Antimicrobial resistance in bacteria isolated from pigs with respiratory clinical signs in Brazil / Resistência a antimicrobianos em bactérias isoladas de suínos com sinais clínicos respiratórios no Brasil

Antimicrobial resistance is a current and important issue to public health, and it is usually associated with the indiscriminate use of antimicrobials in animal production. This study aimed to evaluate the antimicrobial susceptibility profile in bacterial isolates from pigs with clinical respiratory signs in Brazil. One hundred sixty bacterial strains isolated from pigs from 51 pig farms in Brazil were studied. In vitro disk-diffusion method was employed using 14 antimicrobial agents: amoxicillin, penicillin, ceftiofur, ciprofloxacin, enrofloxacin, chlortetracycline, doxycycline, oxytetracycline, tetracycline, erythromycin, tilmicosin, florfenicol, lincomycin, and sulfadiazine/trimethoprim. The majority of isolates were resistant to at least one antimicrobial agent (98.75%; 158/160), while 31.25% (50/160) of the strains were multidrug resistant. Streptococcus suis and Bordetella bronchiseptica were the pathogens that showed higher resistance levels. Haemophilus parasuis showed high resistance levels to sulfadiazine/trimethoprim (9/18=50%). We observed that isolates from the midwestern and southern regions exhibited four times greater chance of being multidrug resistant than the isolates from the southeastern region studied. Overall, the results of the present study showed a great level of resistance to lincomycin, erythromycin, sulfadiazine/trimethoprim, and tetracycline among bacterial respiratory pathogens isolated from pigs in Brazil. The high levels of antimicrobial resistance in swine respiratory bacterial pathogens highlight the need for the proper use of antimicrobials in Brazilian pig farms.(AU)

A resistência antimicrobiana é uma questão atual e muito importante para a saúde pública, geralmente associada ao uso indiscriminado de antimicrobianos na produção animal. Diante disso, foi investigado o perfil de sensibilidade-antimicrobiana em isolados bacterianos de suínos com sinais clínicos respiratórios no Brasil. Foram estudadas 96 isolados provenientes de 51 granjas de suínos do Brasil. O método de disco-difusão foi empregado usando 14 antimicrobianos: amoxicilina, penicilina, ceftiofur, ciprofloxacina, enrofloxacina, clortetraciclina, doxiciclina, oxitetraciclina, tetraciclina, eritromicina, tilmicosina, florfenicol, lincomicina e sulfadiazina/trimetoprim. Streptococcus suis e Bordetella bronchiseptica foram os patógenos que apresentaram maiores níveis de resistência. Haemophilus parasuis apresentou altos níveis de resistência à sulfadiazina/trimetoprim (9/18=50%). Observou-se que isolados das regiões Centro-Oeste e Sul apresentaram quatro vezes mais chance de serem multirresistentes do que os isolados da região Sudeste. A maioria foi resistente a pelo menos um agente antimicrobiano (98,75%; 158/160) e 31,25% (50/160) das estirpes isoladas eram multirresistentes. No geral, os resultados do presente estudo mostraram grande nível de resistência à lincomicina, eritromicina, sulfadiazina/trimetoprim e tetraciclina entre patógenos respiratórios bacterianos isolados de suínos no Brasil. Os altos níveis de resistência antimicrobiana em patógenos bacterianos respiratórios em suínos reforçam a necessidade do uso criterioso de antimicrobianos na suinocultura brasileira.(AU)

Development of Actinobacillus pleuropneumoniae ApxI, ApxII, and ApxIII-specific ELISA methods for evaluation of vaccine efficiency

Among various vaccines against Actinobacillus pleuropneumoniae, subunit vaccines using recombinant proteins of ApxI, ApxII, and ApxIII as vaccine antigens have shown good efficacy in terms of safety and protection. Therefore, subunit vaccines are being applied worldwide and the development of new subunit vaccines is actively being conducted. To evaluate the efficacy of the subunit vaccines, it is important to measure immune responses to each Apx toxin separately. However, the cross-reactivity of antibodies makes it difficult to measure specific immune reactivity to each toxin. In the present study, specific antigen regions among the toxins were identified and cloned to solve this problem. The antigenicity of each recombinant protein was demonstrated by Western blot. Using the recombinant proteins, we developed enzyme-linked immunosorbent assay (ELISA) methods that can detect specific immune responses to each Apx toxin in laboratory guinea pigs. We suggest that the ELISA method developed in this study can be an important tool in the evaluation of vaccine efficiency and vaccine development.

Characterization of Actinobacillus pleuropneumoniae field strains antigenically related to the 3-6-8-15 group from diseased pigs in Japan and Argentina / Caracterización de cepas de Actinobacillus pleuropneumoniae antigénicamente relacionados al grupo 3-6-8-15 obtenidos de cerdos infectados naturalmente en Japón y Argentina

The objectives of this study were to determine the serovar of a collection of Actinobacillus pleuropneumoniae strains within the 3-6-8-15 cross-reacting group and to analyze their phenotypic and genetic properties. Based on the serological tests, forty-seven field strains of Actinobacillus pleuropneumoniae isolated from lungs with pleuropneumonia lesions in Japan and Argentina were found to be serovars belonging to the 3-6-8-15 cross-reacting group. By using a capsule loci-based PCR, twenty-nine (96.7%) and one (3.3%) from Japan were identified as serovars 15 and 8, respectively, whereas seventeen (100%) from Argentina were identified as serovar 8. The findings suggested that serovars 8 and 15 were prevalent within the 3-6-8-15 cross-reacting group, in Argentina and Japan, respectively. Phenotypic analyses revealed that the protein patterns observed on SDS-PAGE and the lipopolysaccharide antigen detected by immunoblotting of the reference and field strains of serovars 8 and 15 were similar to each other. Genetic (16S rDNA, apxIIA, apxIIIA, cps, cpx genes, apx and omlA patterns) analyses revealed that the apxIIA and apxIIIA genes of the field strains of serovars 8 and 15 were similar to those of the reference strains of serovars 3, 4, 6, 8 and 15. The results obtained in the present study may be useful for the development of more effective vaccines against disease caused by A. pleuropneumoniae by including the homologous antigens to the most prevalent serovars in specific geographical areas.

Los objetivos del presente estudio fueron determinar el serovar de una colección de cepas de Actinobacillus pleuropneumoniae pertenecientes al grupo 3, 6, 8, 15 de reacciones cruzadas y analizar sus propiedades fenotípicas y genéticas. En base a técnicas serológicas se determinó que cuarenta y siete cepas de A. pleuropneumoniae aisladas a partir de pulmones con lesiones de pleuroneumonía en Japón y Argentina pertenecen al grupo 3, 6, 8, 15. Mediante el uso de PCR basado en locus capsulares, veintinueve (96.7%) y una (3.3%) de los aislados japoneses fueron identificados como serovar 15 y 8 respectivamente, mientras que diecisiete (100%) de los aislados argentinos resultaron pertenecer al serotipo 8. Este hallazgo sugirió que los serovares 8 y 15 fueron los prevalentes dentro del grupo 3, 6, 8, 15 en Japón y Argentina, respectivamente. El análisis fenotípico reveló que los perfiles proteicos determinados por SDS-PAGE, y de antígenos lipopolisacáridos estudiados por inmunoblot, de las cepas de referencia y de campo de los serovares 8 y 15 fueron similares entre sí. El análisis genético (Í6S rDNA, apxIIA, apxIIA, cps, genes cpx, apx y los perfiles omlA) reveló que los genes apxIIA y apxIIIA de las cepas de campo de los serovares 8 y 15 fueron similares a sus homólogos de las cepas de referencia de los serovares 3, 4, 6, 8 y 15. Los resultados obtenidos en el presente estudio pueden ser útiles para el desarrollo de vacunas más efectivas contra la enfermedad causada por A. pleuropneumoniae, al posibilitar incluir antígenos homólogos a los serovares prevalentes en las áreas geográficas de interés.

Assessment of a segregated medicated early weaning protocol for elimination of Actinobacillus pleuropneumoniae / Avaliação de um protocolo de desmame precoce segregado medicado para a eliminação de Actinobacillus pleuropneumoniae

ABSTRACT: To assess the success of a segregated medicated early weaning protocol for elimination of Actinobacillus pleuropneumoniae, ten consecutive batches of 100 pigs per batch weaned at 7-9 days of age were monitored at 21-22 weeks of age looking for presence of productive cough, presence of antibodies against the agent and pleuropneumonia-compatible lung lesions at slaughter. Also, whole tonsils were processed for A. pleuropneumoniae identification by both bacteriological isolation and nested-PCR using a direct method. Positive serological test (1.4%) and PCR (3.5%) results, suggested that A. pleuropneumoniae could not be eradicated in all batches, even using segregated medicated early weaning protocol.

RESUMO: Para avaliar o sucesso de um protocolo de desmame precoce segregado medicado para a eliminação de Actinobacillus pleuropneumoniae, dez lotes consecutivos de 100 suínos por lote desmamados aos 7-9 dias de idade foram monitorados às 21-22 semanas de idade procurando a presença de tosse produtiva, presença de anticorpos contra o agente e lesões pulmonares compatíveis com pleuropneumonia no abate. Além disso, as tonsilas inteiras foram processadas para a identificação de A. pleuropneumoniae por isolamento bacteriológico e nested PCR usando um método direto. Os resultados serológicos (1,4%) e PCR (3,5%) positivos sugerem que não foi possível erradicar em todos os lotes, mesmo usando um protocolo de desmame precoce segregado medicado.

In silico analysis of putative drug and vaccine targets of the metabolic pathways of Actinobacillus pleuropneumoniae using a subtractive/comparative genomics approach

Actinobacillus pleuropneumoniae is a Gram-negative bacterium that resides in the respiratory tract of pigs and causes porcine respiratory disease complex, which leads to significant losses in the pig industry worldwide. The incidence of drug resistance in this bacterium is increasing; thus, identifying new protein/gene targets for drug and vaccine development is critical. In this study, we used an in silico approach, utilizing several databases including the Kyoto Encyclopedia of Genes and Genomes (KEGG), the Database of Essential Genes (DEG), DrugBank, and Swiss-Prot to identify non-homologous essential genes and prioritize these proteins for their druggability. The results showed 20 metabolic pathways that were unique and contained 273 non-homologous proteins, of which 122 were essential. Of the 122 essential proteins, there were 95 cytoplasmic proteins and 11 transmembrane proteins, which are potentially suitable for drug and vaccine targets, respectively. Among these, 25 had at least one hit in DrugBank, and three had similarity to metabolic proteins from Mycoplasma hyopneumoniae, another pathogen causing porcine respiratory disease complex; thus, they could serve as common therapeutic targets. In conclusion, we identified glyoxylate and dicarboxylate pathways as potential targets for antimicrobial therapy and tetra-acyldisaccharide 4′-kinase and 3-deoxy-D-manno-octulosonic-acid transferase as vaccine candidates against A. pleuropneumoniae.

Prevalence and Characterization of Actinobacillus pleuropneumoniae Isolated from Korean Pigs

Actinobacillus pleuropneumoniae causes porcine pleuropneumoniae which is one of severe threats to the swine industry. In total, 54 strains of Actinobacillus pleuropneumoniae were isolated from 443 pigs between 2012 and 2013 in Korea. Isolates were classified into serotypes 1, 2, 5, 7, 12, and unclassified by multiplex PCR. Genotypes of isolates were divided into three groups according to the sequence of the omlA gene. The antimicrobial resistance rate of serotype 1 was slightly higher than that of serotype 5. In conclusion, to block and treat porcine pleuropneumonia, it is necessary to conduct ongoing characterization of A. pleuropneumoniae isolated from pigs.

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.

Elucidating the role of ApxI in hemolysis and cellular damage by using a novel apxIA mutant of Actinobacillus pleuropneumoniae serotype 10

Exotoxins produced by Actinobacillus (A.) pleuropneumoniae (Apx) play major roles in the pathogenesis of pleuropneumonia in swine. This study investigated the role of ApxI in hemolysis and cellular damage using a novel apxIA mutant, ApxIA336, which was developed from the parental strain A. pleuropneumoniae serotype 10 that produces only ApxI in vitro. The genotype of ApxIA336 was confirmed by PCR, Southern blotting, and gene sequencing. Exotoxin preparation derived from ApxIA336 was analyzed for its bioactivity towards porcine erythrocytes and alveolar macrophages. Analysis results indicated that ApxIA336 contained a kanamycin-resistant cassette inserted immediately after 1005 bp of the apxIA gene. Phenotype analysis of ApxIA336 revealed no difference in the growth rate as compared to the parental strain. Meanwhile, ApxI production was abolished in the bacterial culture supernatant, i.e. exotoxin preparation. The inability of ApxIA336 to produce ApxI corresponded to the loss of hemolytic and cytotoxic bioactivity in exotoxin preparation, as demonstrated by hemolysis, lactate dehydrogenase release, mitochondrial activity, and apoptosis assays. Additionally, the virulence of ApxIA336 appeared to be attenuated by 15-fold in BALB/c mice. Collectively, ApxI, but not other components in the exotoxin preparation of A. pleuropneumoniae serotype 10, was responsible for the hemolytic and cytotoxic effects on porcine erythrocytes and alveolar macrophages.

Development and evaluation of a loop-mediated isothermal amplification (LAMP) assay for rapid detection of Actinobacillus pleuropneumoniae based the dsbE-like gene

This paper reports on the development and validation of a loop-mediated isothermal amplification assay (LAMP) for the rapid and specific detection of Actinobacillus pleuropneumoniae (A. pleuropneumoniae). A set of six primers were designed derived from the dsbE-like gene of A.pleuropneumoniae and validate the assay using 9 A. pleuropneumoniae reference/field strains, 132 clinical isolates and 9 other pathogens. The results indicated that positive reactions were confirmed for all A. pleuropneumoniae strains and specimens by LAMP at 63ºC for 60 min and no cross-reactivity were observed from other non-A.pleuropneumoniae including Haemophilus parasuis, Escherichia coli, Pasteurella multocida, Bordetella bronchiseptica, Streptococcus suis, Salmonella enterica, Staphylococcus, porcine reproductive and respiratory syndrome virus (PRRSV), and Pseudorabies virus. The detection limit of the conventional PCR was 10² CFU per PCR test tube, while that of the LAMP was 5 copies per tube. Therefore, the sensitivity of LAMP was higher than that of PCR. Moreover, the LAMP assay provided a rapid yet simple test of A. pleuropneumoniae suitable for laboratory diagnosis and pen-side detection due to ease of operation and the requirement of only a regular water bath or heat block for the reaction.

Serotype and antimicrobial susceptibility of Actinobacillus pleuropneumoniae isolates from pigs in Korea / 대한수의학회지

Actinobacillus (A.) pleuropneumoniae is the causative agent of pleuropneumonia which is one of the most important respiratory diseases in pigs worldwide. A total of 32 A. pleuropneumoniae isolates from diseased pigs during 2008 to 2010 were serotyped by polymerase chain reaction method. The susceptibility of the isolates to 13 antimicrobial agents were determined by disk diffusion test. In all the 32 isolates examined in this study, serotype 5 (16 isolates: 50%), 1 (7 isolates: 21.9%), 2 (5 isolates: 15.6%) and 12 (1 isolate: 3.1%) were found. Of all tested antimicrobial agents, resistance to oxytetracycline was found in 96.9% of isolates, followed by resistance to amikacin (81.2%), neomycin (68.7%), kanamycin (53.1%), penicillin (50.0%), gentamicin (43.7%), florfenicol (25.0%), ampicillin (18.7%), colistin (9.4%), trimethoprim/sulfamethoxazole, ceftiofur (8.3%), amoxicillin/clavulanic acid (3.1%) and enrofloxacin (0%). Oxytetracycline or florfenicol-resistant isolates were examined for the presence of resistance gene. Among the 31 oxytetracycline-resistant isolates, tetB, tetH and tetO genes were detected in 22 (71%), 8 (26%) and 1 (3%) isolates, respectively. The floR genes were detected in 8 (100%) of the 8 florfenicol-resistant A. pleuropneumoniae isolates.

Generation of transgenic corn-derived Actinobacillus pleuropneumoniae ApxIIA fused with the cholera toxin B subunit as a vaccine candidate

Corn, one of the most important forage crops worldwide, has proven to be a useful expression vehicle due to the availability of established transformation procedures for this well-studied plant. The exotoxin Apx, a major virulence factor, is recognized as a common antigen of Actinobacillus (A.) pleuropneumoniae, the causative agent of porcine pleuropneumonia. In this study, a cholera toxin B (CTB)-ApxIIA#5 fusion protein and full-size ApxIIA expressed in corn seed, as a subunit vaccine candidate, were observed to induce Apx-specific immune responses in mice. These results suggest that transgenic corn-derived ApxIIA and CTB-ApxIIA#5 proteins are potential vaccine candidates against A. pleuropneumoniae infection.

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.

Ocorrência de Actinobacillus pleuropneumoniae biótipo 1 em pulmões de suínos com pleuropneumonia no Norte de Portugal / Occurrence of Actinobacillus pleuropneumoniae biotype 1 in swine with pleuropneumonia in the North of Portugal

The isolation and identification of Actinobacillus pleuropneumoniae in swine lungs with pleuropneumonia in the North of Portugal were reported. A total of 127 swine lungs with and without lesions were examined. The system of lesions classification was based on a semi-quantitative method. Diagnosis was made by isolation and identification of the etiological agent in typical lesions. The occurrence of observed lesions was 75.6 percent and the occurrence of isolation of A. pleuropneumoniae was 19.7 percent. In 25 out of 96 (26.0 percent) lung samples with lesions of pleuropneumonia, A. pleuropneumoniae was isolated.

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.

Padronização de três ELISAs polivalentes com lipopolissacarídeos de cadeia longa dos sorotipos 1 e 5, 2, 3 e 7 ou 10 e 12 de Actinobacillus pleuropneumoniae / Standardization of three polyvalent ELISA based on long chain lipopolysaccharides of serotypes 1 and 5, 2, 3 and 7, or 10 and 12 of Actinobacillus pleuropneumoniae

Três ELISAs polivalentes baseados em lipopolissacarídeos de cadeia longa (LPS-CL) foram estabelecidos para detectar anticorpos para todos os sorotipos prevalentes de Actinobacillus pleuropneumoniae. Foram testadas amostras provenientes do banco de soros de suínos experimentalmente inoculados com todos os sorotipos de A. pleuropneumoniae. Os ELISAs foram sensíveis à detecção de anticorpos contra todos os LPS-CL. Foram observadas reações cruzadas no ELISA polivalente produzido com os sorotipos 1 e 5, com anti-soros específicos para os sorotipos 9 e 11, pois os sorotipos 1, 9 e 11 apresentaram antígenos somáticos comuns. No polivalente com os sorotipos 2, 3 e 7, observaram-se reações com anti-soros dos sorotipos 4, 6 e 8, devido à presença de antígenos somáticos entre os sorotipos 3, 6 e 8 e entre os sorotipos 4 e 7. Amostras de soros de animais infectados com Mycoplasma hyopneumoniae, Mycoplasma flocculare e Haemophilus parasuis, agentes que acometem o sistema respiratório dos suínos, não apresentaram reações cruzadas com os antígenos baseados em LPS-CL.

Three polyvalent ELISA based on long chain lipopolysaccharides (LC-LPS) were established to detect all prevalent serotypes of Actinobacillus pleuropneumoniae. Samples from a serum bank of experimentally inoculated animals with all serotypes of A. pleuropneumoniae were tested. Antibodies specific to LC-LPS of each serotype were detected. Cross-reactions were observed in the polyvalent ELISA produced with serotypes 1 and 5, with specific antisera to serotypes 9 and 11 due to common somatic antigens presence in serotypes 1, 9, and 11. In the polyvalent with serotypes 2, 3 and 7 reactions were observed with antisera of serotypes 4, 6, and 8, due to the presence of somatic antigens in serotypes 3, 6, and 8 and serotypes 4 and 7. Experimentally infected animals with respiratory agents of swine Mycoplasma hyopneumoniae, Mycoplasma flocculare, and Haemophilus parasuis did not present cross-reactions with the antigens based on LC-LPS.

Enhancement of protective immune responses by oral vaccination with Saccharomyces cerevisiae expressing recombinant Actinobacillus pleuropneumoniae ApxIA or ApxIIA in mice

We previously induced protective immune response by oral immunization with yeast expressing the ApxIIA antigen. The ApxI antigen is also an important factor in the protection against Actinobacillus pleuropneumoniae serotype 5 infection; therefore, the protective immunity in mice following oral immunization with Saccharomyces cerevisiae expressing either ApxIA (group C) or ApxIIA (group D) alone or both (group E) was compared with that in two control groups (group A and B). The immunogenicity of the rApxIA antigen derived from the yeast was confirmed by a high survival rate and an ApxIA-specific IgG antibody response (p < 0.01). The highest systemic (IgG) and local (IgA) humoral immune responses to ApxIA and ApxIIA were detected in group E after the third immunization (p < 0.05). The levels of IL-1beta and IL-6 after challenge with an A. pleuropneumoniae field isolate did not change significantly in the vaccinated groups. The level of TNF-alpha increased in a time-dependent manner in group E but was not significantly different after the challenge. After the challenge, the mice in group E had a significantly lower infectious burden and a higher level of protection than the mice in the other groups (p < 0.05). The survival rate in each group was closely correlated to the immune response and histopathological observations in the lung following the challenge. These results suggested that immunity to the ApxIA antigen is required for optimal protection.

Expression of apxIA of Actinobacillus pleuropneumoniae in Saccharomyces cerevisiae

Actinobacillus pleuropneumoniae is an important primary pathogen in pigs, in which it causes a highly contagious pleuropneumoniae. In our previous study, apxIA gene amplified from A. pleuropneumoniae Korean isolate by PCR with primer designed based on the N- and C-terminal of the toxin was cloned in TA cloning vector and sequenced. The nucleotide sequences of apxIA gene was reported to GeneBank with the accession numbers of AF363361. Identity of the Apx IA from the cloned gene in E. coli was proved by SDS-PAGE and Western blot. Yeast has been demonstrated to be an excellent host for the expression of recombinant proteins with uses in diagnostics, therapeutics and vaccine productions. Therefore, to use the yeast as a delivery system in new oral subunit vaccine, apxIA gene was subcloned into Saccharomyces cerevisiae, and ientified the expression of Apx IA protein. First, apxIA gene was amplified by PCR with the primers containing BamHI and SalI site at each end. Second, the DNA digested with BamHI and SalI was ligated into YEpGPD-TER vector, and transformed into S. cerevisiae 2805. Third, after identification of the correctly oriented clone, the 120-kDa of Apx IA protein expressed in S. cerevisiae 2805 was identified by SDS-PAGE and Western blot.

Efeito da infecção por Actinobacillus pleuropneumoniae nos mecanismos da coagulação do sangue de leitões / Effect of Actinobacillus pleuropneumoniae infection on pigs' blood coagulation system

O presente trabalho apresenta os resultados do estudo do efeito da infecção causada por Actínobacíllus pleuropneumoníae no sistema da coagulação do sangue de leitões. Vinte e cinco leitões desmamados, isentos de organismos patogênicos específicos, foram separados, ao acaso, em dois grupos. Dez leitões foram infectados com 5x1 06 CFU de A. pleuropneumoníae sorotipo 1, e quinze foram usados como controle negativo. O tempo parcial ativado de tromboplastina aumentou enquanto que o tempo da protrombina, expressado em porcentagem, diminuiu. As alterações observadas no tempo de trombina e na quantidade de fibrinogênio estão relacionadas com a formação de fibrina no processo de coagulação. Admitimos que estas observações estejam ligadas ao consumo de fatores de coagulação do sangue nos caminhos intrínsecos, extrínsecos e comuns. Assim, a hemorragia pulmonar e a formação de coágulos, que são características da fase aguda dessa doença, podem ser explicadas.

The effect of Actinobacillus pleuropneumoniae infection on pigs' blood coagulation system was studied. Twenty five specific pathogen free weaned piglets were randomly assorted into two groups. Ten of the piglets were infected with 5x10 6 CFU of A. pleuropneumoniaeserotype 1, and 15 used as negative contrais. The prothrombin time, expressed as percentage, decreased. The activated partial thromboplastin time increased and the thrombin time decreased. Concentrations of fibrinogen increased during the trial. These observations were consistent with consumptions of blood coagulation factors in the intrinsic, extrinsic, and common pathways of blood coagulation, producing coagulability. Therefore intrapulmonary haemorrhage and clot formation may be observed in the lungs of piglets infected with A. pleuropneumoniae.