Essential oils isolated from popular medicinal plants and spices as alternative antimicrobial and antibiofilm compounds against the pig pathogen Actinobacilluspleuropneumoniae / Óleos essenciais isolados de plantas medicinais e temperos populares como compostos antimicrobianos e antibiofilme alternativos contra o patógeno de suínosActinobacillus pleuropneumoniae

ABSTRACT: Actinobacilluspleuropneumoniae is the causative agent of swine pleuropneumonia, a contagious respiratory disease associated with high morbidity and economic losses. While antibiotic therapy helps to control the spreading of the pathogen on the farm, resistance to several classes of antibiotics were reported, and treatment can be impaired by the bacterial ability to form biofilms. This increases the need for alternative therapy approaches, including the use of natural compounds with antimicrobial and/or antibiofilm activities. In this research we analyzed, by the broth microdilution method, the inhibitory and bactericidal activities of the essential oils obtained from eighteen Brazilian popular medicinal plants or spices against clinical isolates of Actinobacilluspleuropneumoniae. After that, sub-inhibitory concentrations of active oils were tested for their antibiofilm effects, analyzed by the crystal violet method. Among the eighteen oils tested, eight (extracted from cinnamon, coriander, peppermint, spearmint, thyme, marjoram, eucalyptus, and laurel) presented bacteriostatic and bactericidal activities against all isolates, and subinhibitory concentrations of five of them disrupted up to 80% preformed biofilms, and significantly inhibited biofilm formation. The chemical composition of such oils was assessed by gas chromatography/mass spectrometry (GC/MS) and is presented, indicating that their bactericidal and antibiofilm properties were mostly associated with the presence of monoterpenes and phenylpropanoids. To our knowledge, this is the first report of essential oils with potential to control environmental contamination and animal infection with A. pleuropneumoniae, representing an alternative to increasing levels of antibiotic resistance.

RESUMO: Actinobacillus pleuropneumoniae é o agente causador da pleuropneumonia suína, uma doença respiratória contagiosa associada à alta morbidade e perdas econômicas. Embora a antibioticoterapia ajude a controlar a disseminação do patógeno na fazenda, a resistência a várias classes de antibióticos tem sido relatada e o tratamento pode ser prejudicado pela capacidade bacteriana de formar biofilmes. Isso aumenta a necessidade de abordagens terapêuticas alternativas, que incluem o uso de compostos naturais com atividades antimicrobianas e/ou antibiofilme. Neste trabalho, analisamos, pelo método de microdiluição em caldo nutriente, os efeitos inibitórios e bactericidas dos óleos essenciais obtidos de dezoito plantas medicinais brasileiras populares e/ou temperos contra isolados clínicos de Actinobacillus pleuropneumoniae. Então, concentrações subinibitórias dos óleos ativos foram testados quanto a suas atividades antibiofilme, analisados pelo método do cristal violeta. Dentre os dezoito óleos testados, oito (extraídos da canela, coentro, hortelã-pimenta, hortelã, tomilho, manjerona, eucalipto e louro) apresentaram atividade bacteriostática e bactericida contra todos os isolados, e concentrações subinibitórias de cinco deles romperam biofilmes pré-formados em até 80%, além de inibirem fortemente a formação de biofilmes. A composição química desses óleos foi avaliada por cromatografia gasosa/espectrometria de massa (CG/MS) e é apresentada, indicando que suas propriedades bactericidas e antibiofilme estavam principalmente associadas à presença de monoterpenos e fenilpropanóides. Este é o primeiro relato de óleos essenciais com potencial para controlar a contaminação ambiental e infecção animal por A. pleuropneumoniae, representando uma alternativa contra o crescente aumento de resistência bacteriana aos antibióticos.

Essential oils isolated from popular medicinal plants and spices as alternative antimicrobial and antibiofilm compounds against the pig pathogen Actinobacilluspleuropneumoniae / Óleos essenciais isolados de plantas medicinais e temperos populares como compostos antimicrobianos e antibiofilme alternativos contra o patógeno de suínosActinobacillus pleuropneumoniae

Actinobacilluspleuropneumoniae is the causative agent of swine pleuropneumonia, a contagious respiratory disease associated with high morbidity and economic losses. While antibiotic therapy helps to control the spreading of the pathogen on the farm, resistance to several classes of antibiotics were reported, and treatment can be impaired by the bacterial ability to form biofilms. This increases the need for alternative therapy approaches, including the use of natural compounds with antimicrobial and/or antibiofilm activities. In this research we analyzed, by the broth microdilution method, the inhibitory and bactericidal activities of the essential oils obtained from eighteen Brazilian popular medicinal plants or spices against clinical isolates of Actinobacilluspleuropneumoniae. After that, sub-inhibitory concentrations of active oils were tested for their antibiofilm effects, analyzed by the crystal violet method. Among the eighteen oils tested, eight (extracted from cinnamon, coriander, peppermint, spearmint, thyme, marjoram, eucalyptus, and laurel) presented bacteriostatic and bactericidal activities against all isolates, and subinhibitory concentrations of five of them disrupted up to 80% preformed biofilms, and significantly inhibited biofilm formation. The chemical composition of such oils was assessed by gas chromatography/mass spectrometry (GC/MS) and is presented, indicating that their bactericidal and antibiofilm properties were mostly associated with the presence of monoterpenes and phenylpropanoids. To our knowledge, this is the first report of essential oils with potential to control environmental contamination and animal infection with A. pleuropneumoniae, representing an alternative to increasing levels of antibiotic resistance.

Actinobacillus pleuropneumoniae é o agente causador da pleuropneumonia suína, uma doença respiratória contagiosa associada à alta morbidade e perdas econômicas. Embora a antibioticoterapia ajude a controlar a disseminação do patógeno na fazenda, a resistência a várias classes de antibióticos tem sido relatada e o tratamento pode ser prejudicado pela capacidade bacteriana de formar biofilmes. Isso aumenta a necessidade de abordagens terapêuticas alternativas, que incluem o uso de compostos naturais com atividades antimicrobianas e/ou antibiofilme. Neste trabalho, analisamos, pelo método de microdiluição em caldo nutriente, os efeitos inibitórios e bactericidas dos óleos essenciais obtidos de dezoito plantas medicinais brasileiras populares e/ou temperos contra isolados clínicos de Actinobacillus pleuropneumoniae. Então, concentrações subinibitórias dos óleos ativos foram testados quanto a suas atividades antibiofilme, analisados pelo método do cristal violeta. Dentre os dezoito óleos testados, oito (extraídos da canela, coentro, hortelã-pimenta, hortelã, tomilho, manjerona, eucalipto e louro) apresentaram atividade bacteriostática e bactericida contra todos os isolados, e concentrações subinibitórias de cinco deles romperam biofilmes pré-formados em até 80%, além de inibirem fortemente a formação de biofilmes. A composição química desses óleos foi avaliada por cromatografia gasosa/espectrometria de massa (CG/MS) e é apresentada, indicando que suas propriedades bactericidas e antibiofilme estavam principalmente associadas à presença de monoterpenos e fenilpropanóides. Este é o primeiro relato de óleos essenciais com potencial para controlar a contaminação ambiental e infecção animal por A. pleuropneumoniae, representando uma alternativa contra o crescente aumento de resistência bacteriana aos antibióticos.

Identification of small RNAs associated with RNA chaperone Hfq reveals a new stress response regulator in Actinobacillus pleuropneumoniae.

The RNA chaperone Hfq promotes the association of small RNAs (sRNAs) with cognate mRNAs, controlling the expression of bacterial phenotype. Actinobacillus pleuropneumoniae hfq mutants strains are attenuated for virulence in pigs, impaired in the ability to form biofilms, and more susceptible to stress, but knowledge of the extent of sRNA involvement is limited. Here, using A. pleuropneumoniae strain MIDG2331 (serovar 8), 14 sRNAs were identified by co-immunoprecipitation with Hfq and the expression of eight, identified as trans-acting sRNAs, were confirmed by Northern blotting. We focused on one of these sRNAs, named Rna01, containing a putative promoter for RpoE (stress regulon) recognition. Knockout mutants of rna01 and a double knockout mutant of rna01 and hfq, both had decreased biofilm formation and hemolytic activity, attenuation for virulence in Galleria mellonella, altered stress susceptibility, and an altered outer membrane protein profile. Rna01 affected extracellular vesicle production, size and toxicity in G. mellonella. qRT-PCR analysis of rna01 and putative cognate mRNA targets indicated that Rna01 is associated with the extracytoplasmic stress response. This work increases our understanding of the multilayered and complex nature of the influence of Hfq-dependent sRNAs on the physiology and virulence of A. pleuropneumoniae.

Genotype profiles of Actinobacillus pleuropneumoniae isolated from pigs in Vojvodina, Serbia

Background: Actinobacillus pleuropneumoniae (A. pleuropneumoniae) is one of the most important bacterial respiratory pathogens. It is the only etiological agent of porcine pleuropneumonia (PPP) or it appears as a secondary bacterial infection in the swine respiratory disease complex (PRDC). In Serbia, apart from the identification of serotype 2, no tests have been performed to establish the presence of other A. pleuropneumoniae serotypes in the pig population. The aim of this study was to perform genotyping of A. pleuropneumoniae isolates originating from pig farms in Serbia by apx genes and using multiplex polymerase chain reaction (mPCR). Materials, Methods & Results: Isolates of A. pleuropneumoniae examined in this study were obtained from lungs with macroscopically visible alterations characteristic of a A. pleuropneumoniae. A total of 46 isolates were examined. They were extracted from the lung tissue samples of pig carcasses from 9 farms across different parts of Serbia. Genotyping of isolates was performed in the previously described manner. Briefly, 5 pairs of oligonucleotide primers were used for amplification of 4 different apx genes which encode synthesis of exotoxins (ApxI , ApxII , ApxIII i ApxIV) characteristic for all A. pleuropneumoniae serotypes and biovars. Amplification of appropriate genome parts was performed with a reaction chain polymerase (PCR) in multiplex (m) format using appropriate diagnostic kits to extract DNA from bacteria and perform mPCR reaction. The results of genotyping of 46 isolates of A. pleuropneumoniae indicate the existence of a large number of different serotypes of A. pleuropneumoniae on the studied farms or that different serotypes of this microorganism circulate in the pig population in Serbia. In addition to the detection of dominant serotype 2, which was established on 7 farms, of which in 4 farms it was the only detected serotype, in the examined pig population the presence of serotypes 3, 5, 6, 7 and 9 was also found. Furthermore, the presence of 2 different serotypes of A. Pleuropneumoniae was also detected on 3 farms; on the first farm serotypes 2 and 3, on the second farm serotypes 2 and 6, and on the third farm serotypes 2 and 7. Discussion: Although the research was done with a relatively small number of isolates of A. pleuropneumoniae, comparing the obtained results with the results on the presence and prevalence of appropriate serotypes from other countries, we concluded that there is significant diversity of this pathogen in the pig population in farms of Serbia. Detection of different serotypes of A. pleuropneumoniae in the pig population and the presence of several different serotypes on 1 farm was established for the very first time in Serbia. All isolates from our study can be characterized as highly virulent, considering that the clinical symptoms, pathological findings and the results of bacteriological examination indicated A. pleuropneumoniae to be the cause of animal death. Like in the neighbouring countries, the strongly pathogenic serotype 9 and the less pathogenic serotype 2 are the most frequently identified causative agents of porcine pleuropneumonia in the Autonomous Province of Vojvodina, Republic of Serbia. The necessity to establish the presence of all A. pleuropneumoniae serotypes in the pig population, and in particular to determine the presence of different serotypes on individual farms, is crucial for several reasons: making a definitive e diagnosis; development of prophylactic strategies for medicines; implementation of immunoprophylactic vaccination.

Antimicrobial resistance, biofilm formation and virulence reveal Actinobacillus pleuropneumoniae strains' pathogenicity complexity.

Porcine pleuropneumonia is an important cause of lowered productivity and economic loss in the pig industry worldwide, associated primarily with Actinobacillus pleuropneumoniae infection. Its colonization and persistence within the upper respiratory tract of affected pigs depends upon interactions between a number of genetically controlled virulence factors, such as pore-forming repeats-in-toxin exoproteins, biofilm formation, and antimicrobial resistance. This study investigated correlations between biofilm-forming capacity, antimicrobial resistance, and virulence of A. pleuropneumoniae obtained from clinical outbreaks of disease, using a Galleria mellonella alternative infection model. Results suggest that virulence is diverse amongst the 21 strains of A. pleuropneumoniae examined and biofilm formation correlated with genetic control of antimicrobial resistance.

A computational strategy for the search of regulatory small RNAs in Actinobacillus pleuropneumoniae.

Bacterial regulatory small RNAs (sRNAs) play important roles in gene regulation and are frequently connected to the expression of virulence factors in diverse bacteria. Only a few sRNAs have been described for Pasteurellaceae pathogens and no in-depth analysis of sRNAs has been described for Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, responsible for considerable losses in the swine industry. To search for sRNAs in A. pleuropneumoniae, we developed a strategy for the computational analysis of the bacterial genome by using four algorithms with different approaches, followed by experimental validation. The coding strand and expression of 17 out of 23 RNA candidates were confirmed by Northern blotting, RT-PCR, and RNA sequencing. Among them, two are likely riboswitches, three are housekeeping regulatory RNAs, two are the widely studied GcvB and 6S sRNAs, and 10 are putative novel trans-acting sRNAs, never before described for any bacteria. The latter group has several potential mRNA targets, many of which are involved with virulence, stress resistance, or metabolism, and connect the sRNAs in a complex gene regulatory network. The sRNAs identified are well conserved among the Pasteurellaceae that are evolutionarily closer to A. pleuropneumoniae and/or share the same host. Our results show that the combination of newly developed computational programs can be successfully utilized for the discovery of novel sRNAs and indicate an intricate system of gene regulation through sRNAs in A. pleuropneumoniae and in other Pasteurellaceae, thus providing clues for novel aspects of virulence that will be explored in further studies.

Oral immunization against porcine pleuropneumonia using the cubic phase of monoolein and purified toxins of Actinobacillus pleuropneumoniae.

The main goal of this work was to obtain an orally administered immunogen that would protect against infections by Actinobacillus pleuropneumoniae. The Apx I, II and III toxins were obtained from the supernatants of cultures of serotypes 1 and 3 of A. pleuropneumoniae. The capacity of monoolein gel to trap and protect the Apx toxins, and the effect of their incorporation on the stability of the cubic phase were evaluated. The gel was capable of trapping a 400-µg/ml concentration of the antigen with no effects on its structure. Approximately 60% of the protein molecules were released from the gel within 4h. Four experimental groups were formed, each one with four pigs. All challenges were conducted in a nebulization chamber. Group A: Control (-) not vaccinated and not challenged; Group B: Control (+) not vaccinated but challenged; Group C: vaccinated twice intramuscularly with ToxCom (a commercial toxoid) at an interval of 15 days and then challenged; and Group D: vaccinated orally twice a week for 4 weeks with ToxOral (an oral toxoid) and challenged on day 28 of the experiment with a same dose of 2.0 × 10(4) UFC of A. pleuropneumoniae serotypes 1 and 3. The lesions found in group B covered 27.7-43.1% of the lungs; the pigs in group C had lesions over 12.3-28%; and those in group D over 15.4-32.3%. No lesions were found in the Group A pigs. A. pleuropneumoniae induced macroscopic lesions characteristic of infection by and lesions microscopic detected by histopathology. The etiologic agent was recovered from the infected lungs, tonsils and spleen. The serotypes identified were 1 and 3. An indirect ELISA test identified the antibodies against the Apx toxins in the serum of the animals immunized orally.

Characterization of the omlA gene from different serotypes of Actinobacillus pleuropneumoniae: A new insight into an old approach.

The OmlA protein is a virulence factor of Actinobacillus pleuropneumoniae, an important pathogen in pigs. The polymorphisms present in the omlA gene sequence of 15 reference serotypes of A. pleuropneumoniae and non-serotypable isolates were assessed to determine the possible evolutionary relationship among them and to validate the importance of this gene as a molecular marker for the characterization of this bacterium. Divergence among the 15 serotypes of A. pleuropneumoniae probably resulted initially from two major evolutionary events that led to subsequent differentiation into nine groups. This differentiation makes it possible to characterize most of the serotypes by using bionformatics, thereby avoiding problems with immunological cross-reactivity. A conserved α-helix common to all the serotypes was most likely involved in connecting the protein to the outer membrane and acting as a signal peptide. A previously unknown gene duplication was also identified and could contribute to the genetic variability that makes it difficult to serotype some isolates. Our data support the importance of the omlA gene in the biology of A. pleuropneumoniae and provide a new area of research into the OmlA protein.

Detecção direta de Actinobacillus pleuropneumoniae em órgãos de suídeos do estado de São Paulo pela técnica de reação em cadeia pela polimerase (nested-PCR) / Direct detection of Actinobacillus pleuropneumoniae in suidae organs in São Paulo state, Brazil, by polymerase chain reaction (nested-PCR)

A pleuropneumonia suína, causada pelo Actinobacillus pleuropneumoniae, é uma importante doença respiratória, responsável por prejuízos e queda de produtividade nas criações. Este trabalho teve como objetivo determinar a ocorrência de A. pleuropneumoniae em amostras de campo, mediante a adaptação e emprego de uma técnica de nested-PCR dirigida ao gene Apx IV. Definiu-se a sensibilidade analítica das técnicas de PCR e nested-PCR utilizando a amostra padrão A. pleuropneumoniae sorotipo III, em concentrações de DNA variando entre 30 µg/mL a 0,01 ng/ mL. Um total de trinta e sete amostras de campo encaminhadas ao Instituto Biológico entre 1995 a 2007 foram analisadas pelas técnicas de PCR e nested-PCR. A avaliação da sensibilidade analítica revelou que a PCR possui capacidade de gerar sinal a partir de 2 ng/mL de DNA extraído e a nested-PCR a partir de 0,4 ng/mL. Uma vez que a nested-PCR apresentou sensibilidade analítica cinco vezes maior se comparada à PCR para detecção de A. pleuropneumoniae em amostra padrão, o seu emprego pode minimizar a ocorrência de resultados tipo "falso-negativo". Dentre as amostras testadas, dez foram positivas à nested-PCR, sendo observada a ocorrência de A. pleuropneumoniae em nove diferentes animais, um deles javali. A presente técnica de nested-PCR pode ser utilizada para detecção direta de A. pleuropneumoniae em amostras de campo, mesmo após congelamento da amostra por longos períodos e sem necessidade de isolamento bacteriano prévio.

Porcine pleuropneumonia, caused by Actinobacillus pleuropneumoniae, is an important respiratory disease, responsible for economic losses and reduced productivity. The aim of this study was to determine occurrence of A. pleuropneumoniae in field samples, using an adapted nested-PCR reaction targeting the Apx IV gene. Different DNA concentrations (from 30 µg/mL to 0.01 ng/mL) of A. pleuropneumoniae serotype III reference strain were used to determine the level of sensitivity of first generation and nested-PCR reactions. Thirty-seven field samples sent to Instituto Biológico from 1995 to 2007 were tested by PCR and nested-PCR. Determination of the level of sensitivity showed that PCR could amplify to 2 ng/mL of extracted DNA and nested-PCR to 0.4 ng/mL. Since the nested reaction exhibited a level of sensitivity 5 times greater than the PCR reaction to detect a reference strain, using nested-PCR could minimize the occurrence of false-negative results. Among tested samples, 10 of them were nested-PCR positive, showing occurrence of A. pleuropneumoniae in 9 different animals (including one wild boar). This nested-PCR reaction can be used for direct detection of A. pleuropneumoniae in field samples, even after frozen storage for long periods, without the need for previous bacterial isolation.

DETECÇÃO DIRETA DE ACTINOBACILLUS PLEUROPNEUMONIAE EM ÓRGÃOS DE SUÍDEOS DO ESTADO DE SÃO PAULO PELA TÉCNICA DE REAÇÃO EM CADEIA PELA POLIMERASE (NESTED-PCR)

ABSTRACT Porcine pleuropneumonia, caused by Actinobacillus pleuropneumoniae, is an important respiratory disease, responsible for economic losses and reduced productivity. The aim of this study was to determine occurrence of A. pleuropneumoniae in field samples, using an adapted nested-PCR reaction targeting the Apx IV gene. Different DNA concentrations (from 30 µg/mL to 0.01 ng/mL) of A. pleuropneumoniae serotype III reference strain were used to determine the level of sensitivity of first generation and nested-PCR reactions. Thirty-seven field samples sent to Instituto Biológico from 1995 to 2007 were tested by PCR and nested-PCR. Determination of the level of sensitivity showed that PCR could amplify to 2 ng/mL of extracted DNA and nested-PCR to 0.4 ng/mL. Since the nested reaction exhibited a level of sensitivity 5 times greater than the PCR reaction to detect a reference strain, using nested-PCR could minimize the occurrence of false-negative results. Among tested samples, 10 of them were nested-PCR positive, showing occurrence of A. pleuropneumoniae in 9 different animals (including one wild boar). This nested-PCR reaction can be used for direct detection of A. pleuropneumoniae in field samples, even after frozen storage for long periods, without the need for previous bacterial isolation.

RESUMO A pleuropneumonia suína, causada pelo Actinobacillus pleuropneumoniae, é uma importante doença respiratória, responsável por prejuízos e queda de produtividade nas criações. Este trabalho teve como objetivo determinar a ocorrência de A. pleuropneumoniae em amostras de campo, mediante a adaptação e emprego de uma técnica de nested-PCR dirigida ao gene Apx IV. Definiu-se a sensibilidade analítica das técnicas de PCR e nested-PCR utilizando a amostra padrão A. pleuropneumoniae sorotipo III, em concentrações de DNA variando entre 30 µg/mL a 0,01 ng/ mL. Um total de trinta e sete amostras de campo encaminhadas ao Instituto Biológico entre 1995 a 2007 foram analisadas pelas técnicas de PCR e nested-PCR. A avaliação da sensibilidade analítica revelou que a PCR possui capacidade de gerar sinal a partir de 2 ng/mL de DNA extraído e a nested-PCR a partir de 0,4 ng/mL. Uma vez que a nested-PCR apresentou sensibilidade analítica cinco vezes maior se comparada à PCR para detecção de A. pleuropneumoniae em amostra padrão, o seu emprego pode minimizar a ocorrência de resultados tipo falso-negativo. Dentre as amostras testadas, dez foram positivas à nested-PCR, sendo observada a ocorrência de A. pleuropneumoniae em nove diferentes animais, um deles javali. A presente técnica de nested-PCR pode ser utilizada para detecção direta de A. pleuropneumoniae em amostras de campo, mesmo após congelamento da amostra por longos períodos e sem necessidade de isolamento bacteriano prévio.

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.

Otimização da técnica da PCR para a detecção de Actinobacillus pleuropneumoniae

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 57°C 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.

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 57°C 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.

Otimização da técnica da PCR para a detecção de Actinobacillus pleuropneumoniae

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 57°C 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.

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 57°C 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.

Aspectos recentes da patogênese e diagnóstico da pleuropneumonia suína

Porcine pleuropneumonia is a highly contagious disease caused by the bacterium Actinobacillus pleuropneumoniae, a pathogen responsible for substantial losses worldwide to the pig industry. The disease is characterized by fibrinous pleuritis with hemorrhagic and necrotic lesions in the lungs, and pleural adhesions. Specific and precise methods for the identification of this pathogen are still required. In the present article we review the pathogenesis, as the traditional and the recently developed molecular methods employed for the identification and characterization of A. pleuropneumoniae.

A pleuropneumonia suína, causada por Actinobacillus pleuropneumoniae, é uma doença caracterizada pela apresentação fibrino-hemorrágica com pleurite adesiva. A enfermidade está presente em todos os países produtores de suínos, sendo responsável por prejuízos econômicos elevados. No Brasil e no mundo, diversos grupos vêm conduzindo estudos na busca por um melhor entendimento da doença e de sua epidemiologia. Avanços importantes foram obtidos, entre os quais a caracterização dos fatores de virulência, implicados na apresentação clínica da enfermidade; e a aplicação de novos métodos de diagnóstico. A difusão das técnicas de biologia molecular como ferramenta diagnóstica em Medicina Veterinária tem contribuindo para a identificação de Actinobacillus pleuropneumoniae. Nesta revisão, são abordados os aspectos mais recentes sobre a patogênese e o diagnóstico deste importante patógeno.

Aspectos recentes da patogênese e diagnóstico da pleuropneumonia suína

Porcine pleuropneumonia is a highly contagious disease caused by the bacterium Actinobacillus pleuropneumoniae, a pathogen responsible for substantial losses worldwide to the pig industry. The disease is characterized by fibrinous pleuritis with hemorrhagic and necrotic lesions in the lungs, and pleural adhesions. Specific and precise methods for the identification of this pathogen are still required. In the present article we review the pathogenesis, as the traditional and the recently developed molecular methods employed for the identification and characterization of A. pleuropneumoniae.

A pleuropneumonia suína, causada por Actinobacillus pleuropneumoniae, é uma doença caracterizada pela apresentação fibrino-hemorrágica com pleurite adesiva. A enfermidade está presente em todos os países produtores de suínos, sendo responsável por prejuízos econômicos elevados. No Brasil e no mundo, diversos grupos vêm conduzindo estudos na busca por um melhor entendimento da doença e de sua epidemiologia. Avanços importantes foram obtidos, entre os quais a caracterização dos fatores de virulência, implicados na apresentação clínica da enfermidade; e a aplicação de novos métodos de diagnóstico. A difusão das técnicas de biologia molecular como ferramenta diagnóstica em Medicina Veterinária tem contribuindo para a identificação de Actinobacillus pleuropneumoniae. Nesta revisão, são abordados os aspectos mais recentes sobre a patogênese e o diagnóstico deste importante patógeno.