Molecular serotyping of clinical strains of Haemophilus (Glaesserella) parasuis brings new insights regarding Glässer's disease outbreaks in Brazil.

Glässer's disease (GD) is an important infectious disease of swine caused by Haemophilus (Glaesserella) parasuis. Vaccination with inactivated whole cell vaccines is the major approach for prevention of H. parasuis infection worldwide, but the immunity induced is predominantly against the specific polysaccharide capsule. As a consequence, the available vaccines may not induce adequate protection against the field strains, when the capsules present in the vaccine strains are different from those in strains isolated from the farms. Therefore, it is crucial to map H. parasuis serovars associated with regional outbreaks so that appropriate bacterin vaccines can be developed and distributed for prevention of infection. In this study, 459 H. parasuis field strains isolated from different Glässer's disease outbreaks that occurred in 10 different Brazilian States were analyzed for serotype using PCR-based approaches. Surprisingly, non-typeable (NT) strains were the second most prevalent group of field strains and along with serovars 4, 5 and 1 comprised more than 70% of the isolates. A PCR-based approach designed to amplify the entire polysaccharide capsule locus revealed 9 different band patterns in the NT strains, and 75% of the NT strains belonged to three clusters, suggesting that a number of new serovars are responsible for a substantial proportion of disease. These results indicate that commercially available vaccines in Brazil do not cover the most prevalent H. parasuis serovars associated with GD.

Antimicrobial susceptibility profile of historical and recent Brazilian pig isolates of Pasteurella multocida / Perfil de suscetibilidade a antimicrobianos de isolados históricos e contemporâneos de Pasteurella multocida de suínos no Brasil

Pasteurella (P.) multocida is the causative agent of pneumonic pasteurellosis in swine, which is commonly associated with the final stages of enzootic pneumonia or porcine respiratory disease complex. Although this syndrome is one of the most common and important diseases of pigs, data on antimicrobial susceptibility of P. multocida isolates are uncommon in Brazil. Therefore, the present study was carried out to determine and to compare antimicrobial susceptibility profile of Brazilian P. multocida isolated from pigs with lesions of pneumonia or pleuritis during two-time periods. Historical isolates (period of 1981 to 1997; n=44) and recent isolates (period of 2011 to 2012; n=50) were used to determine the MIC of amoxicillin, enrofloxacin, florfenicol and tetracycline by microbroth dilution. Florfenicol had the lowest level of resistance for both historical and recent isolates (0% and 6%, respectively), while tetracycline had the highest (20.5% and 34%, respectively). Multi-drug resistance (MDR) to amoxicillin/florfenicol/tetracycline was observed in 6% of recent isolates. There was a significant increase (p˂0.05) in resistance for amoxicillin and enrofloxacin in recent isolates compared with historic isolates (3.8% and 18%, respectively), most likely due to the selective pressure of antimicrobial usage to treat and prevent P. multocida infections. The results of this study showed an increase of isolates resistant to important drugs used in treatment of P. multocida infections in pigs, demonstrating the need for the implementation of rational use of antimicrobials in Brazilian swine industry.(AU)

Pasteurella (P.) multocida é o agente da pasteurelose pneumônica em suínos, a qual é comumente associada com o estágio final da pneumonia enzoótica suína ou complexo das doenças respiratórias dos suínos. Apesar de ser uma das doenças mais comuns e importantes na suinocultura, dados sobre suscetibilidade antimicrobiana de isolados de P. multocida são raros no Brasil. Dessa forma, o presente estudo foi realizado para determinar e comparar o perfil de suscetibilidade de isolados de P. multocida de suínos com lesões de pneumonia ou pleurite no Brasil durante dois períodos. Isolados históricos (período de 1981 a 1997; n=44) e contemporâneos (período de 2011 a 2012; n=50) foram usados para determinar a concentração inibitória mínima (CIM) de amoxicilina, enrofloxacina, florfenicol e tetraciclina através do teste de microdiluição em caldo. Florfenicol apresentou o menor nível de resistência para ambos os isolados históricos e contemporâneos (0% e 6%, respectivamente), enquanto que tetraciclina apresentou o maior nível de resistência (20.5% e 34%, respectivamente). Resistência a múltiplos antimicrobianos (amoxicilina, florfenicol e tetraciclina) foi observada em 6% dos isolados recentes. Foi observado aumento significativo (p˂0.05) na resistência a amoxicilina e enrofloxacina em isolados recentes comparado com isolados históricos (3.8% e 18%, respectivamente), provavelmente devido à pressão de seleção de antimicrobianos usados no tratamento e prevenção de infecções causadas por P. multocida. Os resultados deste trabalho demostraram o aumento de isolados resistentes a importantes drogas utilizadas no tratamento de infecções causadas por P. multocida em suínos, evidenciando a necessidade da implementação do uso racional de antimicrobianos na suinocultura brasileira.(AU)

Antimicrobial susceptibility profile of historical and recent Brazilian pig isolates of Pasteurella multocida / Perfil de suscetibilidade a antimicrobianos de isolados históricos e contemporâneos de Pasteurella multocida de suínos no Brasil

Pasteurella (P.) multocida is the causative agent of pneumonic pasteurellosis in swine, which is commonly associated with the final stages of enzootic pneumonia or porcine respiratory disease complex. Although this syndrome is one of the most common and important diseases of pigs, data on antimicrobial susceptibility of P. multocida isolates are uncommon in Brazil. Therefore, the present study was carried out to determine and to compare antimicrobial susceptibility profile of Brazilian P. multocida isolated from pigs with lesions of pneumonia or pleuritis during two-time periods. Historical isolates (period of 1981 to 1997; n=44) and recent isolates (period of 2011 to 2012; n=50) were used to determine the MIC of amoxicillin, enrofloxacin, florfenicol and tetracycline by microbroth dilution. Florfenicol had the lowest level of resistance for both historical and recent isolates (0% and 6%, respectively), while tetracycline had the highest (20.5% and 34%, respectively). Multi-drug resistance (MDR) to amoxicillin/florfenicol/tetracycline was observed in 6% of recent isolates. There was a significant increase (p˂0.05) in resistance for amoxicillin and enrofloxacin in recent isolates compared with historic isolates (3.8% and 18%, respectively), most likely due to the selective pressure of antimicrobial usage to treat and prevent P. multocida infections. The results of this study showed an increase of isolates resistant to important drugs used in treatment of P. multocida infections in pigs, demonstrating the need for the implementation of rational use of antimicrobials in Brazilian swine industry.(AU)

Pasteurella (P.) multocida é o agente da pasteurelose pneumônica em suínos, a qual é comumente associada com o estágio final da pneumonia enzoótica suína ou complexo das doenças respiratórias dos suínos. Apesar de ser uma das doenças mais comuns e importantes na suinocultura, dados sobre suscetibilidade antimicrobiana de isolados de P. multocida são raros no Brasil. Dessa forma, o presente estudo foi realizado para determinar e comparar o perfil de suscetibilidade de isolados de P. multocida de suínos com lesões de pneumonia ou pleurite no Brasil durante dois períodos. Isolados históricos (período de 1981 a 1997; n=44) e contemporâneos (período de 2011 a 2012; n=50) foram usados para determinar a concentração inibitória mínima (CIM) de amoxicilina, enrofloxacina, florfenicol e tetraciclina através do teste de microdiluição em caldo. Florfenicol apresentou o menor nível de resistência para ambos os isolados históricos e contemporâneos (0% e 6%, respectivamente), enquanto que tetraciclina apresentou o maior nível de resistência (20.5% e 34%, respectivamente). Resistência a múltiplos antimicrobianos (amoxicilina, florfenicol e tetraciclina) foi observada em 6% dos isolados recentes. Foi observado aumento significativo (p˂0.05) na resistência a amoxicilina e enrofloxacina em isolados recentes comparado com isolados históricos (3.8% e 18%, respectivamente), provavelmente devido à pressão de seleção de antimicrobianos usados no tratamento e prevenção de infecções causadas por P. multocida. Os resultados deste trabalho demostraram o aumento de isolados resistentes a importantes drogas utilizadas no tratamento de infecções causadas por P. multocida em suínos, evidenciando a necessidade da implementação do uso racional de antimicrobianos na suinocultura brasileira.(AU)

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.

Comparison of phenotypic and genotypic identification methods of Pasteurella multocida serotypes isolated from pigs

Background: Pasteurella multocida serotypes A and D are commonly associated with pneumonia and pleuritis in pigs. Different phenotypic techniques, such as hyaluronidase and acriflavine tests, and genotyping techniques, such as PCR, are used to distinguish between these serotypes. The objective of this study was to compare the capsular identification methods of type A and type D P. multocida isolated from pigs using both phenotypic (hyaluronidase and acriflavine tests) and genotypic (multiplex PCR) techniques. Materials, Methods & Results: A total of 44 lyophilized P. multocida isolates, obtained between 1981 and 1997 from pig farms at Rio Grande do Sul State, Brazil, were analyzed. The isolates were reactivated in Brain Heart Infusion (BHI) broth and cultured in BHI broth and blood agar supplemented with 5% sheep blood. Colony identity was further confirmed by evaluating colony morphology in blood agar and confirming the absence of growth on MacConkey agar. Bacteria in Tryptone Soy Agar (TSA) were used for the Triple Sugar Iron (TSI), Sulfide-Indole-Motility (SIM), and nitrate, glucose, lactose, sucrose and mannitol fermentation tests. For hyaluronidase test, P. multocida colonies were streaked transversally across the entire plate, approximately 3-5mm apart, in order to observe their lines of growth. Following this, a hyaluronidase producing strain of Staphylococcus aureus[...]

Comparison of phenotypic and genotypic identification methods of Pasteurella multocida serotypes isolated from pigs

Background: Pasteurella multocida serotypes A and D are commonly associated with pneumonia and pleuritis in pigs. Different phenotypic techniques, such as hyaluronidase and acriflavine tests, and genotyping techniques, such as PCR, are used to distinguish between these serotypes. The objective of this study was to compare the capsular identification methods of type A and type D P. multocida isolated from pigs using both phenotypic (hyaluronidase and acriflavine tests) and genotypic (multiplex PCR) techniques. Materials, Methods & Results: A total of 44 lyophilized P. multocida isolates, obtained between 1981 and 1997 from pig farms at Rio Grande do Sul State, Brazil, were analyzed. The isolates were reactivated in Brain Heart Infusion (BHI) broth and cultured in BHI broth and blood agar supplemented with 5% sheep blood. Colony identity was further confirmed by evaluating colony morphology in blood agar and confirming the absence of growth on MacConkey agar. Bacteria in Tryptone Soy Agar (TSA) were used for the Triple Sugar Iron (TSI), Sulfide-Indole-Motility (SIM), and nitrate, glucose, lactose, sucrose and mannitol fermentation tests. For hyaluronidase test, P. multocida colonies were streaked transversally across the entire plate, approximately 3-5mm apart, in order to observe their lines of growth. Following this, a hyaluronidase producing strain of Staphylococcus aureus[...](AU)

Aspectos patológicos e microbiológicos das doenças respiratórias em suínos de terminação no Brasil / Pathological and microbiological aspects of respiratory disease in fattening pigs in Brazil

Para avaliação dos aspectos patológicos e microbiológicos de casos clínicos de doenças respiratórias em suínos de terminação foram analisados 75 suínos doentes oriundos de 36 lotes. Suínos que apresentavam sinais clínicos respiratórios evidentes foram necropsiados para avaliação macroscópica e colheita de amostras para análise histopatológica e microbiológica. Foram realizados testes de isolamento bacteriano para as principais bactérias do sistema respiratório dos suínos, PCR para Mycoplasma hyorhinis, imuno-histoquímica para Influenza A, Circovirus suíno tipo 2 e Mycoplasma hyopneumoniae. A sensibilidade antimicrobiana de 24 amostras de Pasteurella multocida tipo A foi avaliada por testes de concentração inibitória mínima para os principais antimicrobianos utilizados em suinocultura. Mycoplasma hyopneumoniae e Pasteurella multocida tipo A foram os agentes infecciosos mais prevalentes. Broncopneumonia supurativa e pleurite foram as principais lesões respiratórias encontradas. Pasteurella multocida tipo A, quando presente, aumentou a extensão das lesões pulmonares. Todas as amostras de Pasteurella multocida testadas foram sensíveis aos antimicrobianos Doxiciclina, Enrofloxacina e Tilmicosina. Em 58% das amostras foi identificado mais de um agente infeccioso, evidenciando a alta prevalência da associação de agentes nas doenças respiratórias de suínos em terminação.(AU)

For pathological and microbiological evaluation of porcine respiratory disease in fattening pigs, seventy five animals showing respiratory distress, fever and/or cough were analyzed. These pigs were necropsied and samples were collected for histological and microbiological analysis. Bacterial isolation procedures were performed aiming to detect major swine bacterial respiratory pathogens. Also, PCR for Mycoplasma hyorhinis, and immunohistochemistry for Influenza A, porcine circovirus type 2, and Mycoplasma hyopneumoniae were carried out. Mycoplasma hyopneumoniae and Pasteurella multocida type A were the most prevalent infectious agents. The antimicrobial sensitivity of 24 samples of P. multocida type A was evaluated by minimum inhibitory concentration tests and all these samples were sensitive to doxycycline, tilmicosin and enrofloxacin. Suppurative bronchopneumonia and pleuritis were main respiratory lesions found. When P. multocida type A was present, the extension of lung lesions was increased. In 58% of the samples more than one infectious agent was identified, suggesting a high prevalence of infectious agents associations in porcine respiratory disease in Brazil.(AU)

Pasteurella multocida type A as the primary agent of pneumonia and septicaemia in pigs / Pasteurella multocida tipo A como agente primário de pneumonia e septicemia em suínos

In order to understand better the pathological aspects and spread of Pasteurella multocida type A as the primary cause of pneumonia in pigs, was made an experiment with intranasal inoculation of different concentrations of inocula [Group (G1): 108 Colony Forming Units (CFU)/ml; G2: 107 CFU/ml; G3: 106 CFU/ml and G4: 105 CFU/ml], using two pigs per group. The pigs were obtained from a high health status herd. Pigs were monitored clinically for 4 days and subsequently necropsied. All pigs had clinical signs and lesions associated with respiratory disease. Dyspnoea and hyperthermia were the main clinical signs observed. Suppurative cranioventral bronchopneumonia, in some cases associated with necrosuppurative pleuropneumonia, fibrinous pericarditis and pleuritic, were the most frequent types of lesion found. The disease evolved with septicaemia, characterized by septic infarctions in the liver and spleen, with the detection of P. multocida type A. In this study, P. multocida type A strain #11246 was the primary agent of fibrinous pleuritis and suppurative cranioventral bronchopneumonia, pericarditis and septicaemia in the pigs. All concentrations of inoculum used (105-108 CFU/ml) were able to produce clinical and pathological changes of pneumonia, pleuritis, pericarditis and septicemia in challenged animals.(AU)

Para entender melhor os aspectos patológicos e disseminação de Pasteurella multocida tipo A como causa primária de pneumonia em suínos foi realizado um experimento com inoculação intranasal de diferentes concentrações de inóculos [Grupo (G1): 108 Unidades Formadoras de Colônias (UFC)/ml; G2: 107 UFC/ml; G3: 106 UFC/ml e G4: 105 UFC/ml], usando dois suínos por grupo. Esses suínos foram obtidos de um rebanho de alto status sanitário. Os animais foram monitorados clinicamente por quarto dias e subsequentemente necropsiados. Todos os suínos apresentaram sinais clínicos e lesões associadas com doença respiratória. Dispneia e hipertermia foram os principais sinais clínicos observados. Broncopneumonia cranioventral supurativa, em alguns casos associados com pleuropneumonia necrossupurativa, pleurites e pericardite fibrinosa foram mais frequentes. A doença evoluiu com septicemia, caracterizada por infartos sépticos no fígado e baço, com detecção de P. multocida. Neste estudo, P. multocida tipo A isolado 11246 foi agente primário de pleurite fibrinosa e broncopneumonia cranioventral supurativa, pericardite fibrinosa e septicemia em suínos. Todas as concentrações de inóculo utilizado (105-108 UFC/ml) foram capazes de produzir sinais clínicos e patológicos de alterações de pneumonia, pleurites, pericardites e septicemia nos animais.(AU)

Pasteurella multocida type A as the primary agent of pneumonia and septicaemia in pigs / Pasteurella multocida tipo A como agente primário de pneumonia e septicemia em suínos

In order to understand better the pathological aspects and spread of Pasteurella multocida type A as the primary cause of pneumonia in pigs, was made an experiment with intranasal inoculation of different concentrations of inocula [Group (G1): 108 Colony Forming Units (CFU)/ml; G2: 107 CFU/ml; G3: 106 CFU/ml and G4: 105 CFU/ml], using two pigs per group. The pigs were obtained from a high health status herd. Pigs were monitored clinically for 4 days and subsequently necropsied. All pigs had clinical signs and lesions associated with respiratory disease. Dyspnoea and hyperthermia were the main clinical signs observed. Suppurative cranioventral bronchopneumonia, in some cases associated with necrosuppurative pleuropneumonia, fibrinous pericarditis and pleuritic, were the most frequent types of lesion found. The disease evolved with septicaemia, characterized by septic infarctions in the liver and spleen, with the detection of P. multocida type A. In this study, P. multocida type A strain #11246 was the primary agent of fibrinous pleuritis and suppurative cranioventral bronchopneumonia, pericarditis and septicaemia in the pigs. All concentrations of inoculum used (105-108 CFU/ml) were able to produce clinical and pathological changes of pneumonia, pleuritis, pericarditis and septicemia in challenged animals...

Para entender melhor os aspectos patológicos e disseminação de Pasteurella multocida tipo A como causa primária de pneumonia em suínos foi realizado um experimento com inoculação intranasal de diferentes concentrações de inóculos [Grupo (G1): 108 Unidades Formadoras de Colônias (UFC)/ml; G2: 107 UFC/ml; G3: 106 UFC/ml e G4: 105 UFC/ml], usando dois suínos por grupo. Esses suínos foram obtidos de um rebanho de alto status sanitário. Os animais foram monitorados clinicamente por quarto dias e subsequentemente necropsiados. Todos os suínos apresentaram sinais clínicos e lesões associadas com doença respiratória. Dispneia e hipertermia foram os principais sinais clínicos observados. Broncopneumonia cranioventral supurativa, em alguns casos associados com pleuropneumonia necrossupurativa, pleurites e pericardite fibrinosa foram mais frequentes. A doença evoluiu com septicemia, caracterizada por infartos sépticos no fígado e baço, com detecção de P. multocida. Neste estudo, P. multocida tipo A isolado 11246 foi agente primário de pleurite fibrinosa e broncopneumonia cranioventral supurativa, pericardite fibrinosa e septicemia em suínos. Todas as concentrações de inóculo utilizado (105-108 UFC/ml) foram capazes de produzir sinais clínicos e patológicos de alterações de pneumonia, pleurites, pericardites e septicemia nos animais...

Aspectos patológicos e microbiológicos das doenças respiratórias em suínos de terminação no Brasil / Pathological and microbiological aspects of respiratory disease in fattening pigs in Brazil

Para avaliação dos aspectos patológicos e microbiológicos de casos clínicos de doenças respiratórias em suínos de terminação foram analisados 75 suínos doentes oriundos de 36 lotes. Suínos que apresentavam sinais clínicos respiratórios evidentes foram necropsiados para avaliação macroscópica e colheita de amostras para análise histopatológica e microbiológica. Foram realizados testes de isolamento bacteriano para as principais bactérias do sistema respiratório dos suínos, PCR para Mycoplasma hyorhinis, imuno-histoquímica para Influenza A, Circovirus suíno tipo 2 e Mycoplasma hyopneumoniae. A sensibilidade antimicrobiana de 24 amostras de Pasteurella multocida tipo A foi avaliada por testes de concentração inibitória mínima para os principais antimicrobianos utilizados em suinocultura. Mycoplasma hyopneumoniae e Pasteurella multocida tipo A foram os agentes infecciosos mais prevalentes. Broncopneumonia supurativa e pleurite foram as principais lesões respiratórias encontradas. Pasteurella multocida tipo A, quando presente, aumentou a extensão das lesões pulmonares. Todas as amostras de Pasteurella multocida testadas foram sensíveis aos antimicrobianos Doxiciclina, Enrofloxacina e Tilmicosina. Em 58% das amostras foi identificado mais de um agente infeccioso, evidenciando a alta prevalência da associação de agentes nas doenças respiratórias de suínos em terminação...

For pathological and microbiological evaluation of porcine respiratory disease in fattening pigs, seventy five animals showing respiratory distress, fever and/or cough were analyzed. These pigs were necropsied and samples were collected for histological and microbiological analysis. Bacterial isolation procedures were performed aiming to detect major swine bacterial respiratory pathogens. Also, PCR for Mycoplasma hyorhinis, and immunohistochemistry for Influenza A, porcine circovirus type 2, and Mycoplasma hyopneumoniae were carried out. Mycoplasma hyopneumoniae and Pasteurella multocida type A were the most prevalent infectious agents. The antimicrobial sensitivity of 24 samples of P. multocida type A was evaluated by minimum inhibitory concentration tests and all these samples were sensitive to doxycycline, tilmicosin and enrofloxacin. Suppurative bronchopneumonia and pleuritis were main respiratory lesions found. When P. multocida type A was present, the extension of lung lesions was increased. In 58% of the samples more than one infectious agent was identified, suggesting a high prevalence of infectious agents associations in porcine respiratory disease in Brazil...

Detection of agents associated with respiratory diseases of swine by real time PCR / Detecção de agentes associados com doenças respiratórias de suínos por PCR em tempo real

Porcine Respiratory Disease Complex (PRDC) is a group of diseases that cause high losses in the swine industry. Several infectious agents are related to PRDC including porcine circovirus 2 (PCV-2), pseudorabies virus (SuHV-1), Haemophilus parasuis (HP), Mycoplasma hypneumoniae (MH) and Pasteurela multocida (PM). The aim of this study was to develop real-time PCRs (qPCR) for the detection of these infectious agents. Oligonucleotides were designed for each specific infectious agent and labeled with different fluorophores to amplify specific parts of the genome. This was done in two groups of reactionsa duplex qPCR for SuHV-1 and PCV-2 and a multiplex qPCR to detect the three bacteria simultaneously. The reactions were tested in 142 pooled samples of swine lymph nodes and lungs with clinical signs of PRDC. There were 135 samples that tested positive for PCV-2, 61 for HP, 29 for PM, 30 for MH and zero for SuHV-1. We recorded 76 cases of co-infection. The qPCRs developed in this study are useful tools in the diagnosis of PRDC.(AU)

Complexo de Doenças Respiratórias de Suínos (CDRS ) é um grupo de doenças que causam grandes perdas na indústria suína. Vários agentes infecciosos estão relacionados com a CDRS , entre eles o circovírus suíno 2 (PCV -2), vírus da pseudo-raiva (SuHV -1) , Haemophilus parasuis (HP) , Mycoplasma hypneumoniae (MH ) e Pasteurela multocida (PM). O objetivo com este estudo foi desenvolver PCR em tempo real (qPCR) para a detecção destes agentes infecciosos. Os oligonucleotídeos foram concebidos para cada agente infeccioso específico e marcado com fluoróforos diferentes para amplificar partes específicas do genoma em dois grupos de reacções , uma qPCR dúplex em SuHV -1 e PCV- 2 e uma qPCR multiplex para detectar as três bactérias simultaneamente. As reações foram testadas em 142 amostras de pools de linfonodos e pulmões de suínos com sinais clínicos de CDRS. Foram detectadas 135 amostras positivas para PCV- 2 , 61 para a HP, 29 para PM e 30 para MH e zero para SuHV -1, dentre esses foram registrados 76 casos de co-infecção . As qPCRs desenvolvidas neste estudo são ferramentas úteis no diagnóstico da CDRS.(AU)

Detection of agents associated with respiratory diseases of swine by real time PCR / Detecção de agentes associados com doenças respiratórias de suínos por PCR em tempo real

Porcine Respiratory Disease Complex (PRDC) is a group of diseases that cause high losses in the swine industry. Several infectious agents are related to PRDC including porcine circovirus 2 (PCV-2), pseudorabies virus (SuHV-1), Haemophilus parasuis (HP), Mycoplasma hypneumoniae (MH) and Pasteurela multocida (PM). The aim of this study was to develop real-time PCRs (qPCR) for the detection of these infectious agents. Oligonucleotides were designed for each specific infectious agent and labeled with different fluorophores to amplify specific parts of the genome. This was done in two groups of reactionsa duplex qPCR for SuHV-1 and PCV-2 and a multiplex qPCR to detect the three bacteria simultaneously. The reactions were tested in 142 pooled samples of swine lymph nodes and lungs with clinical signs of PRDC. There were 135 samples that tested positive for PCV-2, 61 for HP, 29 for PM, 30 for MH and zero for SuHV-1. We recorded 76 cases of co-infection. The qPCRs developed in this study are useful tools in the diagnosis of PRDC.

Complexo de Doenças Respiratórias de Suínos (CDRS ) é um grupo de doenças que causam grandes perdas na indústria suína. Vários agentes infecciosos estão relacionados com a CDRS , entre eles o circovírus suíno 2 (PCV -2), vírus da pseudo-raiva (SuHV -1) , Haemophilus parasuis (HP) , Mycoplasma hypneumoniae (MH ) e Pasteurela multocida (PM). O objetivo com este estudo foi desenvolver PCR em tempo real (qPCR) para a detecção destes agentes infecciosos. Os oligonucleotídeos foram concebidos para cada agente infeccioso específico e marcado com fluoróforos diferentes para amplificar partes específicas do genoma em dois grupos de reacções , uma qPCR dúplex em SuHV -1 e PCV- 2 e uma qPCR multiplex para detectar as três bactérias simultaneamente. As reações foram testadas em 142 amostras de pools de linfonodos e pulmões de suínos com sinais clínicos de CDRS. Foram detectadas 135 amostras positivas para PCV- 2 , 61 para a HP, 29 para PM e 30 para MH e zero para SuHV -1, dentre esses foram registrados 76 casos de co-infecção . As qPCRs desenvolvidas neste estudo são ferramentas úteis no diagnóstico da CDRS.

Detection of agents associated with respiratory diseases of swine by real time PCR

Porcine Respiratory Disease Complex (PRDC) is a group of diseases that cause high losses in the swine industry. Several infectious agents are related to PRDC including porcine circovirus 2 (PCV-2), pseudorabies virus (SuHV-1),Haemophilus parasuis (HP), Mycoplasma hypneumoniae (MH) and Pasteurela multocida (PM). The aim of this study was to develop real-time PCRs (qPCR) for the detection of these infectious agents. Oligonucleotides were designed for each specific infectious agent and labeled with different fluorophores to amplify specific parts of the genome. This was done in two groups of reactionsa duplex qPCR for SuHV-1 and PCV-2 and a multiplex qPCR to detect the three bacteria simultaneously. The reactions were tested in 142 pooled samples of swine lymph nodes and lungs with clinical signs of PRDC. There were 135 samples that tested positive for PCV-2, 61 for HP, 29 for PM, 30 for MH and zero for SuHV-1. We recorded 76 cases of co-infection. The qPCRs developed in this study are useful tools in the diagnosis of PRDC.

Complexo de Doenças Respiratórias de Suínos (CDRS ) é um grupo de doenças que causam grandes perdas na indústria suína. Vários agentes infecciosos estão relacionados com a CDRS , entre eles o circovírus suíno 2 (PCV -2), vírus da pseudo-raiva (SuHV -1) , Haemophilus parasuis (HP) ,Mycoplasma hypneumoniae (MH ) e Pasteurela multocida (PM). O objetivo com este estudo foi desenvolver PCR em tempo real (qPCR) para a detecção destes agentes infecciosos. Os oligonucleotídeos foram concebidos para cada agente infeccioso específico e marcado com fluoróforos diferentes para amplificar partes específicas do genoma em dois grupos de reacções , uma qPCR dúplex em SuHV -1 e PCV- 2 e uma qPCR multiplex para detectar as três bactérias simultaneamente. As reações foram testadas em 142 amostras de pools de linfonodos e pulmões de suínos com sinais clínicos de CDRS. Foram detectadas 135 amostras positivas para PCV- 2 , 61 para a HP, 29 para PM e 30 para MH e zero para SuHV -1, dentre esses foram registrados 76 casos de co-infecção . As qPCRs desenvolvidas neste estudo são ferramentas úteis no diagnóstico da CDRS.

Detection of agents associated with respiratory diseases of swine by real time PCR

Porcine Respiratory Disease Complex (PRDC) is a group of diseases that cause high losses in the swine industry. Several infectious agents are related to PRDC including porcine circovirus 2 (PCV-2), pseudorabies virus (SuHV-1),Haemophilus parasuis (HP), Mycoplasma hypneumoniae (MH) and Pasteurela multocida (PM). The aim of this study was to develop real-time PCRs (qPCR) for the detection of these infectious agents. Oligonucleotides were designed for each specific infectious agent and labeled with different fluorophores to amplify specific parts of the genome. This was done in two groups of reactionsa duplex qPCR for SuHV-1 and PCV-2 and a multiplex qPCR to detect the three bacteria simultaneously. The reactions were tested in 142 pooled samples of swine lymph nodes and lungs with clinical signs of PRDC. There were 135 samples that tested positive for PCV-2, 61 for HP, 29 for PM, 30 for MH and zero for SuHV-1. We recorded 76 cases of co-infection. The qPCRs developed in this study are useful tools in the diagnosis of PRDC.

Complexo de Doenças Respiratórias de Suínos (CDRS ) é um grupo de doenças que causam grandes perdas na indústria suína. Vários agentes infecciosos estão relacionados com a CDRS , entre eles o circovírus suíno 2 (PCV -2), vírus da pseudo-raiva (SuHV -1) , Haemophilus parasuis (HP) ,Mycoplasma hypneumoniae (MH ) e Pasteurela multocida (PM). O objetivo com este estudo foi desenvolver PCR em tempo real (qPCR) para a detecção destes agentes infecciosos. Os oligonucleotídeos foram concebidos para cada agente infeccioso específico e marcado com fluoróforos diferentes para amplificar partes específicas do genoma em dois grupos de reacções , uma qPCR dúplex em SuHV -1 e PCV- 2 e uma qPCR multiplex para detectar as três bactérias simultaneamente. As reações foram testadas em 142 amostras de pools de linfonodos e pulmões de suínos com sinais clínicos de CDRS. Foram detectadas 135 amostras positivas para PCV- 2 , 61 para a HP, 29 para PM e 30 para MH e zero para SuHV -1, dentre esses foram registrados 76 casos de co-infecção . As qPCRs desenvolvidas neste estudo são ferramentas úteis no diagnóstico da CDRS.

Padronização da técnica de nanopartícula de ouro não modificada (AuNPs) para detecção de Actinobacillus pleuropneumoniae em pulmões de suínos / Standardization of unmodified gold nanoparticle (AuNPs) for detection of Actinobacillus pleuropneumoniae in swine lungs

Testes diagnósticos baseados na detecção de ácidos nucleicos sem amplificação prévia através da utilização de nanopartículas de ouro (AuNPs) têm sido descritos para várias enfermidades. Este trabalho teve como objetivo desenvolver uma técnica de AuNPs não modificada para detecção de Actinobacillus pleuropneumoniae (App). Utilizaram-se 70 amostras de pulmão de suínos, 17 sem lesão e 53 com lesões características de pneumonia, objetivando a detecção de App. O oligonucleotídeo utilizado foi baseado no gene ApxIV. O teste de AuNPs apresentou sensibilidade de 93,8 por cento e especificidade de 84,6 por cento quando comparado com a detecção pela PCR. Os resultados mostraram boa concordância entre os testes de AuNPs e a PCR, sendo que a técnica pode ser utilizada como alternativa aos testes convencionais, já que é de fácil e rápida execução e não exige infraestrutura e mão de obra especializada.(AU)

Based on diagnostic tests for the detection of nucleic acids without amplification through the use of gold nanoparticles (AuNPs) have been described for various diseases. This study aimed to develop a technique of unmodified AuNPs to detect Actinobacillus pleuropneumoniae (App). We used 70 lung samples from pigs, 17 with and 53 without characteristic lesions of pneumonia, to detect App. The primer used was based on ApxIV gene. The AuNPs test had a sensitivity of 93.8 percent and specificity of 84.6 percent when compared with PCR detection. The results showed good agreement between AuNPs and PCR testing, and the technique can be used as an alternative to conventional tests, since it is quick and easy, and does not require implementation infrastructure and skilled labor.(AU)

Padronização da técnica de nanopartícula de ouro não modificada (AuNPs) para detecção de Actinobacillus pleuropneumoniae em pulmões de suínos / Standardization of unmodified gold nanoparticle (AuNPs) for detection of Actinobacillus pleuropneumoniae in swine lungs

Testes diagnósticos baseados na detecção de ácidos nucleicos sem amplificação prévia através da utilização de nanopartículas de ouro (AuNPs) têm sido descritos para várias enfermidades. Este trabalho teve como objetivo desenvolver uma técnica de AuNPs não modificada para detecção de Actinobacillus pleuropneumoniae (App). Utilizaram-se 70 amostras de pulmão de suínos, 17 sem lesão e 53 com lesões características de pneumonia, objetivando a detecção de App. O oligonucleotídeo utilizado foi baseado no gene ApxIV. O teste de AuNPs apresentou sensibilidade de 93,8 por cento e especificidade de 84,6 por cento quando comparado com a detecção pela PCR. Os resultados mostraram boa concordância entre os testes de AuNPs e a PCR, sendo que a técnica pode ser utilizada como alternativa aos testes convencionais, já que é de fácil e rápida execução e não exige infraestrutura e mão de obra especializada.

Based on diagnostic tests for the detection of nucleic acids without amplification through the use of gold nanoparticles (AuNPs) have been described for various diseases. This study aimed to develop a technique of unmodified AuNPs to detect Actinobacillus pleuropneumoniae (App). We used 70 lung samples from pigs, 17 with and 53 without characteristic lesions of pneumonia, to detect App. The primer used was based on ApxIV gene. The AuNPs test had a sensitivity of 93.8 percent and specificity of 84.6 percent when compared with PCR detection. The results showed good agreement between AuNPs and PCR testing, and the technique can be used as an alternative to conventional tests, since it is quick and easy, and does not require implementation infrastructure and skilled labor.

Immunological characterization of Mycoplasma hyopneumoniae recombinant proteins.

Mycoplasma hyopneumoniae, the primary pathogen of enzootic pneumonia, is highly prevalent worldwide and causes major economic losses to the pig industry. Commercial vaccines are widely used in the control of this disease, however, they provide only partial protection. The aim of this study was to evaluate 34 recombinant proteins of M. hyopneumoniae expressed in Escherichia coli. Antigenic and immunogenic properties of these proteins were analyzed. For this, the proteins were tested against hyperimmune and convalescent pig sera through ELISA and Western blot. Immunogenicity of the recombinant proteins was evaluated in BALB/c mice following intramuscular inoculation. Most antigens were able to induce a strong immune response and sera from inoculated mice were able to recognize native proteins by cell ELISA and Western blot. Several recombinant proteins were specifically recognized by convalescent pig sera, indicating they are expressed during infection. These data may help to develop more efficacious vaccines against M. hyopneumoniae.

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.

Mycoplasma hyopneumoniae type I signal peptidase: expression and evaluation of its diagnostic potential.

Type I signal peptidase (SPase I) is a membrane-anchored protease of the general secretory pathway, which is encoded by the sipS gene in Mycoplasma hyopneumoniae, the etiological agent of porcine enzootic pneumonia (PEP). In this study, the expression of the M. hyopneumoniae SPase I (MhSPase I) was analyzed in virulent and avirulent strains, and the recombinant protein (rMhSPase I), expressed in Escherichia coli, was evaluated regarding its potential as an immunodiagnostic antigen. It was demonstrated that the sipS coding DNA sequence (CDS) is most likely part of an operon, being co-transcribed along with four other CDSs. Quantitative reverse transcriptase PCR and immunoblot assays showed that MhSPase I is expressed by all three strains analyzed, with no transcriptional difference, but with evidence of a higher protein level in a pathogenic strain (7422), in comparison to another pathogenic (7448) and a non-pathogenic (J) strain. rMhSPase I was strongly immunogenic for mice, and the MhSPase I antigenicity was confirmed. Polyclonal serum anti-rMhSPase I presented no detectable cross-reaction with Mycoplasma flocculare and Mycoplasma hyorhinis. Moreover, phylogenetic analysis demonstrated a low conservation between MhSPase I and orthologous proteins from other porcine respiratory disease complex-related bacteria, Firmicutes and other Mycoplasma species. The potential of an rMhSPase I-based ELISA for PEP immunodiagnosis was demonstrated. Overall, we investigated the expression of sipS and the encoded MhSPase I in three M. hyopneumoniae strains and showed that this protein is a good antigen for use in PEP serodiagnosis and possibly vaccination, as well as a potential target for antibiotic development.

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.