RESUMO

Background: The pleuropneumonia caused by Actinobacillus pleuropneumoniae is one the most important swine respiratory diseases. Biochemical and serological tests are widely applied for the diagnosis and characterization of this bacterium. However, in some isolates, confl icting results are found. There are at least 15 serotypes with signifi cant differencesin virulence that have been identifi ed until now. Moreover, cross reactions between serotypes are not uncommon. Theserotype determination from isolates occurring in outbreaks is an important procedure in prophylaxis and control of thedisease. The present work focuses on the application of an ERIC-PCR technique for genotyping and differentiation ofA. pleuropneumoniae isolates.Materials, Methods & Results: Fifteen reference strains for the recognized A. pleuropneumoniae serotypes were analyzedin this work, alongside with 27 fi eld isolates that had been previously characterized regarding biochemical, serological andmolecular features. Total DNA from each sample was purifi ed and subjected to PCR amplifi cation using ERIC-specifi cprimers (ERIC1R and ERIC2). The resulting amplicons were analyzed by agarose gel electrophoresis and their sizes wereestimated from the gel images. Bands with similar sizes were identifi ed and used to construct a binary matrix that tookinto account the presence or absence of individual bands in all lanes. Pair-wise similarity coeffi cients were computed fromthe binary matrix and the similarity matrices obtained were utilized to construct an UPGMA-based dendrogram. The amplicons obtained from the A. pleuropneumoniae reference strains generated a very distinctive pattern for each one of thetested strains. Moreover, all samples presented a large enough number of amplicons (bands) as to enable an unequivocaldifferentiation of each sample. Reproducibility of the developed ERIC-PCR method was assessed by means of duplicate...

Assuntos

RESUMO

Background: The pleuropneumonia caused by Actinobacillus pleuropneumoniae is one the most important swine respiratory diseases. Biochemical and serological tests are widely applied for the diagnosis and characterization of this bacterium. However, in some isolates, confl icting results are found. There are at least 15 serotypes with signifi cant differencesin virulence that have been identifi ed until now. Moreover, cross reactions between serotypes are not uncommon. Theserotype determination from isolates occurring in outbreaks is an important procedure in prophylaxis and control of thedisease. The present work focuses on the application of an ERIC-PCR technique for genotyping and differentiation ofA. pleuropneumoniae isolates.Materials, Methods & Results: Fifteen reference strains for the recognized A. pleuropneumoniae serotypes were analyzedin this work, alongside with 27 fi eld isolates that had been previously characterized regarding biochemical, serological andmolecular features. Total DNA from each sample was purifi ed and subjected to PCR amplifi cation using ERIC-specifi cprimers (ERIC1R and ERIC2). The resulting amplicons were analyzed by agarose gel electrophoresis and their sizes wereestimated from the gel images. Bands with similar sizes were identifi ed and used to construct a binary matrix that tookinto account the presence or absence of individual bands in all lanes. Pair-wise similarity coeffi cients were computed fromthe binary matrix and the similarity matrices obtained were utilized to construct an UPGMA-based dendrogram. The amplicons obtained from the A. pleuropneumoniae reference strains generated a very distinctive pattern for each one of thetested strains. Moreover, all samples presented a large enough number of amplicons (bands) as to enable an unequivocaldifferentiation of each sample. Reproducibility of the developed ERIC-PCR method was assessed by means of duplicate...(AU)

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Background: The infection with Mycoplasma (M.) hyopneumoniae is a major cause of pneumonia in pigs worldwide, but the interaction between M. hyopneumoniae and Pasteurella (P.) multocida has been described with low frequency and histopathological characteristics of this association have not been described. In Brazil this interaction is considered very important and is found in most cases of pigs with pneumonia and pleuritis at slaughter. The objective of this work was to examine 41 samples of swine lungs collected at slaughter, from which P. multocida had been isolated and to study the co-infection with M. hyopneumoniae by histopathology and immunohistochemistry (IHC). Materials, Methods & Results: Among lung tissue samples obtained from necropsy in swine with clinical signs of pneumonia, 41 that had shown positive results on bacteriological examination for P. multocida were selected. The histopathological findings of bronchopneumonia were classified in different degrees according to the extension of the lesion as follow: absent (0), mild (+), moderate (+ +) and severe (+ + +). Hyperplasia of bronchial and bronchiolar epithelium was classified as absent (0) and present (+). The bronchus associated lymphoid tissue (BALT) hyperplasia lesions were classified as absent (0), (+) when the peribronchial and peribronchiolar lymphocytic infiltrate were discrete, (+ +) when the peribronchial and peribronchiolar lymphocytic infiltrate was moderate and/or the presence of a few lymphoid nodules; (+ + +) highest amount of lymphoid nodules, and (+ + ++) when a large amount of lymphoid nodules compressing the bronchi and bronchioles were observed. All samples were submitted to IHC technique for M. hyopneumoniae p-36 protein. The main findings in histological examination were BALT hyperplasia (97.6%), purulent bronchopneumonia (92.7%) and bronchial and bronchiolar epithelial hyperplasia (51.2%). BALT hyperplasia was mild (+) in 43.9%; moderate (++) in 31.7%; accentuated (+++) in 12.2%; accentuated with bronchium and bronchiole obstruction (++++) in 9.8% and absent (-) in 2.4%. The classification of purulent bronchopneumonia was moderate (++) in 36.6%; accentuated (+++) in 34.1%; mild (+) in 22.0% and absentee (-) in 7.3%. Lesions compatible with a combined infection between P. multocida and M. hyopneumoniae were found in 97.6% of the samples by histopathology and in 61.0% by IHC. Discussion: There was a high frequency of lesions consistent with infection by M. hyopneumoniae in samples of lungs from which P. multocida was isolated, being detected at the same time by histopathology in 97.6% of samples, and 61% by IHC. However, 15 samples (39.0%) had microscopic lesions of BALT hyperplasia in different degrees and/or epithelial hyperplasia with no labeling in IHC M. hyopneumoniae. This could be due to the absence of the agent in chronic infections. On histopathological examination, the presence of BALT hyperplasia is considered very suggestive of infection with M. hyopneumoniae. In the study only one lung (2.3%) did not have this injury, and did not show labeling in IHC. Among samples positive for P. multocida in bacteriological examination, only three showed no lesion suggestive of the infection with the agent in histopathology, which could be due to a recent bacterial infection or its presence as a commensal in the lungs. Collectively, bacteriological examination, histopathology and IHC confirmed the co-infection between M. hyopneumoniae and P. multocida in 97.2% of pneumonic lungs analyzed, suggesting the importance of the relationship between the agents in Brazil. Moreover, it was possible to determine the histopathological and immunohistochemical characteristics of the association between both bacteria.

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Scrapie is a transmissible spongiform encephalopathy of sheeps and goats, associated with the deposition of a isoform of the prion protein (PrPsc). This isoform presents an altered conformation that leads to aggregation in the host's central nervous and lymphoreticular systems. Predisposition to the prion agent infection can be influenced by specific genotypes related to mutations in amino acids of the PrPsc gene. The most characterized mutations occur at codons 136, 154 and 171, with genotypes VRQ being the most susceptible and ARR the most resistant. In this study we have analyzed polymorphisms in 15 different codons of the PrPsc gene in sheeps from a Suffolk herd from Brazil affected by an outbreak of classical scrapie. Amplicons from the PrPsc gene, encompassing the most relevant altered codons in the protein, were sequenced in order to determine each animal's genotype. We have found polymorphisms at 3 of the 15 analyzed codons (136, 143 and 171). The most variable codon was 171, where all described alleles were identified. A rare polymorphism was found at the 143 codon in 4 percent of the samples analyzed, which has been described as increasing scrapie resistance in otherwise susceptible animals. No other polymorphisms were detected in the remaining 12 analyzed codons, all of them corresponding to the wild-type prion protein. Regarding the risk degree of developing scrapie, most of the animals (96 percent) had genotypes corresponding to risk groups 1 to 3 (very low to moderate), with only 4 percent in the higher risks group. Our data is discussed in relation to preventive measures involving genotyping and positive selection to control the disease.(AU)

Scrapie é uma encefalopatia espongiforme transmissível de ovinos e caprinos, associado a deposição da isoforma da proteína priônica (PrPsc). Essa isoforma apresenta uma alteração conformacional que leva ao acúmulo da proteína no sistema nervoso central e linforeticular do hospedeiro. A predisposição a infecção pelo agente priônico pode ser influenciado por genótipos específicos relacionados a mutações na sequência de aminoácidos do gene PrPsc. As principais mutações caracterizadas ocorrem nos códons 136, 154 e 171, sendo o genótipo VRQ o mais suscetível e o genótipo ARR o mais resistente. Nesse estudo nós analisamos os polimorfismos de 15 códons diferentes da gene PrPsc em ovinos de um rebanho da raça Suffolk no Brasil afetado com scrapie clássico. Os amplicons do gene da PrPsc, que contem os códons mais frequentemente encontrados foram sequenciados para determinar o genótipo de cada animal. Nós encontramos 3 polimorfismos do 15 códons analisados (136, 143 e 171). O códon que mais teve variações foi o códon 171, onde todos os alelos foram identificados. Um polimorfismo raro foi encontrado no códon 143, em 4 por cento das amostras analisadas, o qual tem sido descrito por aumentar a resistência a scrapie em animais suscetíveis. Nenhum outro polimorfismo foi detectado nos 12 códons restantes, todos então, correspondendo à proteína priônica selvagem. De acordo com a grau de risco a desenvolver scrapie, a maioria dos animais (96 por cento) tiveram genótipo correspondentes aos grupos de risco 1 a 3 (muito baixo a moderado), e somente 4 por cento no grupo de risco alto. Nossos dados discutem a relação das medidas de prevenção envolvendo a genotipagem e a seleção positiva para o controle da doença.(AU)

Assuntos

RESUMO

Scrapie is a transmissible spongiform encephalopathy of sheeps and goats, associated with the deposition of a isoform of the prion protein (PrPsc). This isoform presents an altered conformation that leads to aggregation in the host's central nervous and lymphoreticular systems. Predisposition to the prion agent infection can be influenced by specific genotypes related to mutations in amino acids of the PrPsc gene. The most characterized mutations occur at codons 136, 154 and 171, with genotypes VRQ being the most susceptible and ARR the most resistant. In this study we have analyzed polymorphisms in 15 different codons of the PrPsc gene in sheeps from a Suffolk herd from Brazil affected by an outbreak of classical scrapie. Amplicons from the PrPsc gene, encompassing the most relevant altered codons in the protein, were sequenced in order to determine each animal's genotype. We have found polymorphisms at 3 of the 15 analyzed codons (136, 143 and 171). The most variable codon was 171, where all described alleles were identified. A rare polymorphism was found at the 143 codon in 4 percent of the samples analyzed, which has been described as increasing scrapie resistance in otherwise susceptible animals. No other polymorphisms were detected in the remaining 12 analyzed codons, all of them corresponding to the wild-type prion protein. Regarding the risk degree of developing scrapie, most of the animals (96 percent) had genotypes corresponding to risk groups 1 to 3 (very low to moderate), with only 4 percent in the higher risks group. Our data is discussed in relation to preventive measures involving genotyping and positive selection to control the disease.

Scrapie é uma encefalopatia espongiforme transmissível de ovinos e caprinos, associado a deposição da isoforma da proteína priônica (PrPsc). Essa isoforma apresenta uma alteração conformacional que leva ao acúmulo da proteína no sistema nervoso central e linforeticular do hospedeiro. A predisposição a infecção pelo agente priônico pode ser influenciado por genótipos específicos relacionados a mutações na sequência de aminoácidos do gene PrPsc. As principais mutações caracterizadas ocorrem nos códons 136, 154 e 171, sendo o genótipo VRQ o mais suscetível e o genótipo ARR o mais resistente. Nesse estudo nós analisamos os polimorfismos de 15 códons diferentes da gene PrPsc em ovinos de um rebanho da raça Suffolk no Brasil afetado com scrapie clássico. Os amplicons do gene da PrPsc, que contem os códons mais frequentemente encontrados foram sequenciados para determinar o genótipo de cada animal. Nós encontramos 3 polimorfismos do 15 códons analisados (136, 143 e 171). O códon que mais teve variações foi o códon 171, onde todos os alelos foram identificados. Um polimorfismo raro foi encontrado no códon 143, em 4 por cento das amostras analisadas, o qual tem sido descrito por aumentar a resistência a scrapie em animais suscetíveis. Nenhum outro polimorfismo foi detectado nos 12 códons restantes, todos então, correspondendo à proteína priônica selvagem. De acordo com a grau de risco a desenvolver scrapie, a maioria dos animais (96 por cento) tiveram genótipo correspondentes aos grupos de risco 1 a 3 (muito baixo a moderado), e somente 4 por cento no grupo de risco alto. Nossos dados discutem a relação das medidas de prevenção envolvendo a genotipagem e a seleção positiva para o controle da doença.

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Samples of intestine with necrotic enteritis from 63 pigs naturally infected with porcine circovirus type 2 (PCV2) were studied. Colon was the main target of PCV2 associated necrotic enteritis in 60 cases. Immunohistological investigations were carried out to detect the presence of PCV2 in necrotic lesions and to identify the type of cells infected by the virus. Crypt epithelial cells had positive labelling for PCV2 in 17 cases. Depletion of goblet cells occurred in 10 cases. In 24 necrotic enteritis cases, co-infection of PCV2 and Salmonella was identified. An increased rate of apoptosis in the crypt epithelial cells of the large intestine from PCV2 of naturally infected pigs was observed. Immunohistochemical findings confirmed the presence of PCV2 within cells from necrotic intestinal tissue, suggesting that PCV2 may play a role in the development of those lesions. Diagnosis of necrotic enteritis associated with PCV2 should be based on the detection of PCV2 antigen or DNA in the necrotizing lesions. However, bacteriological examination should be performed to rule out the presence of bacterial agents, since co-infections are likely to occur in PCV2 affected pigs.

Foram selecionadas amostras intestinais com enterite necrótica de 63 suínos naturalmente infectados pelo circovírus suíno tipo 2 (PCV2). Enterite necrótica associada com PCV2 ocorreu principalmente no cólon, em 60 casos. Análise imuno-histoquímica foi realizada para identificar a presença de PCV2 em lesões necróticas e o tipo de células infectadas pelo vírus. Células epiteliais das criptas apresentaram marcação positiva para PCV2 em 17 casos. Depleção de células caliciformes ocorreu em 10 casos. Em 24 casos de enterite necrótica, observou-se co-infecção por PCV2 e Salmonella. Foi observado um aumento no índice de apoptose nas células das criptas do intestino grosso de suínos naturalmente infectados com PCV2. Os achados imuno-histoquímicos e histopatológicos sugerem que a infecção por PCV2 das células do tecido intestinal pode ocasionar enterite necrótica. O diagnóstico de enterite necrótica associada com PCV2 deve ser baseado na detecção do antígeno ou do DNA viral nas lesões necróticas. Contudo, análise bacteriológica deve ser realizada para descartar a presença de agente bacteriano, já que co-infecções são comuns.

RESUMO

Mycoplasma hyopneumoniae is the etiological agent of porcine enzootic pneumonia (PEP), a major problem for the pig industry. The mechanisms of M. hyopneumoniae pathogenicity allow to predict the existence of several classes of virulence factors, whose study has been essentially restricted to the characterization of adhesion-related and major antigenic proteins. The now available complete sequences of the genomes of two pathogenic and one non-pathogenic strain of M. hyopneumoniae allowed to use a comparative genomics approach to putatively identify virulence genes. In this preliminary survey, we were able to identify 118 CDSs encoding putative virulence factors, based on specific criteria ranging from predicted cell surface location or variation between strains to previous functional studies showing antigenicity or involvement in host-pathogen interaction. This survey is expected to serve as a first step towards the functional characterization of new virulence genes/proteins that will be important not only for a better comprehension of M. hyopneumoniae biology, but also for the development of new and improved protocols for PEP vaccination, diagnosis and treatment.