validate the DE technique, 300 serum samples from patients with PCM clinical suspicion (probable and possible cases) were employed, and these results were compared with those of DI. Results: The DE assay showed sensitivity of 91%, specificity of 95.4%, positive predictive value of 96%, negative predictive value of 98.2%, accuracy of 93%, and great precision (k = 0.93). In addition, the nitrocellulose membranes have proved to be viable for using at least 90 days after P. brasiliensis B-339 antigen sensitization. Conclusion: Dot-ELISA method was found to be an extremely promising tool as serologic screening technique, because of its high sensitivity. Furthermore, Dot-ELISA shows the prospect of being transferred to laboratories of mycoserology including those with fewer resources or even to be used directly in the field. It has an excellent shelf life ­ membranes coated with antigen can be used for testing without changes in the pattern of reactivity among laboratories ­ and presents reliable values of sensitivity, specificity, predictive values, accuracy and a high correlation with the serological standard methodology. Based on the present findings, it possible to state that this technique constitutes a remarkable option to be used in routine diagnosis for public health centers.(AU)

Preparation of monoclonal antibodies against gamma-type phospholipase A2 inhibitors and immunodetection of these proteins in snake blood

Background: Paracoccidioidomycosis (PCM) is a neglected systemic mycosis caused by a dimorphic fungus of the Paracoccidioides genus. The standard diagnosis is based on isolation of the fungi in culture, and by microscopic visualization of characteristic multiple budding yeast cells in biological samples. However, in some situations, access to the site of injury prevents the collection of biological material. A variety of immuno-serological techniques has proven useful for allowing inferring diagnosis with a certain degree of certainty, thus optimizing time. The aim of this study was to standardize and validate the Dot-ELISA (DE) assay, comparing it with the serological standard, double immunodiffusion (DI). Methods: In order to standardize the DE assay, 143 serum samples were used. Out of those, 23 were from apparently healthy patients, 77 were from patients with confirmed PCM and 43 were from patients with other lung infections (tuberculosis, aspergillosis and histoplasmosis). To validate the DE technique, 300 serum samples from patients with PCM clinical suspicion (probable and possible cases) were employed, and these results were compared with those of DI. Results: The DE assay showed sensitivity of 91%, specificity of 95.4%, positive predictive value of 96%, negative predictive value of 98.2%, accuracy of 93%, and great precision (k = 0.93). In addition, the nitrocellulose membranes have proved to be viable for using at least 90 days after P. brasiliensis B-339 antigen sensitization. Conclusion: Dot-ELISA method was found to be an extremely promising tool as serologic screening technique, because of its high sensitivity. Furthermore, Dot-ELISA shows the prospect of being transferred to laboratories of mycoserology including those with fewer resources or even to be used directly in the field. It has an excellent shelf life ­ membranes coated with antigen can be used for testing without changes in the pattern of reactivity among laboratories ­ and presents reliable values of sensitivity, specificity, predictive values, accuracy and a high correlation with the serological standard methodology. Based on the present findings, it possible to state that this technique constitutes a remarkable option to be used in routine diagnosis for public health centers.(AU)

Aplicação da proteômica de Leptospira e ferramentas de imunoinformática para a identificação de candidatos ao desenvolvimento de uma vacina contra leptospirose

A leptospirose é uma zoonose causada por espiroquetas do gênero Leptospira, que tem um impacto significativo em saúde pública com mais de um milhão de novas infecções e 59 mil mortes por ano, além de grandes perdas econômicas na agropecuária. A falta de uma vacina totalmente eficaz contra a doença é um dos principais fatores que comprometem o seu controle, por isso é emergencial o desenvolvimento de novas vacinas que seja capaz de superar as limitações das vacinais disponíveis no mercado atualmente, e que possa ser empregada em populações humanas globalmente. Por isso, o objetivo deste estudo foi realizar uma avaliação completa do perfil proteico de Leptospira interrogans sorovar Copenhageni cepa M20, com caracterização in silico das proteínas desconhecidas e das proteínas com capacidade de gerar resposta imune para compor uma vacina multiepítopo. Para a realização da proteômica foi desenvolvido um protocolo simplificado de obtenção do extrato proteico seguido pela espectrometria de massas LTQ-Orbitrap Velos acoplado à cromatografia líquida EASY-nLC II. As proteínas encontradas passaram por uma avaliação ampla, como análise taxonômica para identificação das proteínas comuns à outras espécies e sorovares da bactéria, descrição dos grupos funcionais, design da estrutura tridimensional de proteínas importantes no estudo da Leptospira e caracterização in silico das proteínas não caracterizadas e das proteínas já identificadas como geradoras de resposta imune. A partir daí foram utilizados diversos servidores para realização das análises de imunoinformática, visando a identificação e de seleção das proteínas com maior potencial para alvo vacinal e subsequente seleção dos melhores epítopos, capazes de estimular resposta imune humoral e celular. As proteínas Loa 22, LipL32, Flagelina, Fator de elongação Tu e Fator de elongação Ts, tiveram sua estrutura tridimensional desenvolvida e validada. Dentre as proteínas selecionadas com maior potencial para alvo vacinal, a maioria correspondeu a proteínas não caracterizadas até então. Este estudo também identificou epítopos de células B, de Linfócito T Citotóxico, de Linfócito T Helper, de células TCD4 e indutores de IFN-, que foram reunidos através de um design racional para constituir em uma proteína quimérica multiepítopo a ser empregada como alvo vacinal com potencial para superar as limitações da bacterina utilizada atualmente. O estudo do perfil proteico de Leptospira associado a triagem de alvos vacinais, por meio da imunoinformática, resultou na construção de uma proteína quimérica, com características desejáveis para um imunógeno no processo de desenvolvimento da vacina.

Leptospirosis is a zoonosis caused by spirochetes of the Leptospira genus, which causes a significant impact on public health, with more than one million new infections and 59,000 deaths each year, moreover major economic losses in agriculture. Lack of an fully effective vaccine against the disease is one of the main factors that compromise its control, so it is urgent to develop new vaccines that is able to overcoming the limitations of vaccines currently available, that can be used in human populations globally. Therefore, the aim of this study was to fully evaluate the protein profile of Leptospira interrogans serovar Copenhageni strain M20, with in silico characterization of uncharacterized proteins and proteins that is able to induce immune response and compose a subunit vaccine. To perform proteomics, a simplified protocol for obtaining protein extract was developed followed by mass spectrometry LTQ-Orbitrap Velos coupled to liquid chromatography EASY-nLC II. The proteins found undego a full evaluation, like a taxonomic analysis to identify common proteins to other species and serovars of the bacterium; description of functional categories; design of the three-dimensional structure of important proteins in the Leptospira study and in silico characterization of uncharacterized proteins and proteins already identified as induce an immune response. Then, many servers were used to perform immunoinformatics analysis, aiming to identify and select the proteins with the major potential for vaccine target and subsequent selection of the best epitopes able to stimulating humoral and cellular immunity. Proteins Loa 22, LipL32, Flagellin, Elongation fator Tu and Elongation fator Ts had their three-dimensional structure developed and validated. Among the selected proteins with the major vaccine target potential, most are uncharacterized proteins until then. This study also identified epitopes of B cell, of cytotoxic T lymphocyte, of helper T lymphocyte, of TCD4 cell and of IFN- inducers. This epitopes have been assembled through rational design to constitute a multi-epitope chimeric protein that can be used as a vaccine target with potential for overcome the bacterin limitations. The detailed study of Leptospira protein profile associated with the screening of vaccine targets by immunoinformatics resulted in the design of a chimeric protein, with desirable characteristics for an immunogen in the vaccine development process.

Caracterização molecular do vírus da cinomose canina, aspectos epizootiológicos, clínicos e do diagnóstico em cães naturalmente infectados

O vírus da cinomose canina (CDV) é um dos principais agentes infecciosos de cães domésticos e carnívoros silvestres, causando uma doença infecciosa multissistêmicas de alta mortalidade que acomete animais de diferentes faixas etárias, raças, machos e fêmeas e, tanto vacinados quanto não vacinados. A vacinação é o principal método de controle desta doença, no entanto, estudos vêm mostrando a possível existência de falhas vacinais em decorrência de diferença genética entre estirpes circulantes e estirpes vacinais. Dados referentes à epizootiologia são desconhecidos em Goiânia, poucos estudos comparam métodos diagnósticos em diferentes amostras em uma população de cães sintomáticos e às características moleculares do CDV em Goiás ainda não foram documentados. Este estudo objetivou determinar as características epizootiológicas do CDV, comparar os métodos diagnósticos de imunocromatografia (IC) e RT-PCR para a identificação do agente etiológico em diferentes amostras biológicas [swab conjuntival, sangue, urina, fluido cerebroespinal (FCE) e pool de amostras] e identificar as estirpes do CDV circulantes em cães naturalmente infectados e sintomáticos, no município de Goiânia, GO, Brasil, durante os anos de 2017 e 2020. Dados epizootiológicos foram coletados de 74 cães com sinais clínicos extraneurais e/ou neurais compatíveis com cinomose. Destes cães, 46 (62%) foram positivos por meio do teste de imunocromatografia (31%) ou pela técnica de RT-PCR (53%). Cães machos e fêmeas, de diferentes raças e idades foram acometidos, mas houve predominância em cães sem raça definida, adultos de um a seis anos e não vacinados. No entanto, aproximadamente 10,9% dos cães infectados e sintomáticos eram vacinados. Os sinais extraneurais predominantes foram hiporexia ou anorexia, secreção nasal e/ou ocular, desidratação e hiperqueratose de coxins e trufa. Dentre os sinais clínicos neurais os mais frequentes foram alteração de estado mental, déficit motor e mioclonias. Foi observada letalidade de aproximadamente 89% entre os animais com acometimento neurológico. As amostras de swab conjuntival, urina, pool e FCE tiveram maior frequência de positividade no teste de IC enquanto as amostras de urina e sangue, na RT-PCR. Do total de 149 amostras (sangue, urina e FCE) destinadas ao RT-PCR (gene N), o vírus foi identificado em 64 amostras (42,95%) e foram selecionadas 54 positivas para a RT-PCR seguida por Nested PCR para amplificação parcial do gene hemaglutinina (H). A amplificação parcial do gene H foi realizada e oito amostras foram sequenciadas e caracterizadas por meio da análise filogenética como pertencentes ao genótipo América do Sul 1/ Europa. Foram encontrados diferentes locais de substituição de aminoácidos no fragmento do gene H analisado das sequências identificadas e uma estirpe apresentou a mutação Y549H, típica de animais silvestres. Outros locais do fragmento apresentaram substituições em epítopos (resíduos 367, 376, 379, 381, 386 e 388) que podem interferir com a habilidade da vacina em promover proteção adequada contra a infecção pelo CDV, reduzindo sua efetividade. As estirpes isoladas foram geneticamente distante das estirpes vacinais, pertencentes ao genótipo América do Norte 1, com aproximadamente 10% de diferença nucleotídica. A partir da análise do genótipo América do Sul 1/ Europa, foram definidos dez subgenótipos. Esses achados evidenciam a relevância da infecção de cães por cinomose no Brasil, demonstrando a predominância do genótipo América do Sul 1/ Europa na região central do país e suportam a necessidade da realização de estudos epizootiológicos e de identificação molecular do CDV em outras regiões do país, em decorrência de sua grande extensão geográfica, da variabilidade de espécies animais acometidas, das diferenças moleculares entre as estirpes e da alta taxa de letalidade entre os animais infectados sintomáticos.

The canine distemper virus (CDV) is one of the main infectious agents of domestic dogs and wild carnivores, causing a multisystemic infectious disease with high mortality that affects animals of different age groups, breeds, males and females, and both vaccinated and unvaccinated. Vaccination is the main method of controlling this disease, however, studies have shown the possible existence of vaccine failures due to a genetic difference between circulating strains and vaccine strains. Data regarding epizootiology are unknown in Goiânia, few studies compare diagnostic methods in different samples in a population of symptomatic dogs and the molecular characteristics of CDV in Goiás have not yet been documented. This study aimed to determine the epizootiological characteristics of CDV, compare the diagnostic methods of immunochromatography (IC) and RT-PCR for the identification of the etiological agent in different biological samples [conjunctival swab, blood, urine, cerebrospinal fluid (CSF), and sample pool] and identify the circulating CDV strains in naturally infected and symptomatic dogs in the municipality of Goiânia, GO, Brazil, during 2017 and 2020. Epizootiological data were collected from 74 dogs with extraneural and/or neural clinical signs compatible with distemper. Of these dogs, 46 (62%) were positive using the immunochromatography test (31%) or by the RT-PCR technique (53%). Male and female dogs of different breeds and ages were affected, but there was a predominance in mixed breed dogs, adults aged one to six years and not vaccinated. However, approximately 10.9% of infected and symptomatic dogs were vaccinated. Predominant extraneural signs were hyporexia or anorexia, nasal and/or ocular discharge, dehydration, and nasal and digital hyperkeratosis. Among the neural clinical signs, the most frequent were changes in mental status, motor deficit, and myoclonus. A mortality rate of approximately 89% was observed among animals with neurological involvement. Conjunctival swab, urine, pool, and CSF samples had a higher frequency of positivity in the CI test, while urine and blood samples in RT-PCR. From a total of 149 samples (blood, urine, and FCE) destined for RT-PCR (N gene), the virus was identified in 64 samples (42.95%) and 54 positives were selected for RT-PCR followed by Nested PCR for partial amplification of the hemagglutinin (H) gene. Partial amplification of the H gene was performed and eight samples were sequenced and characterized by phylogenetic analysis as belonging to the South America 1/Europe genotype. Different amino acid substitution sites were found in the analyzed H gene fragment of the identified sequences and one strain presented the Y549H mutation, typical of wild animals. Other fragment sites had epitope substitutions (residues 367, 376, 379, 381, 386, and 388) that may interfere with the vaccine's ability to provide adequate protection against CDV infection, reducing its effectiveness. The isolated strains were genetically distant from the vaccine strains, belonging to the North America 1 genotype, with approximately 10% nucleotide difference. From the analysis of the South America 1/ Europe genotype, ten subgenotypes were defined. These findings highlight the relevance of canine distemper infection in Brazil, demonstrating the predominance of the South America 1/ Europe genotype in the central region of the country and support the need to conduct epizootiological and molecular identification studies of CDV in other regions of the country, due to its large geographic extension, the variability of animal species affected, the molecular differences between the strains and the high lethality rate among symptomatic infected animals.

GENETIC VARIABILITY OF NEW BRAZILIAN PCV2D STRAINS AND THEIR RELATION TO COMMERCIAL VACCINES

No presente estudo 246 amostras clínicas oriundas de granjas de suínos comerciais nas regiões sul, sudeste e centr-oeste do Brasil, previamente vacinadas contra PCV2 e que apresentavam sinais clínicos compatíveis com PCVD foram submetidas para genotipagem de PCV2. O tipo de material enviado para diagnóstico foram amostras de fluido oral, soro e pool de órgãos linfoides. Das amostras analisadas, 106 (43.09% - 106/246) foram genotipadas, sendo 75.47% (80/106) identificadas como PCV2d e 22.64% (24/106) identificadas como PCV2b. Em 2/106 (1.89%) amostras foram verificadas co-infecção de PCV2b e PCV2d. Não houve detecção do genótipo PCV2a nas amostras analisadas. Dentre as amostras clínicas analisadas, o fluido oral obteve um maior número de amostras genotipadas (40/106-37.74%), seguido de pool de órgãos linfóides e soro, com o mesmo número de amostras genotipadas (33/106- 31.13%). Adicionalmente, 51 amostras clínicas brasileiras coletadas em 2019 foram enviadas para sequenciamento genético do gene ORF2, que codifica a proteína do capsídeo viral (Cap). Destas, 25 apresentaram boa qualidade para análise, sendo 8 genotipadas como PCV2b e 17 como PCV2d, confirmando que no Brasil, como descrito mundialmente, houve o aumento do genótipo PCV2d. Para analisar a distribuição temporal dos genótipos e flutuação mundial dos genótipos desde 1993 à 2019, 3,544 sequencias disponíveis no GenBank foram analisadas, demonstrando um considerável aumento do genótipo PCV2d a partir de 2006, sendo hoje, o genótipo mais prevalente no mundo. Para estimar a variabilidade da Cap protein de nossas sequencias de PCV2d, comparamos nossas amostras com outras 1,300 sequencias de PCV2d disponíveis no GenBank. Com o objetivo de avaliar a possível pressão vacinal sobre o o genótipo PCV2d, separamos as sequencias em 4 grupos: (1) Pre vacinação (PreVacD) - sequencias de amostras coletadas antes de 2006; (2) Pós vacinação (PosVacD) - sequencias de amostras coletadas entre 2007 e 2020; (3) sequencias de suínos selvagens ou de vida livre(WboarD) que não sofreram pressão vacinal; e (4) nossas sequencias de PCV2d (TestD), analisadas no presente estudo.. Devido às possíveis falhas vacinais observadas nas granjas de suínos comerciais no Brasil, as amostras de PCV2d sequenciadas no presente estudo foram comparadas com as sequencias da ORF2 disponíveis de três vacinas comercias utilizadas no Brasil, uma contendo o genótipo PCV2b e duas contendo o genótipo PCV2a. Grande parte dos resíduos de aminoácidos que se encontram dentro de regiões de epítopo são conservadas, sugerindo uma boa reação cruzada entre as amostras vacinais e as amostras de PCV2d analisadas. Contudo, em alguns resíduos de aminoácidos em sítios antigênicos, essencias para ligação de anticorpos e, consequentemente, neutralização viral, foram detectadas mutações entre as sequencias de PCV2d e as sequencias vacinais. Este resultado sugere que estas mutações possam ter importancia em relação à resposta. Porém, mais estudos devem ser realizados para elucidar essas questões, principalmente a implementação de processos de vigilância de genótipos de Circovirus suínos tipo 2 circulantes no Brasil.

In the present study, 246 clinical samples from commercial pig farms located in the south, southeast and central-west regions of Brazil previously vaccinated against PCV2 and showing clinical signs compatible with PCVD were submitted for PCV2 genotyping. The type of material sent for diagnosis were samples of oral fluid, serum and lymphoid organs pool. Of the analyzed samples, 106 (43.09% - 106/246) were genotyped, with 75.47% (80/106) identified as PCV2d and 22.64% (24/106) as PCV2b. In 2/106 (1.89%) samples, coinfection of PCV2b and PCV2d was observed. There was no detection of the PCV2a genotype in the analyzed samples. Among the analyzed clinical samples, oral fluid obtained the greater number of genotyped samples (40/106- 37.74%), followed by pool of lymphoid organs and serum, with the same number of genotyped samples (33/106-31.13%) . Additionally, 51 Brazilian clinical samples collected in 2019 were sent for genetic sequencing of the ORF2 gene, widely used for genomic/protein analysis, which encodes for the viral capsid protein (Cap). Of these, 25 had good quality for analysis, and 8 were genotyped as PCV2b and 17 as PCV2d, corroborating many other publications around the world that demonstrated the increase of the PCV2d genotype. To analyze the temporal distribution of genotypes and fluctuation of genotypes from 1993 to 2019 worldwide, 3,544 sequences available in GenBank were analyzed, demonstrating a considerable increase in the PCV2d genotype since 2006, today the most prevalent genotype in the world. To estimate the Cap protein variability of our PCV2d sequences, our results were compared to other 1,300 PCV2d sequences available in GenBank. In order to assess the possible pressure of the vaccine against the PCV2d genotype population infection, four groups of samples were defined: (1) Prevaccination (PreVacD) - sequences from samples collected before 2006; (2) Post-vaccination (PosVacD) - sequences of samples collected between 2007 and 2020; (3) sequences from wild or feral pigs (WboarD) that were not vaccinated, and (4) our PCV2d sequences (TestD) analyzed in the present study. Due to possible vaccine failures observed in commercial pig farms in Brazil, the PCV2d sequenced samples in the present study were compared to the ORF2 amino acid sequences available from three commercial vaccines used in Brazil, one containing the PCV2b genotype and 2 containing the PCV2a genotype. Most of the amino acid residues found within epitope regions were conserved, suggesting a good cross-reaction between vaccines and the analyzed PCV2d samples. However, in some amino acid residues in antigenic sites essential for antibody binding and consequently viral neutralization, mutations were detected in PCV2d sequences and in vaccine sequences. This result may suggest that these mutations might be involved in the vaccine immune response. However, further studies must be carried out to elucidate these aspects, especially the implementation of surveillance processes of Porcine Circovirus type 2 genotypes circulating in Brazil.

Characterization of an IgM-like immunoglobulin from silver catfish (Rhamdia quelen) serum and its use for the production of polyclonal antibodies and development of immunoassays / Caracterização de imunoglobulina similar a IgM em soros de jundiás (Rhamdia quelen) e sua utilização para a produção de soro policlonal e desenvolvimento de imunoensaios

Knowledge on fish immunoglobulin (Ig) characteristics and the availability of monoclonal or polyclonal antibodies to fish Igs are essential to evaluate the humoral immune response and the Ig distribution on leukocyte cells. We demonstrated that silver catfish serum Ig is composed of one immunodominant H chain with approximately 75k Da and one L chain with approximately 28 kDa, similar to human IgM. Rabbit polyclonal antibodies to the catfish IgM-like Ig recognized both the H and L chain and were useful in developing an indirect ELISA to measure the production of antibodies in fish immunized with bovine serum albumin. Dot blot and western blot cross-reactivity studies indicated a wide degree of epitope sharing amongst Ig from several Siluriformes and Characiformes fish indigenous to Brazilian rivers. In these fish species, polyclonal antibodies reacted mostly with the H chain. The results presented here are central to the development of tools and strategies to investigate the antibody production to inoculated antigens and tissue distribution of Ig molecules in native fish species. Furthermore, because of the wide range of cross-reactivity, polyclonal antibodies to silver catfish IgM-like Ig might be used to develop immunoassays to measure the humoral immune response in other fish species.(AU)

Informações sobre as características das imunoglobulinas (Ig) de peixes e a disponibilidade de anticorpos mono ou policlonais são essenciais para avaliar a resposta imune humoral e a distribuição leucocitária de Igs. Nesse trabalho nós demonstramos que a Ig do soro de jundiás é composta por uma cadeia pesada (H) imunodominante, de aproximadamente 75kDA e de uma cadeia leve (L) de aproximadamente 28 kDa, similar à IgM humana. Anticorpos policlonais produzidos contra a Ig do jundiá reconheceram a cadeia H e L e permitiram o desenvolvimento de um ELISA indireto para mensurar a produção de anticorpos em peixes imunizados com albumina sérica bovina. Estudos de reatividade cruzada, por meio de Dot blot e western blot, indicaram um alto grau de compartilhamento de epitopos entre as Igs de diversos peixes Siluriformes e Caraciformes nativos do Brazil. Nestas espécies de peixes, os anticorpos policlonais reconheceram principalmente a cadeia H. Os resultados deste estudo são fundamentais para o desenvolvimento de ferramentas e estratégias para investigar a produção de anticorpos subsequente à imunização e a distribuição tecidual de Igs em peixes nativos. Além disso, devido ao compartilhamento de epitopos entre as espécies de peixes avaliadas, os anticorpos policlonais anti Ig do jundiá poderão ser usados para desenvolver ensaios imunoenzimáticos para avaliar a resposta imune humoral nestas espécies.(AU)

Characterization of an IgM-like immunoglobulin from silver catfish (Rhamdia quelen) serum and its use for the production of polyclonal antibodies and development of immunoassays / Caracterização de imunoglobulina similar a IgM em soros de jundiás (Rhamdia quelen) e sua utilização para a produção de soro policlonal e desenvolvimento de imunoensaios

Knowledge on fish immunoglobulin (Ig) characteristics and the availability of monoclonal or polyclonal antibodies to fish Igs are essential to evaluate the humoral immune response and the Ig distribution on leukocyte cells. We demonstrated that silver catfish serum Ig is composed of one immunodominant H chain with approximately 75k Da and one L chain with approximately 28 kDa, similar to human IgM. Rabbit polyclonal antibodies to the catfish IgM-like Ig recognized both the H and L chain and were useful in developing an indirect ELISA to measure the production of antibodies in fish immunized with bovine serum albumin. Dot blot and western blot cross-reactivity studies indicated a wide degree of epitope sharing amongst Ig from several Siluriformes and Characiformes fish indigenous to Brazilian rivers. In these fish species, polyclonal antibodies reacted mostly with the H chain. The results presented here are central to the development of tools and strategies to investigate the antibody production to inoculated antigens and tissue distribution of Ig molecules in native fish species. Furthermore, because of the wide range of cross-reactivity, polyclonal antibodies to silver catfish IgM-like Ig might be used to develop immunoassays to measure the humoral immune response in other fish species.(AU)

Informações sobre as características das imunoglobulinas (Ig) de peixes e a disponibilidade de anticorpos mono ou policlonais são essenciais para avaliar a resposta imune humoral e a distribuição leucocitária de Igs. Nesse trabalho nós demonstramos que a Ig do soro de jundiás é composta por uma cadeia pesada (H) imunodominante, de aproximadamente 75kDA e de uma cadeia leve (L) de aproximadamente 28 kDa, similar à IgM humana. Anticorpos policlonais produzidos contra a Ig do jundiá reconheceram a cadeia H e L e permitiram o desenvolvimento de um ELISA indireto para mensurar a produção de anticorpos em peixes imunizados com albumina sérica bovina. Estudos de reatividade cruzada, por meio de Dot blot e western blot, indicaram um alto grau de compartilhamento de epitopos entre as Igs de diversos peixes Siluriformes e Caraciformes nativos do Brazil. Nestas espécies de peixes, os anticorpos policlonais reconheceram principalmente a cadeia H. Os resultados deste estudo são fundamentais para o desenvolvimento de ferramentas e estratégias para investigar a produção de anticorpos subsequente à imunização e a distribuição tecidual de Igs em peixes nativos. Além disso, devido ao compartilhamento de epitopos entre as espécies de peixes avaliadas, os anticorpos policlonais anti Ig do jundiá poderão ser usados para desenvolver ensaios imunoenzimáticos para avaliar a resposta imune humoral nestas espécies.(AU)

Análise molecular de Carnivore protoparvirus 1 proveniente de cepas vacinais e cepas selvagens de carnívoros domésticos

Causador da parvovirose canina, enfermidade altamente contagiosa e prevalente na população canina mundial, Canine parvovirus 2 (CPV-2) é uma das quatro cepas pertencentes à espécie Carnivore protoparvovirus 1. CPV-2 foi descrito pela primeira vez nos anos 70 e, desde então, mutações em resíduos de aminoácidos da principal proteína antigênica VP2, vêm sendo reportadas. Tais alterações resultaram no estabelecimento das variantes antigênicas CPV-2a, CPV-2b e CPV-2c. Recentemente, CPV-2-like, new CPV-2a e new CPV-2b vêm sendo relatadas. Uma vez que a taxa de mutações genéticas de CPV-2 é expressiva, e que alterações em resíduos de aminoácidos em VP2 geram variantes com diferentes propriedades biológicas e antigênicas associadas ao escape do vírus do sistema imunológico, não se pode descartar a hipótese de que as variantes não sejam geradoras de resposta imune cruzada entre si. Dessa maneira, se faz necessário estudos moleculares a fim de verificar possíveis discrepâncias genéticas entre cepas e variantes selvagens e vacinais de CPV-2. Portanto, este estudo teve como objetivo realizar a análise molecular de variantes do CPV-2 infectando carnívoros domésticos na Zona da Mata e Região Metropolitana de Minas Gerais e de cepas do CPV-2 presentes em sete marcas de vacinas comercializadas no Brasil. As amostras analisadas foram caracterizadas como CPV-2b (16/17) e CPV-2-like (1/17). Duas vacinas comerciais apresentaram diferenças notáveis frente as cepas obtidas neste trabalho, com alterações não sinônimas em resíduos de aminoácidos: Asp413Asn, Lys570Glu e Lys575Arg, em Vac7 e Tyr573Phe, em Vac3, sendo 575 e 573 posições pertencentes à regiões de epítopos.

Causative of canine parvovirosis, a highly contagious and prevalent disease in the canine population worldwide, Canine parvovirus 2 (CPV-2) is one of four strains belonging to the species Carnivore protoparvovirus 1. CPV-2 was first described in the 1970s and since then, mutations in amino acid residues of the main antigenic protein VP2 have been reported. Such alterations resulted in the establishment of the antigenic variants CPV-2a, CPV-2b and CPV-2c. Recently, CPV-2-like, new CPV-2a and new CPV-2b have been reported. CPV-2 possesses a high mutation rate, and changes in amino acid residues in VP2 generate variants with different biological and antigenic properties associated with virus escape from the immune system. Thus, the hypothesis that variants do not generate a cross-immune response may be investigated. Hence, molecular studies are needed to verify possible genetic discrepancies between CPV-2 from wild and vaccine strains. Therefore, this study aimed to perform the molecular analysis of CPV-2 variants infecting domestic carnivores in the Zona da Mata and Metropolitan Region of Minas Gerais and CPV-2 strains present in seven brands of vaccines marketed in Brazil. The analyzed samples were characterized as CPV-2b (16/17) and CPV-2-like (1/17). Two (2/7) commercial vaccines showed notable differences against the strains obtained in this study, with non-synonymous mutations in amino acid residues: Asp413asn, Lys570Glu, and Lys575Arg, in Vac7 and Tyr573Phe, in Vac3. Importantly, 575 and 573 positions belong to epitope regions.

Avaliação da resposta imune e análise de linfonodos de ovinos deslanados (Ovis aries) imunizados com as sub unidades recombinantes rsncsag4 e rsncgra1

O Neospora caninum é um protozoário parasita intracelular cujo interesse tem aumentado principalmente devido a sua distribuição cosmopolita e pelo impacto econômico relacionado a infecção no rebanho bovino. As principais perdas relacionadas à neosporose bovina estão associadas a abortos e também a redução dos índices produtivos dos animais portadores. Atualmente, não há tratamento ou vacina disponível para o controle desse agente. Dentre as possíveis metodologias de controle, a vacinação é considerada a melhor alternativa para deter o avanço dessa doença, sendo que o desenvolvimento de vacinas de subunidades tem sido visto como uma das tecnologias mais promissoras. Dentre os principais antígenos imunogênicos estão as proteínas NcGRA1 e NcSAG4. Neste estudo foi utilizada a imunização em modelo ovino para avaliar parcialmente a resposta imune de duas subunidades proteicas recombinantes (rsNcSAG4 e rsNcGRA1) construídas através da metodologia de genética reversa de epítopos antigênicos. A resposta a cada imunógeno foi avaliada pela mensuração da cinética de anticorpos, histologia e imunohistoquímica dos linfonodos após 3 imunizações. Análises histológicas confirmaram a imunogenicidade e capacidade de apresentação dos peptídeos pela formação de centros germinativos, hiperplasia de cordões medulares e presença de células positivas para imuno-histoquimica. Pôde-se também observar, através de ELISA, que a formulação conjunta foi capaz de induzir a produção de anticorpos, o que nos leva a inferir que podem ser usados como candidatos vacinais anti N. caninum.

Neospora caninum is an intracellular parasitic protozoan whose interest has increased mainly due to its cosmopolitan distribution and the economic impact related to infection in the bovine herd. The main losses related to bovine neosporosis are associated with abortions and also the reduction of the productive indexes of the carrier animals. There is currently no treatment or vaccine available to control this agent. Among the possible control methodologies, vaccination is considered the best alternative to stop the progress of this disease, and the development of subunit vaccines has been seen as one of the most promising technologies. Among the main immunogenic antigens are the proteins NcGRA1 and NcSAG4. In this study, immunization in a sheep model was used to partially assess the immune response of two recombinant protein subunits (rsNcSAG4 and rsNcGRA1) constructed using the antigenic epitope reverse genetics methodology. The response to each immunogen was assessed by measuring the kinetics of antibodies, histology and immunohistochemistry of the lymph nodes after 3 immunizations. Histological analyzes confirmed the immunogenicity and the ability to present peptides through the formation of germinal centers, spinal cord hyperplasia and the presence of positive cells for immunohistochemistry. It was also possible to observe, through ELISA, that the joint formulation was able to induce the production of antibodies, which leads us to infer that they can be used as vaccine candidates against N. caninum.

DESENVOLVIMENTO DE ESTRATÉGIAS DE CULTURA PARA ISOLAMENTO DE LEPTOSPIRAS FASTIDIOSAS E USO DE BIOINFORMÁTICA PARA DESCOBERTA DE NOVOS ALVOS VACINAIS E DROGÁVEIS PARA CONTROLE DA LEPTOSPIROSE ANIMAL

A leptospirose, causada por espiroquetas do gênero Leptospira, é uma zoonose globalmente disseminada, negligenciada e emergente. O isolamento de leptospiras é o primeiro passo para sua caracterização molecular e genotipagem, no entanto, para os sorovares mais fastidiosos, a cultura e isolamento são controversos e laboriosos. Com relação ao controle e tratamento da leptospirose, muitos estudos são realizados, mas ainda não existe uma vacina eficaz contra todas as espécies patogênicas ou variedade de antimicrobianos com atuação sobre o quadro de leptospirúria. Assim o presente estudo visou primeiramente desenvolver estratégias de cultura e isolamento para as leptospiras fastidiosas e utilizar métodos rápidos de diagnóstico molecular para caracterização e tipificação dos sorovares. Posteriormente, a partir do isolamento de novas estirpes, o sequenciamento genômico foi realizado com a finalidade de obtenção de dados de genômica comparativa com outras estirpes disponíveis no GenBank para uma busca in silico de alvos drogáveis e vacinais. No primeiro estudo, três formulações de meios de cultura líquidos básicos foram produzidas para isolar e manter leptospiras. Em cada formulação (A, B e C) foram adicionados diferentes suplementos para ajudar no crescimento de leptospiras fastidiosas: piruvato de sódio, enzima superóxido dismutase e soro fetal bovino. Durante o período aproximado de 55 dias, adotou-se estratégias de troca entre as três formulações de acordo com a observação em microscopia de campo escuro e ao final desse período houve sucesso no isolamento de três estirpes do sorovar Hardjo (dois genotipos, Hardjobovis e Hardjoprajitno) com adaptação total ao meio de cultura. Posteriormente, estas culturas foram avaliadas com uso da microscopia eletrônica e notou-se diferenças na morfologia e viabilidade de acordo com a composição do meio. No segundo estudo, os genomas desses dois genotipos foram sequenciados, montados e depositados no GenBank. A partir desses dados e também de outros genomas de outras espécies de Leptospira foi possível realizar uma abordagem de com base na vacinologia reversa, genômica comparativa e de docking molecular com o uso de ferramentas de bioinformática. Para os alvos vacinais, os resultados com base nas características da probabilidade de adesão, TMHMM e densidade de epítopos, sugerem que cinco alvos são bons candidatos e podem ser testados rapidamente em novas formulações de vacinas e posteriormente testados in vivo. Para a etapa de docking molecular, oito proteínas preditas como citoplasmáticas e identificadas como essenciais para a sobrevivência de leptospiras foram consideradas bons alvos para novos medicamentos. Destacando a proteína de divisão celular FtsZ, que foi o alvo identificado com as melhores características de ligação e, por meio de uma triagem virtual, o ZINC04259719 foi identificado como a melhor molécula para ligar a essa proteína, a fim de inibir sua função eliminando o patógeno. A vacinologia reversa e o docking molecular são poderosas ferramentas de bioinformáticas que possibilitam uma busca rápida e sem procedimentos laboriosos, por alvos proteicos específicos que possam ser candidatos a uma nova vacina ou medicamento no controle da leptospirose.

Leptospirosis, caused by spirochetes of the genus Leptospira, is a globally widespread, neglected and emerging zoonosis. The leptospires isolation is the first step towards its molecular characterization and genotyping, however, for the most fastidious serovars, the culture and isolation are controversial and laborious. Regarding the control and treatment of leptospirosis, many studies have been carried out, but there is not yet an efficient vaccine against all pathogenic species or more variety of antimicrobials acting on leptospiruria. Thus, the present study aimed primarily to develop culture and isolation strategies for fastidious leptospires and using rapid molecular diagnostic methods for characterization and typing of serovars. Subsequently, from the isolation of new strains, genomic sequencing was carried out in order to obtain comparative genomic data with other strains available at GenBank for an in silico search for drug and vaccine targets. In the first study, three formulations of basic liquid culture media were produced to isolate and maintain leptospires. In each formulation (A, B and C) different supplements were added to help the growth of fastidious leptospires: sodium pyruvate, superoxide dismutase enzyme and fetal bovine serum. During the approximate period of 55 days, exchange strategies were adopted between the three formulations according to the observation in dark field microscopy and at the end of this period, there was success in the isolation of three strains of serovar Hardjo (two genotypes, Hardjobovis and Hardjoprajitno) with total adaptation to the culture medium. Subsequently, these culture was evaluated through electron microscopy and differences in morphology and viability were noted according to the composition of the medium. In the second study, the genomes of these two genotypes were sequenced, assembled and deposited on GenBank. From these data and also from other genomes of Leptospira species, it was possible to carry out an approach based on reverse vaccinology, genomic comparative and molecular docking with the use of bioinformatics tools. For vaccine targets, results based on characteristics of probability of adherence, TMHMM and epitope density, suggest that five targets are good candidates and can be tested quickly in new vaccine formulations and subsequently tested in vivo. For the molecular docking step, eight proteins predicted as cytoplasmic and considered essential for the survival of leptospires were considered good targets for new drugs. Highlighting the cell division protein FtsZ, which was the target identified with the best binding characteristics and through a virtual screening the ZINC04259719 was identified as the best molecule for linked to this protein in order to inhibit its function and consequently eliminating the microorganism. Reverse vaccinology and molecular docking are powerful bioinformatics tools that enable a quick discovery, without laborious procedures, for specific protein targets that may be candidates for a new vaccine or therapeutic target to leptospirosis control.

Sequenciamento, anotação e análise do genoma de Mycobacterium pinnipedii e genômica comparativa de espécies do Complexo Mycobacterium tuberculosis

O Complexo Mycobacterium tuberculosis (MTBC) causa tuberculose em humanos e animais e é composto de 12 espécies bacterianas com diferentes tropismos por hospedeiros e perfis de virulência. O MTBC se originou na África, e evoluiu clonalmente por meio de mutações pontuais e deleções genéticas de até 12Kb. Apesar das variações fenotípicas, sequências homólogas de genomas do MTBC são 99,95% idênticas e transferências horizontais de genes são consideradas ausentes. Estudos compreensivos de genômica comparativa incluindo todas as espécies de MTBC a fim de identificar os determinantes genéticos dessas diferenças fenotípicas não estão disponíveis. Paralelamente, a recente descoberta de uma estirpe relacionada a Mycobacterium pinnipedii infectando múmias peruanas pré-colombianas, isto é, antes da introdução europeia da tuberculose nas Américas, levantou dúvidas quanto ao papel do M. pinnipedii na evolução do MTBC e sobre a co-evolução desses patógenos com populações humanas. Desta forma, esta dissertação tem como objetivos: (i) sequenciar e anotar os genomas completos de estirpes de M. pinnipedii isoladas de um leão marinho no Brasil e compará-los com outras estirpes da mesma espécie depositadas em bancos de dados públicos; (ii) produzir uma avaliação de genômica comparativa de todos as espécies descritas do MTBC a fim de identificar genes relacionados à virulência e à adaptabilidade ao hospedeiro. No primeiro estudo, foi realizado o sequenciamento dos genomas completos de dois isolados de M. pinnipedii obtidos da carcaça de um leão marinho (Otaria flavescens) encontrada no Rio Grande do Sul. Análises comparativas das mutações encontradas nesses genomas indica que esse animal estava infectado por duas estirpes diferentes desta bactéria. Este achado constitui o primeiro relato de infecção mista por M. pinipedii nesta espécie hospedeira e sugere que o patógeno é altamente endêmico na população de origem deste animal. Em contraste com estirpes de M. tuberculosis, a genômica comparativa entre estirpes modernas e antigas de M. pinnipedii depositadas em bancos de dados públicos indica proteomas altamente conservados e a ocorrência de um decaimento gênico através de deleções e pseudogenização, em um processo ativo de redução do genoma que vem ocorrendo há, pelo menos, 1.000 anos. Por fim, duas linhagens filogenéticas, modernas de M. pinnipedii foram detectadas globalmente, circunscritas por geografia e, consequentemente, por espécies hospedeiras. No segundo estudo, o objetivo foi investigar a presença e conservação de epítopos de células T e fatores de virulência no genoma core, acessório e espécie específico de 205 estirpes do MTBC. A estrutura do pan-genoma do MTBC mostra que a maioria das proteínas (832.234/866.916, 96,00%) está no genoma core, que é composto por proteínas importantes para a sobrevivência bacteriana. A análise da arquitetura do genoma acessório de MTBC com a distribuição dos epítopos mostrou, pela primeira vez, um mecanismo de variação antigênica de epítopos de células T baseado na perda de genes. Adicionalmente, foi possível observar variações significativas na quantidade de genes relacionados aos fatores de virulência, principalmente em estirpes de M. pinnipedii e M. africanum, sugerindo que a perda gênica relacionada à virulência possui importante papel nos fenótipos bacterianos. Em conclusão, os resultados apresentados contribuem para o entendimento da interação patógeno-hospedeiro de M. pinnipedii e outros membros do MTBC, sugerindo marcadores genéticos importantes dessa interação, e elucidando efeitos de pressões seletivas na determinação dos fenótipos de adaptabilidade hospedeira e virulência do grupo clonal MTBC.

The Mycobacterium tuberculosis Complex (MTBC) causes tuberculosis in humans and animals and is composed of 12 bacterial species with different host tropisms and virulence profiles. MTBC originated in Africa and evolved clonally through point mutations and genetic deletions of up to 12Kb. Despite phenotypic variations, homologous sequences of MTBC genomes are 99.95% identical and horizontal gene transfers are considered absent. Comprehensive comparative genomics studies including all MTBC species to identify the genetic determinants of these phenotypic differences are not available. At the same time, the recent discovery of a strain related to Mycobacterium pinnipedii infecting pre-Columbian Peruvian mummies, i.e. prior to the European introduction of tuberculosis in the Americas, raised doubts about the role of M. pinnipedii in the evolution of MTBC and the co-evolution of these pathogens with human populations. Thus, the aims of this study were to: (i) sequence and annotate the complete genomes of M. pinnipedii strains isolated from a sea lion in Brazil and compare them with other strains of the same species deposited in public databases; (ii) produce a comparative genomic evaluation of all described MTBC species to identify genes related to virulence and host adaptability. In the first study we sequenced the complete genomes of two M. pinnipedii isolates obtained from the carcass of a sea lion (Otaria flavescens) found in Rio Grande do Sul. Comparative analysis of mutations found in these genomes indicates that this animal was infected with two different strains of this bacterium. This finding is the first report of mixed M. pinipedii infection in this host species and suggests that the pathogen is highly endemic in the source population of this animal. In contrast to M. tuberculosis strains, the comparative genomics of ancient and modern M. pinnipedii strains deposited in public databases indicate highly conserved proteomes and the occurrence of gene decay through deletions and pseudogenization in an active process of genome reduction occurring for at least 1,000 years. Finally, two modern phylogenetic strains of M. pinnipedii were detected globally, circumscribed by geography and, consequently, by host species. In the second study, the aim was to investigate the presence and conservation of T cell epitopes and virulence factors in the core, accessory and strain-specific genomes of 205 MTBC strains. The MTBC pan-genome structure shows that most proteins (832,234/866,916, 96.00%) are in the core genome, which is composed of proteins important for bacterial survival. Analysis of the MTBC accessory genome architecture with epitope distribution showed, for the first time, a mechanism of antigenic T cell epitope variation based on gene loss. Additionally, it was possible to observe significant variations in the number of genes related to virulence factors, mainly in M. pinnipedii and Mycobacterium africanum strains, suggesting that virulence-related gene loss plays an important role in determining bacterial phenotypes. In conclusion, the results presented contribute to the understanding of the pathogen-host interaction of M. pinnipedii and other MTBC members, suggesting important genetic markers of this interaction, and elucidating the effects of selective pressures in the determination of host adaptability and virulence phenotypes of the clonal group MTBC.

Production of egg yolk antibody (IgY) against recombinant porcine epidemic diarrhea virus COE protein

Background: Porcine epidemic diarrhea (PED) is a highly contagious disease of pigs, and is characterized by a series ofclinical symptoms, such as severe diarrhea, vomiting and dehydration. Partial protective antigen gene (COE gene) of Sprotein possessing the main B cell epitope, is able to encode proteins with reactogenicity to induce the production of neutralizing antibodies. IgY was found to reduce the mortality in piglets after challenge exposures. Anti-COE IgY antibodyhas never been reported before, here it is described a method for the production of anti-COE IgY, which could be appliedin the treatment for the porcine epidemic diarrhea virus (PEDV) infection.Materials, Methods & Results: A PEDV strain was isolated from a clinical sample. The COE ORF (Open reading frame,ORF)was amplifi ed by PCR and iserted into the pMD18-T clone vector. The isolated was defi ned as Porcine epidemic diarrheavirus strain JS-HZ2012 subtype by sequencing, the clinical sample was defi ned as the nucleic acid sequence has a 99.5%homology with that of PEDV CV777 strain. And then the COE ORF was subcloned into pET-32a by T4 DNA ligase andintroduced into the E.coli Bal21 (DE3). COE protein was produced by the induction of the E.coli Bal21 containing pET32a-COE with isopropyl-β-D-1-thiogalactopyrannoside (IPTG). Expression of the recombinant COE protein (rCOE)fused with His-tag was analyzed by SDS-PAGE and detected by western-blotting using anti-His monoclonal antibody.The rCOE was purifi ed by Ni+ affi nity purifi cation chromatography under denature condition and dialyzed against PBS.The concentration of the rCOE was determined by BCA method. After immunnizing the chickens with rCOE , All animalhandling procedures were performed under veterinary supervision and following the recommendations of the local lawsand regulations on Animal Experimentation. Anti-COE IgY was isolated by chloroform extraction and...(AU)

Production of egg yolk antibody (IgY) against recombinant porcine epidemic diarrhea virus COE protein

Background: Porcine epidemic diarrhea (PED) is a highly contagious disease of pigs, and is characterized by a series ofclinical symptoms, such as severe diarrhea, vomiting and dehydration. Partial protective antigen gene (COE gene) of Sprotein possessing the main B cell epitope, is able to encode proteins with reactogenicity to induce the production of neutralizing antibodies. IgY was found to reduce the mortality in piglets after challenge exposures. Anti-COE IgY antibodyhas never been reported before, here it is described a method for the production of anti-COE IgY, which could be appliedin the treatment for the porcine epidemic diarrhea virus (PEDV) infection.Materials, Methods & Results: A PEDV strain was isolated from a clinical sample. The COE ORF (Open reading frame,ORF)was amplifi ed by PCR and iserted into the pMD18-T clone vector. The isolated was defi ned as Porcine epidemic diarrheavirus strain JS-HZ2012 subtype by sequencing, the clinical sample was defi ned as the nucleic acid sequence has a 99.5%homology with that of PEDV CV777 strain. And then the COE ORF was subcloned into pET-32a by T4 DNA ligase andintroduced into the E.coli Bal21 (DE3). COE protein was produced by the induction of the E.coli Bal21 containing pET32a-COE with isopropyl-β-D-1-thiogalactopyrannoside (IPTG). Expression of the recombinant COE protein (rCOE)fused with His-tag was analyzed by SDS-PAGE and detected by western-blotting using anti-His monoclonal antibody.The rCOE was purifi ed by Ni+ affi nity purifi cation chromatography under denature condition and dialyzed against PBS.The concentration of the rCOE was determined by BCA method. After immunnizing the chickens with rCOE , All animalhandling procedures were performed under veterinary supervision and following the recommendations of the local lawsand regulations on Animal Experimentation. Anti-COE IgY was isolated by chloroform extraction and...

DESENVOLVIMENTO DE UM TESTE SOROLÓGICO BASEADO EM UM ANTÍGENO ORIENTADO POR ANTICORPO MONOCLONAL PARA DIAGNÓSTICO DE Mycoplasma hyopneumoniae

Autor: Rafaela Luiza Klein Orientador: Rafael Frandoloso A pneumonia enzoótica suína (PES) é uma doença infectocontagiosa que há longa data é destaque na cadeia suinícola. A PES é causada pelo Mycoplasma hyopneumoniae (Mhyo), uma bactéria específica de suínos que pode ser transmitida através de contato direto e indireto. A doença acomete suínos de todas as idades, porém a clínica é comumente observada na fase de crescimento e terminação de suínos. Mhyo pode produzir doença pulmonar de forma independente, no entanto, sua infecção facilita a ocorrência de inúmeras outras infecções secundárias causadoras de pneumonias e de doenças sistêmicas. O diagnóstico da circulação de Mhyo é realizada através de testes sorológicos em combinação com técnicas moleculares, e a prevenção, mediante o uso de vacinas clássicas aplicadas pela via intramuscular ou intradérmica. Em razão do diagnóstico sorológico ser de vital importância para o posicionamento correto de vacinas a partir do entendimento da cinética de anticorpos naturais adquiridos através do colostro, ou mesmo, desenvolvidos após um processo de infecção, desenvolver insumos imunológicos genuínos e capazes de detectar indiretamente ou diretamente o Mhyo são indispensáveis para o Brasil. Nesta dissertação, apresentamos o desenvolvimento e a caracterização de um anticorpo monoclonal (AcMo) contra Mycoplasma hyopneumoniae. Posteriormente, o AcMo foi utilizado para desenvolver um ELISA moderno, baseado em células inteiras de M. hyopneumoniae corretamente orientadas no interior da placa de ELISA pelo AcMo. Brevemente, células de mieloma murino P3x63Ag8.653 foram fusionadas com esplenócitos obtidos de camundongos BALB/c imunizados com uma suspensão de M. hyopneumoniae cepa ATCC25095. O AcMo obtido, nomeado de MAb 4A2, foi caracterizado por meio de Western Blot, Dot Assay, Citometria de Fluxo e Teste de Avidez. O MAb 4A2 é uma imunoglobulina da subclasse IgG1 com duas cadeias leves do tipo lambda que reconhece um epitopo linear comum presente na estrutura de duas proteínas superficiais de M. hyopnuemoniae, as quais possuem pesos moleculares de 46 e 100 kDa. O índice de avidez do MAb 4A2 foi de 0.5 M de tiocionato de amônia e reações cruzadas com Glaesserella parasuis não foram evidenciadas pelo ensaio de Dot Assay. A análise de citometria de fluxo mostrou a capacidade do MAb 4A2 de reconhecer três diferentes cepas de M. hyopneumoniae, no entanto, a cepa ATCC®25617TM foi significativamente (p<0.01) menos reconhecidas que as cepas ATCC®25095TM e ATCC 25088. Posteriormente, utilizamos o MAb 4A2 para desenhar um moderno ELISA baseado em antígenos corretamente orientados para detectar IgG de suínos contra M. hyopneumoniae. Nossos resultados preliminares monstraram que este ELISA tem maior sensibilidade em comparação com um ELISA comercial globalmente utilizado para a detecção sorológia de IgGs anti M. hyopneumoniae; consequentemente, nosso ELISA possui melhor capacidade de detectar animais com baixos niveis de IgG reativas ao microrganismo. Este novo ELISA consiste numa promissora plataforma de diagnóstico ultra-sensível para detectar anticorpos contra Mycoplasma hyopneumoniae.

Development of a novel antigen-oriented ELISA for Mycoplasma hyopneumoniae diagnosis Author: Rafaela Luiza Klein Advisor: Rafael Frandoloso Swine enzootic pneumonia (EP) is an infectious disease globally disseminated in the pig industry. EP is caused by Mycoplasma hyopneumoniae, a pig-specific bacterium that can be transmitted through direct and indirect. The disease affects pigs of all ages, but the clinical manifestation is commonly observed in the pig growth and finishing phase. M. hyopnuemoniae can produce pulmonary disease independently, however, its infection facilitates the occurrence of other secondary infections that can causing pneumonia and systemic diseases. The diagnosis of M. hyopneumoniae circulation is performed through serological tests in combination with molecular techniques, and the its prevention is carrying out mainly by the use of classic vaccines applied by the intramuscular or intradermal route. Serological diagnosis has a pivotal role for the correct positioning of vaccines based on the understanding of the kinetics of natural antibodies acquired through colostrum, or even, developed after an infection process. So, develop a genuine immunological test capable of detecting M. hyopenumoniae is essential for Brazil. In this dissertation, we present the development, characterization and use of a new monoclonal antibody to design a novel ELISA based on oriented-antigen to detect antibodies reactive to Mycoplasma hyopneumoniae. Monoclonal antibody (MAb) against Mycoplasma hyopneumoniae was obtained by the fusion of P3x63Ag8.653 murine myeloma cells and spleen cells from BALB/c mice immunized with a suspension of M. hyopneumoniae strain ATCC®25095TM. One MAb showed strong reactivity in ELISA and was further characterized using Western blot, Dot Assay, Flow Cytometry and Avidity test. MAb 4A2 is an IgG1 paired with a lambda light chain molecule that recognize a common linear epitope displayed on the structure of two proteins with a molecular mass of 46 and 100 kDa expressed on the surface of M. hyopneumoniae. The avidity index of MAb 4A2 was 0.5 M of ammonium thiocyanate and cross-reactivity with Glaesserella parasuis by Dot Assay was not observed. Flow cytometry analysis showed the capacity of MAb to recognize three different strains of M. hyopneumoniae, however, the strain ATCC®25617TM was significantly less recognized than ATCC®25095TM and ATCC 25088. Further we used the MAb 4A2 to designing a novel antigen-oriented ELISA to detect pig IgG against M. hyopneumoniae. Our preliminary results showed that this ELISA has higher sensitivity than a current commercial ELISA Kit and consequently, better capacity to detect animals with low level of IgG against this microorganism. This novel ELISA is a promising and reliable serological diagnostic for M. hyopneumoniae.

Immunologic cross-reactivity between Muscovy duck parvovirus and goose parvovirus on the basis of epitope prediction

Through bioinformatic prediction, between Muscovy duck parvovirus (MDPV) and goose parvovirus (GPV), there were one epitope AA503-509 (RANEPKE) on non-structural protein and three epitopes AA426-430 (SQDLD), 540-544 (DPYRS), 685-691 (KENSKRW) on structural protein might cross-react with each other. Furthermore, the four epitops were expressed in Escherichia coli. All the four recombinant proteins could react with GPV-antisera and MDPV-antisera in Western blot. (AU)