Pathogenic variability among Pasteurella multocida type A isolates from Brazilian pig farms.

BACKGROUND: Pasteurella multocida type A (PmA) is considered a secondary agent of pneumonia in pigs. The role of PmA as a primary pathogen was investigated by challenging pigs with eight field strains isolated from pneumonia and serositis in six Brazilian states. Eight groups of eight pigs each were intranasally inoculated with different strains of PmA (1.5 mL/nostril of 10e7 CFU/mL). The control group (n = 12) received sterile PBS. The pigs were euthanized by electrocution and necropsied by 5 dpi. Macroscopic lesions were recorded, and swabs and fragments of thoracic and abdominal organs were analyzed by bacteriological and pathological assays. The PmA strains were analyzed for four virulence genes (toxA: toxin; pfhA: adhesion; tbpA and hgbB: iron acquisition) by PCR and sequencing and submitted to multilocus sequence typing (MLST). RESULTS: The eight PmA strains were classified as follows: five as highly pathogenic (HP) for causing necrotic bronchopneumonia and diffuse fibrinous pleuritis and pericarditis; one as low pathogenic for causing only focal bronchopneumonia; and two as nonpathogenic because they did not cause injury to any pig. PCR for the gene pfhA was positive for all five HP isolates. Sequencing demonstrated that the pfhA region of the HP strains comprised four genes: tpsB1, pfhA1, tpsB2 and pfhA2. The low and nonpathogenic strains did not contain the genes tpsB2 and pfhA2. A deletion of four bases was observed in the pfhA gene in the low pathogenic strain, and an insertion of 37 kb of phage DNA was observed in the nonpathogenic strains. MLST clustered the HP isolates in one group and the low and nonpathogenic isolates in another. Only the nonpathogenic isolates matched sequence type 10; the other isolates did not match any type available in the MLST database. CONCLUSIONS: The hypothesis that some PmA strains are primary pathogens and cause disease in pigs without any co-factor was confirmed. The pfhA region, comprising the genes tpsB1, tpsB2, pfhA1 and pfhA2, is related to the pathogenicity of PmA. The HP strains can cause necrotic bronchopneumonia, fibrinous pleuritis and pericarditis in pigs and can be identified by PCR amplification of the gene pfhA2.

Extracellular Proteins of Mycoplasma synoviae.

Mycoplasma synoviae is a Gram positive bacteria lacking of cell wall that affects chickens and turkeys causing infection in the upper respiratory tract and in some cases arthritis, with economical impact to broiler breeders. Treatment and prevention of avian synovitis depend on knowledge of the infectious process. Secreted or surface-exposed proteins play a critical role in disease because they often mediate interactions between host and pathogen. In the present work, we sought to identify possible M. synoviae secreted proteins by cultivating the bacteria in a modified protein-free Frey medium. Using this approach, we were able to detect in the cell-free fraction a number of proteins that have been shown in other organisms to be secreted, suggesting that they may also be secreted by M. synoviae.

Antibacterial activity of vegetal extracts against serovars of Salmonella / Atividade antibacteriana de extratos vegetais sobre sorovares de Salmonella

in vitro antibacterial activity of 21 hydroethanolic vegetal extracts was assessed against 20 serovars of Salmonella. Regarding the tested extracts, 85.7 percent of them presented antibacterial activity. The six active extracts which showed activity on the largest number of serovars and the extract of Eucalyptus sp. were submitted to the determination of Minimum Inhibitory Concentration (MIC) and Minimum Bactericidal Concentration (MBC). Of these, six extracts showed bacteriostatic and bactericidal activity with MIC and MBC for Punica granatum (pomegranate) from 20 and 60mg mL-1, for Eugenia jambolana (rose apple) from 40 and 240mg mL-1, Eugenia uniflora (surinam cherry) from 80 and 240mg mL-1, Caryophyllus aromaticus (clove) from 10 and 60mg mL-1, Psidium araca from 30 and 320mg mL-1 and Eucalyptus sp. from 40 and 160mg mL-1. Achyrocline satureioides (macela) presented only bacteriostatic potential and MIC from 160mg mL-1. Caryophyllus aromaticus, Eucalyptus sp., and Psidium araca presented the best results for bactericidal activity, inhibiting, respectively, 84.2 percent, 42.1 percent, and 17.6 percent of Salmonella's serovars. The activity of each extract varied for different serovars; S. London presented resistance to the six extracts in MBC, while S. Pullorum was the most susceptible serovar.

A atividade antibacteriana de 21 extratos hidroetanólicos vegetais foi avaliada in vitro frente a 20 sorovares de Salmonella. Dos extratos testados, 85,7 por cento apresentaram atividade antibacteriana. Os seis extratos que evidenciaram atividade sobre o maior número de sorovares e Eucalyptus sp. foram submetidos à determinação da Concentração Inibitória Mínima (CIM) e Concentração Bactericida Mínima (CBM). Destes, seis extratos apresentaram atividade bacteriostática e bactericida com MIC para Punica granatum (romã) a partir de 20 e 60mg mL-1, Eugenia jambolana (jambolão) de 40 e 240mg mL-1, Eugenia uniflora (pitanga) de 80 e 240mg mL-1, Caryophyllus aromaticus (cravo) de 10 e 60mg mL-1, Psidium araca (araçá) 30 e 320mg mL-1 e Eucalyptus sp. (eucalipto) de 40 e 160mg mL-1. Achyrocline satureioides (macela) apresentou apenas atividade bacteriostática e MIC a partir de 160mg mL-1. Caryophyllus aromaticus, Eucalyptus sp. e Psidium araca apresentaram os melhores resultados para a atividade bactericida, inativando, respectivamente, 84,21 por cento, 42,1 por cento e 17,64 por cento dos sorovares de Salmonella. A atividade de cada extrato variou para diferentes sorovares. Nenhum dos seis extratos avaliados evidenciou atividade bactericida frente a S. London, enquanto S. Pullorum foi o sorovar mais sensível.

Ocurrence of staphylococcus aureus and multiplex pcr detection of classic enterotoxin genes in cheese and meat products / Ocorrência de Staphylococcus aureus e detecção por PCR multiplex de genes de enterotoxinas clássicas em queijo e derivados cárneos

Multiplex PCR was used to investigate the presence of enterotoxins genes (sea, seb, sec, sed and see) and femA gene (specific for Staphylococcus aureus) in coagulase-positive staphylococci (CPS) isolated from cheese and meat products. From 102 CPS isolates, 91 were positive for femA, 10 for sea, 12 for sed and four for see.

PCR multiplex foi empregado para investigar a presença de genes de enterotoxinas estafilocócicas (sea, seb, sec, sed e see) e do gene femA, específico para S.aureus, em cepas de estafilococos coagulase positiva (ECP) isoladas de queijos e derivados cárneos. De 102 cepas, 91 foram positivas para femA, 10 para sea, 12 para sed e 4 para see.

Ocurrence of Staphylococcus aureus and multiplex pcr detection of classic enterotoxin genes in cheese and meat products.

Multiplex PCR was used to investigate the presence of enterotoxins genes (sea, seb, sec, sed and see) and femA gene (specific for Staphylococcus aureus) in coagulase-positive staphylococci (CPS) isolated from cheese and meat products. From 102 CPS isolates, 91 were positive for femA, 10 for sea, 12 for sed and four for see.

Otimização da técnica da PCR para a detecção de Actinobacillus pleuropneumoniae / Optimization of PCR technique for detection of Actinobacillus pleuropneumoniae

A utilização de métodos moleculares baseados em PCR é fundamental na detecção do Actinobacillus pleuropneumoniae, sendo capaz de identificar a infecção antes do estabelecimento da doença no rebanho. Estes métodos apresentam maior sensibilidade quando comparados com métodos tradicionais de isolamento bacteriano, mas podem sofrer influência de substâncias que reduzem a especificidade do teste e proporcionam o aparecimento de amplificações inespecíficas. No intuito de reduzir as amplificações inespecíficas, observadas quando aplicada a PCR para o gene cpx em amostras de tecido tonsilar, procedeu-se a otimização da técnica, na qual foram analisados o efeito do pré-cultivo bacteriano e as diferentes temperaturas de anelamento dos iniciadores e foi introduzido, no protocolo, um anticorpo que se liga na enzima Taq DNA Polimerase, aumentando a especificidade do teste. Paralelamente, foi realizado um experimento para verificar o efeito inibidor do tecido tonsilar sobre os resultados da PCR. Para isso, porções de tonsila de animais negativos para A. pleuropneumoniae foram contaminadas artificialmente com a amostra referência do sorotipo 5B. A adição do anticorpo para a enzima Taq DNA Polimerase e o aumento da temperatura de anelamento dos iniciadores para 57ºC diminuiu o aparecimento de amplificações inespecíficas. Os resultados obtidos no experimento demonstraram que o tecido tonsilar possui efeito inibidor nas amplificações da PCR. Além disso, a amplificação depende de, no mínimo, 675 UFC presentes na alíquota da amostra usada na PCR (equivalente a 1,35 x 10(5) UFC mL-1), assim, amostras de fragmentos de tecido de infecções iniciais e/ou com poucas células podem apresentar resultados falsos negativos.

The use of molecular methods based on PCR is important in Actinobacillus pleuropneumoniae detection, being able to identify the infection before the establishment of the disease in the herd. These methods have larger sensitivity when compared with traditional methods of bacteriological isolation, but they can suffer influence of substances that reduce the specificity of the test and resulting in inespecific amplifications. In order to reduce inespecific amplifications, observed when applied the PCR technique for the gene cpx in tonsil's tissue samples, the optimization was performed, in which different annealing temperatures were analyzed and introduced, in the technique, an antibody that binds to the enzyme Taq DNA Polimerase, increasing its specificity. In parallel, an experiment was performed in order to verify the inhibiting effect of the tonsil's tissue on the PCR results. For that, portions of tonsil from animals negative to the A. pleuropneumoniae were artificially contaminated with the reference sample of the sorotype 5B. The addition antibody for the enzyme Taq DNA Polimerase and the increase of the primers anneling temperature to 57ºC reduced the inespecific amplifications. The results obtained in the experiment demonstrated a possible inhibiting effect of the tonsil's tissue in the PCR amplifications. Besides, amplifications depend on at least 675 UFC present in the aliquot of samples that will be used in PCR (equivalent to 1.35 x 10(5) UFC mL-1), therefore, samples tissue's fragments in initial infections and/or with few cells can result in false-negative.

Reação em Cadeia da Polimerase (PCR) baseada no gene cpx para detecção de Actinobacillus pleuropneumoniae em suínos natural e experimentalmente infectados / Polymerase Chain Reaction (PCR) based on the cpx gene for detection of Actinobacillus pleuropneumoniae in natural and experimentally infected pigs

A pleuropneumonia suína é uma das mais importantes doenças respiratórias dos suínos, estando presente em todos os países produtores. Para o controle e o monitoramento da pleuropneumonia, é necessário o desenvolvimento de métodos rápidos e acurados de diagnóstico. Com o objetivo de validar a técnica da PCR, baseada no gene cpx de Actinobacillus pleuropneumoniae, em suínos sabidamente positivos, primeiramente foi realizada inoculação experimental com amostras de A. pleuropneumoniae sorotipo 5B e coletadas amostras por meio de suabe de tonsila, biópsia de tonsila e sangue para realização da técnica de PCR, isolamento bacteriológico e teste de ELISA, respectivamente. Posteriormente, estas técnicas foram aplicadas em suínos naturalmente infectados, em três rebanhos com diferentes situações sanitárias quanto à apresentação clínica da doença. De cada rebanho, foram analisados cinco grupos de suínos com idades diferentes, sendo coletado de cada animal biópsia de tonsila para isolamento bacteriológico e PCR e sangue para determinação do perfil sorológico. Os resultados obtidos na inoculação experimental confirmaram que, mesmo com o estabelecimento da infecção comprovada pelo isolamento bacteriológico, após o período de 45 dias, não foi possível detectar o agente pela técnica de PCR. Em animais naturalmente infectados, a técnica de PCR apresentou maior sensibilidade quando comparado com o isolamento. A associação entre PCR e ELISA demonstrou ser uma boa alternativa para definir a situação sanitária do rebanho quanto à infecção por A. pleuropneumoniae.

Swine pleuropneumonia is one of the most important pig respiratory diseases and has been found in all producer countries. For control and monitoring of pleuropneumonia, it is necessary the development of fast and specific methods of diagnosis. To validate PCR based on the cpx gene of Actinobacillus pleuropneumoniae in positive pigs, an experimental infection with A. pleuropneumoniae serotype 5B was performed and samples were obtained by tonsil swab, tonsil biopsy and blood for PCR, bacterial isolation and ELISA, respectively. These tests were then performed in naturally infected pigs from three herds with different sanitary situations of clinical disease. In each herd, five groups of different ages were analyzed. Tonsil biopsy for bacterial isolation and PCR and blood to determine the herd serological status was collected. The results obtained in the experimental infection confirmed that, even with the infection establishment, proved with bacterial isolation, it was not possible to detect the agent by PCR 45 days after infection. In naturally infected animals, PCR was more sensitive than bacterial isolation. The association between PCR and ELISA is a good alternative to define the herd sanitary status regarding the infection with A. pleuropneumoniae.

Aspectos fenotípicos, genotípicos e de diagnóstico da bactéria A. pleuropneumoniae / Phenotypic, genotypic and diagnostic aspects of the bacterium A. pleuropneumoniae

A pleuropneumonia suína (PPS) provoca prejuízos significativos na suinocultura no mundo. O agente etiológico é a bactéria Actinobacillus pleuropneumoniae (App), que apresenta 15 sorotipos descritos, os quais variam consideravelmente em relação a sua patogenicidade. Nesse sentido, a precisa caracterização patotípica desta bactéria é de grande importância para a adoção de medidas de controle e profilaxia. O diagnóstico e a sorotipificação deste patógeno são realizados pelas técnicas microbiológicas convencionais. Entretanto, problemas nestes esquemas podem ser observados, especialmente em isolados de rebanhos sem histórico de PPS. No Brasil, diversos esforços vêm sendo aplicados no sentido de desenvolver técnicas moleculares que auxiliem no diagnóstico da infecção crônica ocasionada por este agente, principalmente em rebanhos presumidamente sadios e com infecção subclínica. Nesta revisão, são discutidos os resultados obtidos na caracterização de isolados de A. pleuropneumoniae e espécies relacionadas provenientes tanto de suínos com PPS, como de animais presumidamente isentos da infecção. Apresentamos, ainda, perspectivas para o desenvolvimento de metodologias que possibilitem o diagnóstico precoce e a melhor compreensão dos mecanismos de virulência deste patógeno.

Evaluation of PCR based on gene apxIVA associated with 16S rDNA sequencing for the identification of Actinobacillus pleuropneumoniae and related species.

The pleuropneumonia caused by Actinobacillus pleuropneumoniae (App) is one the most important swine respiratory diseases. Biochemical and serological tests are widely applied for App diagnosis and characterization. However, in some isolates, conflicting results are found. The present work focus on the characterization of 29 isolates biochemically classified as A. pleuropneumoniae, collected from swine in herds with or without a clinical history of pleuropneumonia. Sixteen isolates were from healthy swine, initially classified as nonserotypable A. pleuropneumoniae; they displayed differences in the molecular characterization patterns of App (genes cpx and apxI, II, and III). Those bacteria that could not be serotyped were submitted to rDNA 16S sequencing. All 29 isolates were analyzed by PCR for the presence of the apxIVA gene. Thirteen isolates (45%) were confirmed to be A. pleuropneumoniae by PCR, nine being from diseased animals (31%) and four from healthy animals (14%) with conclusive serotyping. The rDNA 16S sequencing was used to classify the other 16 isolates in related species other than A. pleuropneumoniae, resulting in eleven A. minor, three A. porcinus, and two Pasteurella sp. Because of conflicting results between biochemical tests and rDNA 16S sequencing, the biochemical characterization was repeated, and the new results were in agreement with the rDNA 16S sequencing data. Biochemical characterization proved to be efficient for the majority of the A. pleuropneumoniae isolates. Nevertheless, conventional tests can render conflicting results, and other methodologies, such as amplification of A. pleuropneumoniae specific apxIVA gene and rDNA 16S sequencing, are very useful for improved classification. We also observed a great variety in rDNA 16S sequences from different A. minor isolates.

Detection of Actinobacillus pleuropneumoniae by PCR on field strains from healthy and diseased pigs.

We investigated whether primers able to specifically amplify a 0.7-kb DNA fragment from the conserved cpx genes could be applied to analyze A. pleuropneumoniae field isolates. The specific cpx primers were tested on 120 strains of A. pleuropneumoniae and other NAD-dependent field isolates from healthy and diseased animals to analyze A. pleuropneumoniae isolates from pigs in Brazil. We found that PCR and hybridization were able to discriminate between isolates of A. pleuropneumoniae and other bacteria. The 0.7-kb cpx DNA fragments were amplified from all 63 A. pleuropneumoniae isolates from herds with clinical symptoms and were isolated from lesions of acute cases of swine pleuropneumonia, both serotypable and nonserotypable. The PCR was also applied to 57 field isolates obtained from animals of apparently healthy herds, and the amplified cpx product was present in four serotypable and only two out of eleven A. pleuropneumoniae nonserotypable isolates. All nonserotypable A. pleuropneumoniae isolates revealed the apxA amplification pattern compatible with previously known serotypes. Some nonserotypable isolates might represent a population of isolates that originally were serotypable but lost the ability to react with serotype-specific antisera or might belong to novel serotypes. The PCR method applied is highly sensitive for serotypable A. pleuropneumoniae strains and for nonserotypable strains isolated from acute cases of swine pleuropneumoniae in Brazil.