Desenvolvimento de métodos para o diagnóstico e diferenciação dos pestivírus

Os pestivírus compreendem agentes causais de doenças que têm um impacto econômico negativo para a agropecuária no mundo. O gênero Pestivirus compreende três espécies, nomeadas de acordo com o hospedeiro do qual eles foram isolados: o vírus da peste suína clássica (CSFV), o vírus da diarréia vírica dos bovínos (BVDV) e o vírus da doença da fronteira (BDV) dos ovínos. Esta tese teve como objetivos gerais o desenvolvimento e aplicação de testes para o diagnóstico e diferenciação dos pestivírus. O Artigo 1 descreveu o desenho e a utilização de um teste de transcrição reversa seguida de reação em cadeia pela polimerase (RT-PCR) que permitiu a detecção e a diferenciação do CSFV dos pestivírus de ruminantes através do tamanho do fragmento amplificado de DNA. Foi amplificado um segmento de DNA de 200 pares de bases (pb), quando se utilizou material proveniente de culturas celulares infectadas com dez linhagens do CSFV, e de 260 pb, quando se utilizou material proveniente de culturas celulares infectadas com 23 linhagens do BVDV e duas linhagens do BDV. A especificidade dos fragmentos amplificados foi confirmada por clivagem com enzimas de restrição. A sensibilidade foi igual ou um pouco menor do que o teste de peroxidase ligada à anticorpos. O Artigo 2 descreveu o estabelecimento de um teste de ELISA para a detecção de anticorpos anti-BVDV em soros de bovínos e a sua utilização na determinação da freqüência de bovínos soropositivos para o BVDV em 19 propriedades do Rio Grande do Sul e uma da Argentina. Também foi sequenciada a região 5' não traduzida do genoma de isolados da mesma região para a determinação dos tipos do BVDV presentes nesta área. O método de preparo do antígeno e o formato do ELISA descrito foram mais simples do que os descritos na literatura. O ELISA desenvolvido possui uma especificidade de 99,2% e uma sensibilidade de 97,5% quando comparado ao teste de soro neutralização (SN). A freqüência de 56% ± 15,1% dos animais com anticorpos antiBVDV foi similar aos valores encontrados na literatura para outros países. A análise dos genótipos indicou que existem BVDV de tipo I e de tipo II nesta região. O Anexo 1 descreveu a caracterização dos genes e a produção e purificação das proteínas Em\ E2 e NS3 da linhagem R1935/72 do BVDV no sistema de baculovírus, e sua utilização em testes preliminares de ELISA para detectar anticorpos anti-BVDV no soro de bovinos. Os testes de ELISA desenvolvidos com as proteínas recombinantes foram comparados com o que utiliza extratos de células infectadas como antígeno (Artigo 2). O seqüenciamento dos genes que codificam para as proteínas Erns, E2 e NS3 da linhagem R1935/72 demonstrou que esta linhagem é um BVDV de tipo I. Resultados preliminares em um ELISA indireto indicaram que as três proteínas recombinantes poderão ser utilizadas em testes para a detecção de anticorpos anti-BVDV em soros de bovinos.Pestiviruses represent causative agents of diseases that have a significant negative financial impact for the livestock industry worldwide. The genus Pestivirus comprises three species named according to the host from which they were isolated and where they cause disease: classical swine fever virus (CSFV), bovine viral diarrhea virus (BVDV), and border disease vírus (BDV) of sheep. This thesis had the general objective of developing and applying tests for the diagnosis and differentiation of pestiviruses. Article 1 described a reverse transcription followed by polymerase chain reaction test designed to detect and differentiate CSFV from ruminant pestivirus by the expected size of the amplified fragments of DNA. CSFV infected cultures (10 strains) amplified a fragment of an expected size of 200 bp; BVDV cultures (23 strains) or BDV (2 strains) amplified a fragment of an expected size of 260 bp. The specificity of the amplified fragments was confirmed by restriction enzyme analysis. The threshold of sensitivity was 100 TCID₅₀ for CSFV and 1 TCID₅₀ for BVDV. Article 2 described an ELISA based on cell culture antigens of bovine viral diarrhea virus (BVDV) was designed to detect anti-BVDV antibodies. This ELISA has a sensitivity of 97.5% and specificity of 99.2% compared to serum neutralization. Sera from 430 adult cattle on 19 farms of the State of Rio Grande do Sul (Brazil) and 1 farm from Corrientes (Argentina) were tested in the ELISA. The resulting prevalence of 56% ± 15,1% corresponds to reports for other countries. Two isolates of BVDV from the same region were amplified in the 5' untranslated region of the genome by Reverse Transcription followed by Polymerase Chain Reaction (RT-PCR). The amplification products were sequenced and compared to available Pestivirus sequence data. The phylogenetic analyses showed that BVDV type I and type II are present in these area. Annex 1 describes the characterization of the genes and the production and purification of the proteins Ems, E2 and NS3 from strain R1935/72 of BVDV in the baculovirus system in order to use them in ELISA tests to detect anti-BVDV antibodies from cattle. The ELISA tests developed with the recombinant proteins were compared with the ELISA test from Article 2. The sequences obtained from genes to proteins Ems, E2 and NS3 showed that strain R1935/72 is a BVDV type I. Preliminary results in indirect ELISAS indicate that the three recombinant proteins can be used in tests for the detection of antibody positive cattle to BVDV

Detecção de Salmonella sp. em emas (Rhea americana) através de suabes cloacais

A ema ( Rhea americana) é uma ratita nativa da América do Sul que está sendo criada comercialmente em fazendas no sul do Brasil pela sua carne e penas. Devido ao potencial que as aves têm de transmitir sorovares de Salmonella sp capazes de causar toxinfecção alimentar, torna-se fundamental conhecer o estado sanitário das emas em relação a este patógeno. Com este objetivo, procurou-se verificar a viabilidade da utilização do suabe cloacal para o isolamento de Salmonella sp. em emas. Vísceras e suabes cloacais foram coletadas de 26 aves abatidas em um frigorífico no Rio Grande do Sul. Utilizando-se o isolamento em fígado e/ou ceco como método diagnóstico padrão, determinou-se alta especificidade (80%) e valor preditivo positivo (90,91%); porém, verificou-se baixa sensibilidade (47,62%) e valor preditivo negativo (26,7%). Das 11 cepas isoladas a partir de suabes, duas (18,2%) foram identificadas como S. enterica enterica rugosa, três (27,3%) como S. Newport e seis (54,5%) como S. Typhimurium. Através desse estudo, verificou-se que a utilização de suabe cloacal para detecção de Salmonella sp. em emas deve ser criteriosa, uma vez que o número de falsos negativos é elevado

Caracterização de Salmonella typhimurium isoladas de suínos no Rio Grande do Sul por fagotipificação, perfil de resistência a antimicrobianos e REP-PCR

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Influência do nível de aminoácidos sulfurados (AAS) na dieta de frangos de corte submetidos a estímulo imunológico

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Perfil de resistência antimicrobiana de amostras de Salmonella typhimirium isoladas em emas (Rhea americana) abatidas no Rio Grande do Sul

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Phylogenetic Analysis: Basic Concepts and Its Use as a Tool for Virology and Molecular Epidemiology

Background: Phylogenetic analyses are an essential part in the exploratory assessment of nucleic acid and amino acid sequences. Particularly in virology, they are able to delineate the evolution and epidemiology of disease etiologic agents and/or the evolutionary path of their hosts. The objective of this review is to help researchers who want to use phylogenetic analyses as a tool in virology and molecular epidemiology studies, presenting the most commonly used methodologies, describing the importance of the different techniques, their peculiar vocabulary and some examples of their use in virology.Review: This article starts presenting basic concepts of molecular epidemiology and molecular evolution, emphasizing their relevance in the context of viral infectious diseases. It presents a session on the vocabulary relevant to the subject, bringing readers to a minimum level of knowledge needed throughout this literature review. Within its main subject, the text explains what a molecular phylogenetic analysis is, starting from a multiple alignment of nucleotide or amino acid sequences. The different software used to perform multiple alignments may apply different algorithms. To build a phylogeny based on amino acid or nucleotide sequences it is necessary to produce a data matrix based on a model for nucleotide or amino acid replacement, also called evolutionary model. There are a number of evolutionary models available, varying in complexity according to the number of parameters (transition, transversion, GC content, nucleotide position in the codon, among others). Some papers presented herein provide techniques that can be used to choose evolutionary models. After the model is chosen, the next step is to opt for a phylogenetic reconstruction method that best fits the available data and the selected model. Here we present the most common reconstruction methods currently used, describing their principles, advantages and disadvantages. Distance methods, for example, are simpler and faster, however, they do not provide reliable estimations when the sequences are highly divergent. The accuracy of the analysis with probabilistic models (neighbour joining, maximum likelihood and bayesian inference) strongly depends on the adherence of the actual data to the chosen development model. Finally, we also explore topology confidence tests, especially the most used one, the bootstrap. To assist the reader, this review presents figures to explain specific situations discussed in the text and numerous examples of previously published scientific articles in virology that demonstrate the importance of the techniques discussed herein, as well as their judicious use.Conclusion: The DNA sequence is not only a record of phylogeny and divergence times, but also keeps signs of how the evolutionary process has shaped its history and also the elapsed time in the evolutionary process of the population. Analyses of genomic sequences by molecular phylogeny have demonstrated a broad spectrum of applications. It is important to note that for the different available data and different purposes of phylogenies, reconstruction methods and evolutionary models should be wisely chosen. This review provides theoretical basis for the choice of evolutionary models and phylogenetic reconstruction methods best suited to each situation. In addition, it presents examples of diverse applications of molecular phylogeny in virology

Comparision of different cell cultures for replication of infectious laryngotracheitis virus from chickens

The propagation of infectious laryngotracheitis virus (ILTV) has been described using primary cell cultures derived from chicken embryo liver and kidney or embryonated eggs, but these cultures use Specific Pathogen Free (SPF) eggs that are time and cost expensive. Since cell line cultures are easier to maintain in laboratory conditions, the growth of ILTV was evaluated in five different cell cultures: chicken embryo related cells (CER), a cell hybrid derived from chicken embryo fibroblasts cells and BHK-21; Vero, from African green monkey kidney cells; HD11, a chicken macrophage cell line; CEC-32, an avian fibroblast cell line and a primary cell culture of chicken embryo fibroblasts (CEF). Cytophatic effect was observed until 96 hours following inoculation and the detection of the viral DNA was performed by PCR. The HD11 and CEC-32 cell lines did not support the virus growth but CEF and Vero, as already described were permissive cultures for propagation of ILTV.The results also showed that the CER cell line can be used for primary isolation and replication of ILTV

Prevalência de suínos portadores de Salmonella sp. ao abate e contaminação de embutidos tipo frescal

O abate de suínos portadores de  Salmonella sp. é considerado o primeiro ponto crítico para a contaminação do produto final. O risco representado por esses animais tende a aumentar quando a bactéria está presente em porções da carcaça que chegam até o consumidor. No presente estudo, buscou-se verificar a associação da prevalência de suínos portadores de Salmonella sp. ao abate e a contaminação da massa utilizada na fabricação de embutidos tipo frescal, produzida com matériaprima proveniente destes animais. Numa primeira etapa, foram realizadas três visitas a um frigorífico, onde foram coletados “pools” de linfonodos submandibulares/tonsilas (LT) e conteúdo intestinal (CI) de 16 animais em cada oportunidade. No dia subseqüente ao abate, foram coletadas 99 porções da massa, produzida com a carne dos animais abatidos, imediatamente antes do embutimento. Encontrou-se uma prevalência média de 83,33% dos suínos portadores de Salmonella sp. ao abate, enquanto que 93,94% das amostras de massa de embutimento foram positivas. Os sorovares mais freqüentemente isolados foram Panama, Bredeney e Typhimurium. Numa segunda etapa, quantificou-se a bactéria em amostras positivas para Salmonella sp. Duas amostras, escolhidas aleatoriamente na quarta coleta, apresentaram 93 NMP/g e 150 NMP/g de S. Bredeney. Por outro lado, na quinta coleta, as duas amostras escolhidas apresentara

Use of a modified AFLP protocol to discriminate Salmonella enterica subsp.enterica serovar Enteritidis isolates

Salmonella enterica  subsp. enterica (S.) serovar Enteritidis is one of the main pathogens involved in food-borne diseases worldwide. In epidemiological investigations of food-related salmonellosis, subtyping is necessary to improve preventive and control measures. Single-enzyme amplified fragment length polymorphism (SE-AFLP) analysis is a modified AFLP that uses only one restriction enzyme to produce DNA fragments that are selectively amplified by PCR. In order to assess the applicability of SE-AFLP in S. Enteritidis typing, one hundred and eight strains isolated from poultry, swine and also from human salmonellosis outbreaks in Southern Brazil were analyzed. Strains from other countries and six different S. enterica serovars were also included as controls. SE-AFLP was able to distinguish S. Enteritidis from the other S. enterica serovars analyzed. However, most of S. Enteritidis strains isolated from poultry, salmonellosis outbreaks and most of the strains from other countries shared the same predominant pattern. The low genetic diversity identified in S. Enteritidis suggests that the strains analyzed are clonally related and one predominant SE-AFLP genotype is widely spread in Southern Brazil

In vitro method to evaluate virus competition between BVDV-1 and BVDV-2 strains using the PrimeFlow RNA assay

Bovine viral diarrhea viruses (BVDV), segregated in BVDV-1 and BVDV-2 species, lead to substantial economic losses to the cattle industry worldwide. It has been hypothesized that there could be differences in level of replication, pathogenesis and tissue tropism between BVDV-1 and BVDV-2 strains. Thus, this study developed an in vitro method to evaluate virus competition between BVDV-1 and BVDV-2 strains. To this end the competitive dynamics of BVDV-1a, BVDV-1b, and BVDV-2a strains in cell cultures was evaluated by a PrimeFlow RNA assay. Similar results were observed in this study, as was observed in an earlier in vivo transmission study. Competitive exclusion was observed as the BVDV-2a strains dominated and excluded the BVDV-1a and BVDV-1b strains. The in vitro model developed can be used to identify viral variations that result in differences in frequency of subgenotypes detected in the field, vaccine failure, pathogenesis, and strain dependent variation in immune responses