Condenações de carcaça suínas devido a doenças respiratórias revisão bibliográfica / Pork carcass condemnation due to respiratory diseases bibliographic review / Condena de canales de cerdo por enfermedades respiratorias revisión bibliográfica

As doenças respiratórias são um problema significativo na produção suína e podem levar à condenação de carcaças no abate. Entre os agentes causadores dessas doenças destacam-se o Actinobacillus pleuropneumoniae, Mycoplasma hyopneumoniae e a Pasteurella multocida. O Actinobacillus pleuropneumoniae é um patógeno altamente contagioso, que ocasiona hemorragia, pleuropneumonia purulenta e fibrosa. A Pleuropneumonia é amplamente distribuída e gera graves prejuízos para a suinocultura. O Mycoplasma hyopneumoniae ocasionador da pneumonia por micoplasma, doença respiratória crônica. As infecções originadas podem regular negativamente o sistema imunológico do hospedeiro e aumentar a infecção e assim a replicação de outros patógenos. A Pasteurella multocida é o agente causador de uma ampla gama de infecções levando a alto impacto econômico. Patógeno comensal e oportunista da boca, nasofaringe e trato respiratório superior. A identificação precoce e o manejo adequado desses agentes causadores de doenças respiratórias são fundamentais para minimizar a incidência de carcaças suínas. A adoção de medidas preventivas, como a vacinação e práticas de manejo adequadas, pode ajudar a prevenir a propagação dessas doenças e garantir a produção de carne suína segura e de alta qualidade para o consumo humano.(AU)

Respiratory diseases are a significant problem in pork production and can lead to condemnation of carcasses at slaughter. Among the causative agents of these diseases are Actinobacillus pleuropneumoniae, Mycoplasma hyopneumoniae and Pasteurella multocida. Actinobacillus pleuropneumoniae is a highly contagious pathogen that causes hemorrhage, purulent and fibrous pleuropneumonia. Pleuropneumonia is widely distributed and causes serious damage to pig farming. Mycoplasma hyopneumoniae causes mycoplasma pneumonia, a chronic respiratory disease. Originating infections can down-regulate the host's immune system and increase infection and thus replication of other pathogens. Pasteurella multocida is the causative agent of a wide range of infections leading to high economic impact. Commensal and opportunistic pathogen of the mouth, nasopharynx and upper respiratory tract. Early identification and proper management of these agents that cause respiratory diseases are essential to minimize the incidence of swine carcasses. Adopting preventive measures, such as vaccination and proper management practices, can help prevent the spread of these diseases and ensure the production of safe, high-quality pork for human consumption.(AU)

Las enfermedades respiratorias son un problema importante en la producción porcina y pueden provocar el decomiso de las canales en el matadero. Entre los agentes causantes de estas enfermedades se encuentran Actinobacillus pleuropneumoniae, Mycoplasma hyopneumoniae y Pasteurella multocida. Actinobacillus pleuropneumoniae es un patógeno altamente contagioso que causa hemorragia, pleuroneumonía purulenta y fibrosa. La pleuroneumonía está ampliamente distribuida y causa graves daños a la cría de cerdos. Mycoplasma hyopneumoniae causa neumonía por micoplasma, una enfermedad respiratoria crónica. Las infecciones que se originan pueden regular a la baja el sistema inmunitario del huésped y aumentar la infección y, por lo tanto, la replicación de otros patógenos. Pasteurella multocida es el agente causal de una amplia gama de infecciones que tienen un alto impacto económico. Patógeno comensal y oportunista de la boca, nasofaringe y tracto respiratorio superior. La identificación temprana y el manejo adecuado de estos agentes causantes de enfermedades respiratorias son fundamentales para minimizar la incidencia de las canales porcinas. La adopción de medidas preventivas, como la vacunación y prácticas de manejo adecuadas, puede ayudar a prevenir la propagación de estas enfermedades y garantizar la producción de carne de cerdo segura y de alta calidad para el consumo humano.(AU)

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.

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.

Isolation of Actinobacillus seminis from a goat with clinical epididymo-orchitis in Brazil

The present study reports the first isolation of Actinobacillus seminis from a goat in Brazil. A four-year-old Moxotó breeding goat in a flock of 70 goats and 65 sheep reared together in the county of Patos, semiarid region of Northeastern Brazil, showed clinical signs of unilateral orchitis and epididymitis. Diagnosis of A. seminis infection was confirmed by association of clinical findings, bacterial isolation and 16S rRNA gene sequencing. This result suggests that A. seminis may be an additional cause of infertility in goats, and that sheep may be the source of infection because the mixed farming system allows the contact between sheep and goats in the semiarid region of Northeastern Brazil.

Periodontopathogen profile of healthy and oral lichen planus patients with gingivitis or periodontitis / 国际口腔科学杂志·英文版

Oral lichen planus (OLP) is a chronic inflammatory disease that is frequently detected in oral tissues. The aim of our study was to identify the prevalence of the detection of periodontopathogenic microorganisms (Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia and Treponema denticola in OLP patients and to compare with this prevalence of periodontopathogenic microorganisms in healthy non-OLP patients. Our study included 27 (18 chronic periodontitis (OLPP) and 9 gingivitis (OLPG)) patients diagnosed with OLP along with 26 (13 chronic periodontitis (HP) and 13 gingivitis (HG)) healthy non-OLP patients. The multiplex polymerase chain reaction (PCR) with subsequent reverse hybridization method (micro-IDent) was used for identifying periodontopathogenic microorganisms present in subgingival plaque samples. The percentages of detection for A. actinomycetemcomitans, P. gingivalis, P. intermedia, T. forsythia and T. denticola in subgingival plaque samples taken from OLP patients (OLPG and OLPP) were 18.5%, 85.1%, 81.4%, 88.8% and 74%, respectively. Meanwhile, in the non-OLP patients (HG and HP), these values were 7.6%, 50%, 46.1%, 73% and 57.7%, respectively. Thus, comparing the non-OLP groups with the OLP groups, the periodontopathogens' percentages of detection in the OLP groups were higher than those in the non-OLP groups. According to our study results, OLP patients have higher levels of infection with A. actinomycetemcomitans, P. gingivalis, P. intermedia, T. forsythia and T. denticola than non-OLP patients. We argue that the high percentages in patients with OLP may help identify the importance of periodontopathogenic microorganisms in the progress of periodontal diseases of OLP.

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.

Construction and characterization of Actinobacillus pleuropneumoniae serovar 7 live attenuated vaccine strain co-expressing ApxIA / 生物工程学报

Actinobacillus pleuropneumoniae (A. pleuropneumoniae), the causative agent of porcine contagious pleuropneumonia (PCP), is a significant pathogen of the world pig industry, vaccination is potentially an effective tool for the prevention of PCP. The purpose of present study was to enhance the immunogenicity of A. pleuropneumoniae live vaccine strain HB04C- (serovar 7), which was unable to express ApxIA, and to develop effective multivalent vaccines for the respiratory pathogens based on the attenuated A. pleuropneumoniae. We introduced a shuttle vector containing intact apxIA gene into HB04C-, generating HB04C2, an A. pleuropneumoniae serovar 7 live attenuated vaccine strain co-expressing ApxIA. Then we investigated the biological characteristics of HB04C2. We found that the shuttle vector expressing ApxIA was stable in HB04C2, and the growth ability of HB04C2 was not affected by the shuttle vector. We observed that HB04C2 elicited detectable antibodies against ApxIA and ApxIIA when it was administrated intratracheally as a live vaccine in pigs, and all immunized pigs were protected from heterologous virulent A. pleuropneumoniae (serovar 1) challenge. In conclusion, we demonstrated that A. pleuropneumoniae live vaccine could be used as a vector for expression of heterologous antigens.

Osteomyelitis of the mandible due to Aggregatibacter (Actinobacillus) actinomycetemcomitans.

Aggregatibacter (Actinobacillus) actinomycetemcomitans is a capnoic gram negative coccobacilli known to produce juvenile periodontitis. This organism was isolated in pure culture from an unusual case of osteomyelitis of the mandible. The patient was treated with tetracycline, which is the drug of choice for A. actinomycetemcomitans and the clinical response improved. From our limited review of the literature, it appears that this is the first case of osteomyelitis due to A.actinomycetemcomitans reported in India.

Distribution of biotypes and leukotoxic activity of Aggregatibacter actinomycetemcomitans isolated from Brazilian patients with chronic periodontitis / Distribuição de biótipos e atividade leucotóxica de Aggregatibacter actinomycetemcomitans isolados de pacientes brasileiros com periodontite crônica

Aggregatibacter actinomycetemcomitans is an important etiologic agent of the periodontitis and is associated with extra-oral infections. In this study, the detection of the ltxA gene as well as the ltx promoter region from leukotoxic A. actinomycetemcomitans isolated from 50 Brazilian patients with periodontitis and 50 healthy subjects was performed. The leukotoxic activity on HL-60 cells was also evaluated. Leukotoxic activity was determined using a trypan blue exclusion method. The 530 bp deletion in the promoter region was evaluated by PCR using a PRO primer pair. A. actinomycetemcomitans was detected by culture and directly from crude subgingival biofilm by PCR using specific primers. By culture, A. actinomycetemcomitans was detected in nine (18 percent) of the periodontal patients and one (2 percent) healthy subject. However, by PCR, this organism was detected in 44 percent of the periodontal patients and in 16 percent of the healthy subjects. It was verified a great discrepancy between PCR detection of the ltx operon promoter directly from crude subgingival biofilm and from bacterial DNA. Only one periodontal sample harbored highly leukotoxic A. actinomycetemcomitans. Moreover, biotype II was the most prevalent and no correlation between biotypes and leukotoxic activity was observed. The diversity of leukotoxin expression by A. actinomycetemcomitans suggests a role of this toxin in the pathogenesis of periodontal disease and other infectious diseases

Aggregatibacter actinomycetemcomitans é um importante agente etiológico da periodontite e produz infecções extra-bucais. Neste estudo, foram detectados os biótipos, o gene ltxA associado à produção de leucotoxina e o promotor ltx em A. actinomycetemcomitans de pacientes com e sem periodontite. A atividade leucotóxica sobre células HL-60 também foi avaliada. A atividade leucotóxica foi determinada através do método de exclusão do azul de tripam. A deleção de 530 bp no promotor ltx foi avaliada usando-se o par de iniciadores PRO. A. actinomycetemcomitans foi detectado por cultura e por PCR. Por cultura, A. actinomycetemcomitans foi detectado em nove pacientes com periodontite (18 por cento) e em um indivíduo sadio (2 por cento). Por PCR esse microrganismo foi detectado em 44 por cento dos pacientes com periodontite e em 16 por cento dos saudáveis. Verificou-se diferença estatística entre a detecção do promotor do operon ltx, por PCR, diretamente do biofilme subgengival e do DNA bacteriano. Somente uma amostra clínica apresentou A. actinomycetemcomitans altamente leukotóxico. O biótipo II foi o mais prevalente e não foi observada correlação biótipo-atividade leucotóxica. A expressão da leucotoxina por A. actinomycetemcomitans na doença periodontal e outras doenças infecciosas necessita ser avaliado.

Transmission of Aggregatibacter actinomycetemcomitans between Brazilian women with severe chronic periodontitis and their children

This study evaluated the transmission of Aggregatibacter actinomycetemcomitans (Aa) in women with severe chronic periodontitis and their children. Thirty women (mean age = 36.1±6.0 years) who were mothers of at least one child aged 7 to 16 years were enrolled. In order to investigate mother-child transmission of Aa, the children were also evaluated when their mothers were colonized by the bacterium. Subgingival plaque samples of each woman were collected from 3 sites (mean probing depth of 7.3±1.2 mm and mean clinical attachment level of 7.9±1.5 mm) and pooled in reduced transport fluid (RTF). These samples were processed, inoculated onto TSBV-agar selective medium and incubated at 37°C in microaerophilic atmosphere for 5 days. Aa was identified on the basis of colony morphology, Gram staining, catalase and oxidase reactions. Aa was found in 8 out of 30 women. Therefore, 8 children from these women (mean age= 12 ± 3.7 years) were evaluated, but Aa was found only in 2 of them. Aa strains of the two mother-child pairs were evaluated by arbitrarily-primed polymerase chain reaction (AP-PCR), although it was not found similarity between the amplitypes of each pair. No Aa transmission was found between Brazilian women with severe chronic periodontitis and their children.

Este estudo avaliou a transmissão de Aggregatibacter actinomycetemcomitans (Aa) entre mulheres com periodontite crônica severa e seus filhos. A amostra constituiu-se de 30 mulheres com idade média de 36,1 ± 6,0 anos, mães de filhos com idade entre 7 e 16 anos. Apenas crianças cujas mães haviam sido colonizadas por Aa foram incluídas. Amostras de placa dentária subgengival foram colhidas de três sítios com profundidade de sonagem média de 7,3 ± 1,2 mm e perda de inserção clínica média de 7,9 ±1,5 mm e agrupadas em fluido de transporte reduzido (RTF). Estas amostras foram processadas e semeadas em meio seletivo ágar TSBV e incubados a 37ºC em atmosfera de microaerofilia por 5 dias. Aa foi identificado baseado na morfologia colonial, coloração de Gram e testes da catalase e oxidase. Aa foi detectado em 8 das 30 mulheres . Assim, 8 filhos destas mulheres, com idade média de 12 ± 3,7 anos foram investigados, mas Aa foi detectado em apenas 2 deles. Cepas de Aa dos 2 pares de mães e filhos foram submetidos a análise pela técnica de reação em cadeia de polimerase usando primers arbitrários (AP-PCR), mas os amplitipos de cada par não demonstraram similaridade. Portanto, não foi encontrada transmissão de Aa entre mulheres brasileiras com periodontite crônica severa e seus filhos.

Association between infection of different strains of Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans in subgingival plaque and clinical parameters in chronic periodontitis / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

<p><b>OBJECTIVE</b>The aim of this study was to investigate subgingival infection frequencies of Porphyromonas gingivalis and Actinobacillus actinomycetemcomitans strains with genetic variation in Chinese chronic periodontitis (CP) patients and to evaluate its correlation with clinical parameters.</p><p><b>METHODS</b>Two multiplex polymerase chain reaction (PCR) assays were developed to detect the 16SrDNA, collagenase (prtC) and fimbria (fimA) genes of P. gingivalis and the 16SrDNA, leukotoxin (lktA) and fimbria-associated protein (fap) genes of A. actinomycetemcomitans in 60 sulcus samples from 30 periodontal healthy subjects and in 122 subgingival plaque samples from 61 patients with CP. The PCR products were further T-A cloned and sent for nucleotide sequence analysis.</p><p><b>RESULTS</b>The 16SrDNA, prtC and fimA genes of P. gingivalis were detected in 92.6%, 85.2% and 80.3% of the subgingival plaque samples respectively, while the 16SrDNA, lktA and fap genes of A. actinomycetemcomitans were in 84.4%, 75.4% and 50.0% respectively. Nucleotide sequence analysis showed 98.62%~100% homology of the PCR products in these genes with the reported sequences. P. gingivalis strains with prtC+/fimA+ and A. actinomycetemcomitans with lktA+ were predominant in deep pockets (>6 mm) or in sites with attachment loss > or =5 mm than in shallow pockets (3~4 mm) or in sites with attachment loss < or =2 mm (P<0.05). P. gingivalis strains with prtC+/fimA+ also showed higher frequency in gingival index (GI)=3 than in GI=1 group (P<0.05).</p><p><b>CONCLUSION</b>Infection of P. gingivalis with prtC+/fimA+ and A. actinomycetemcomitans with lktA+ correlates with periodontal destruction of CP in Chinese. Nonetheless P. gingivalis fimA, prtC genes and A. actinomycetemcomitans lktA gene are closely associated with periodontal destruction, while A. actinomycetemcomitans fap gene is not.</p>

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.

Prevalence of leukotoxic genotypes of Actinobacillus actinomycetemcomitans in Brazilians with chronic periodontitis

Actinobacillus actinomycetemcomitans is considered a major etiologic agent of aggressive periodontitis but this species has also been associated with other forms of periodontal disease. Further, highly leukotoxic strains are related to severity of disease. This investigation determined the prevalence of A. actinomycetemcomitans and the occurrence of the leukotoxin gene 530-bp deletion in Brazilian subjects with chronic periodontitis. Twenty periodontally healthy and 20 chronic periodontitis subjects were selected. Full-mouth clinical examination was carried out at 6 sites/tooth. Subgingival biofilm samples were collected from the 3 deepest sites and 3 healthy sites from periodontitis subjects, as well as from 3 sites of individuals with periodontal health. A. actinomycetemcomitans and the genetic deletion were determined by the polymerase chain reaction. Significant differences were sought by Mann-Whitney, Chi-square, and Wilcoxon sign tests. Periodontitis subjects presented a higher prevalence (75 percent) of A. actinomycetemcomitans than individuals with health (45 percent) (p = 0.053). A mean frequency of 57.5 percent of A. actinomycetemcomitans - positive sites was observed in the periodontitis group. Of those, 75 percent were diseased, whereas 40 percent were healthy sites (p = 0.0001). Healthy subjects showed a mean frequency of 35 percent of positive sites. In contrast, the genetic deletion was detected only in 4 diseased sites from 2 chronic periodontitis patients. A high prevalence of A. actinomycetemcomitans was observed in Brazilians with chronic periodontitis. However, the leukotoxin gene 530-bp deletion was rarely detected in the subgingival biofilm of these subjects.

Actinobacillus actinomycetemcomitans tem sido associado com diferentes formas de doenças periodontais, mas tal espécie é considerada o principal agente etiológico da doença periodontal agressiva. Algumas cepas de A. actinomycetemcomitans apresentam uma deleção de 530 pb na região promotora do operon do gene da leucotoxina, produzindo assim maiores quantidades desta toxina. Tal fato pode ter um importante papel na patogênese das doenças periodontais. A proposta do presente estudo foi determinar a prevalência do A. actinomycetemcomitans em amostras de biofilme subgengival de indivíduos brasileiros com periodontite crônica e saúde periodontal; bem como avaliar a distribuição do tipo genético leucotóxico desta espécie nestes indivíduos. Vinte pacientes com periodontite crônica e 20 controles com saúde periodontal foram selecionados. O exame clínico periodontal foi realizado em 6 sítios/dente em todos os dentes. Amostras de biofilme subgengival foram coletadas de 3 sítios com a maior profundidade de bolsa e 3 sítios sem doença dos pacientes com periodontite, assim como de 3 sítios aleatórios dos controles. A detecção do A. actinomycetemcomitans e a ocorrência da deleção genética foram realizadas utilizando a técnica de PCR diretamente nas amostras de biofilme. Pacientes com periodontite crônica apresentaram uma alta prevalência de A. actinomycetemcomitans (75 por cento) quando comparados aos indivíduos sem doença periodontal (45 por cento), porém essa diferença não foi significativa (p = 0.053). Foi observada uma freqüência média de 57.5 por cento de sítios com A. actinomycetemcomitans no grupo com periodontite. Destes sítios, 75 por cento eram sítios com doença, enquanto 40 por cento eram sítios saudáveis (p = 0.0001). Indivíduos sem doença periodontal apresentaram uma freqüência média de 35 por cento de sítios com A. actinomycetemcomitans. A deleção de 530-pb foi encontrada somente em 4 sítios doentes de 2 pacientes com periodontite crônica. Entretanto...

Analysis of leukotoxin gene types of Actinobacillus actinomycetemcomitans in brazilians with aggressive periodontitis

Actinobacillus actinomycetemcomitans (Aa) tem sido associado com diferentes formas de doenças periodontais, mas tal espécie é considerada o principal agente etiológico da doença periodontal agressiva. Algumas cepas de Aa apresentam uma deleção de 530 pb na região promotora do operon do gene da leucotoxina, produzindo assim maiores quantidades de leucotoxina. Tal fato pode ter um importante papel na patogênese das doenças periodontais. A proposta do presente estudo foi determinar a prevalência do Aa e a ocorrência da deleção genética da leucotoxina em pacientes com periodontite agressiva generalizada (PAG) de uma amostra da população brasileira. Trinta indivíduos com saúde periodontal e 29 pacientes com PAG participaram do estudo. Profundidade de bolsa à sondagem (PBS), nível clínico de inserção (NCI), presença de placa supragengival (PL) e sangramento à sondagem (SAS) foram avaliados em 6 sítios/dente de todos os pacientes. Amostras de saliva foram coletadas para isolamento do DNA bacteriano. A detecção do Aa e a ocorrência da deleção genética foram realizadas através da técnica de PCR diretamente nas amostras. Diferenças nos parâmetros clínicos e microbiológicos entre os grupos foram avaliadas através dos testes de Mann-Whitney, Fisher e Qui-quadrado. Associações entre os parâmetros clínicos e microbiológicos foram testadas através do teste de Pearson. Aa foi detectado com maior frequência em pacientes com PAG (96,6 por cento) do que pacientes saudáveis (76,7 por cento) (c2 = 4,9; p < 0,05). A deleção genética foi observada em 16 dos 28 (57,1 por cento) pacientes com PAG que foram positivos para Aa. Porém, nenhuma das amostras de indivíduos com saúde periodontal apresentaram a deleção (c2 = 19,15; p < 0,001). Correlações significantes entre a presença da deleção e os parâmetros clínicos PBS (r=0,312, p<0,05), NCI (r=0,406, p<0,01), PL (r=0,278, p<0,05) e SAS (r=0,409, p<0,01) foram observadas. Uma alta prevalência de Aa foi observada em brasileiros com PAG e...

Aspectos clínicos, radiográficos e microbianos de uma família com expressiva prevalência de doença periodontal / Clinical, radiographics and microbiological aspects of a family with expressive prevalence of periodontal disease

A periodontite agressiva é caracterizada por uma rápida perda de inserção conjuntiva e destruição óssea, podendo ocorrer na forma localizada ou generalizada. Na periodontite agressiva localizada ocorre o comprometimento, principalmente, dos dentes incisivos e primeiros molares permanentes. É a forma de doença periodontal mais associada com Actinobacillus actinomycetemcomitans. De modo distinto, a forma generalizada da periodontite agressiva afeta vários dentes em um ou ambos os arcos dentários. O objetivo do presente estudo foi estabelecer através de exames clínicos, radiográficos e microbianos o diagnóstico de uma família com expressiva prevalência de doença periodontal. Foram incluídos 5 indivíduos da mesma família, os quais foram submetidos à exame clínico periodontal e exame radiográfico de todos os dentes. Para a análise microbiológica foram selecionados no mínimo dois dentes para cada indivíduo, e a presença de Actinobacillus actinomycetemcomitans foi determinada pelo método de cultura bacteriana e reação em cadeia da polimerase. Dos indivíduos examinados, dois receberam o diagnóstico de periodontite incipiente, dois de periodontite agressiva localizada e um de periodontite agressiva generalizada. O Actinobacillus actinomycetemcomitans de máxima leucotoxicidade foi detectado em todos os indivíduos. Com base nestes resultados observou-se que os indivíduos apresentavam a mesma característica microbiana e diferenças na expressão e severidade da doença periodontal, sugerindo que outros fatores poderiam estar presentes modificando a manifestação clínica da doença.

Study on immunogenicity of the N-terminal polypeptide of RTX toxin I of Actinobacillus pleuropneumoniae / 生物工程学报

ApxI is one of the most important virulence factors of Actinobacillus pleuropneumoniae (APP). To study the immunogenicity of the ApxI, the complete coding sequence (3146bp) and its 5'-terminal 1140 bp fragment of the apxIA gene were separately cloned into the prokaryotic expression vector pET-28a, and expressed in the E. coli BL21 (DE3) with induction by IPTG. The expression products, rApxIA and rApxIAN, were present in a form of inclusion bodies and showed the same immunological reactivity as natural ApxI (nApxI) in Western-blot analysis. BALB/c mice were intraperitoneally immunized with the rApxIA, rApxIAN and nApxI respectively. The serum antibody levels of the rApxIAN immunized mice were significantly lower than those immunized with rApxIA or nApxI in an ApxI-specific ELISA, but serum neutralization test demonstrated that immunized mice with rApxIAN, rApxIA and nApxI could generate similar levels of antibodies neutralizing the hemolytic activity of the natural ApxI. The rApxIAN was able to elicite 80% protection rate against APP serovar 1 and 100% against serovar 2 when challenged at a dose of one LD50 after 2 weeks of boost immunization.

Acute toxicity and immunoprotection of recombinant apxI toxin of Actinobacillus pleuropneumoniae in mice / 生物工程学报

Acute toxicity and immunoprotection of Actinobacillus pleuropneumoniae (APP) ApxI toxin recombinant proteins (include crude inclusion bodies and refolded recombinant protein) were evaluated in mice, and compared with the natural ApxI extracted from culture supernatant of APP serotype 10. In the acute toxicity experiment, the three proteins were intraperitoneally injected into Kunming mice in a dose of 200microg per mouse. The body and organ weight, heamatological and biochemical indexes were examined at 24h, 7 days and 14 days post administration. There was no death after the intraperitoneal administration of the three proteins, and no significant change was found in the body weight, organ indexes, heamatological and biochemical indexes. To study their immunoprotection, the three proteins were emulsified with Freund's adjuvant respectively and vaccinated the mice twice with a 2-week of interval. Two weeks after the second vaccination, the mice were challenged intraperitoneally with a lethal dose of APP serotype 10 (1.09 x 10(8) cfu), and serums were examined by an ApxI-specific ELISA. The results revealed that the recombinant protein had a good immunogenicity and could induce protection immune reaction.

Un patógeno periodontal virulento: Actinobacillus actinomycetemcomitans / A virulent periodontal pathogen: Actinobacillus actinomycetemcomitans

Actinobacillus.actinomycetemcomitans es un bacilo Gram negativo, un patógeno virulento capaz de invadir las células epitelialesgingivales.Es agente etiológico de enfermedades periodontales destructivas y responsable de la Periodontitis Juvenil Localizada, aunque nola única causa de esta enfermedad. Es importante conocer el diagnóstico microbiológico para su estudio así como el tratamientoy terapia más eficiente para su destrucción.

Actinobacillus actinomycetemcomitans, is a Gram negative rod, a virulent pathogen able to invade the gingival epithelial cells.It is the cause of destructives periodontal diseases and responsible of Localized Juvenile Periodontitis, although it is not the onlycause of this disease.It is important to know its microbiological diagnostic ,and also the most efficient treatment and therapy to destroy it.

Study on the detection of P. gingivalis, A. actinomycetemcomitans and T. denticola and the correlation between coinfections of the microbes and levels of chronic periodontitis lesion / 中华流行病学杂志

<p><b>OBJECTIVE</b>To establish a 16S rDNA multiplex polymerase chain reaction (PCR) for simultaneously detecting P. gingivalis, A. actinomycetemcomitans and T. denticola in clinical specimens of chronic periodontitis and to study the correlation between different modes of infection and severity of the disease.</p><p><b>METHODS</b>Periodontal pocket specimens from 152 patients with mild, moderate or advanced chronic periodontitis and gingival sulcus specimens from 30 periodontally healthy individuals were collected and placed in 200 microl lysis buffer. The specimens were incubated in 100 degrees C for 10 min and 10 microl of the supernatant was directly used as PCR template. DNAs from P. gingivalis strain ATCC33277, A. actinomycetemcomitans strain Y4, T. denticola strain FM and E. coli strain DH5alpha were used as positive and negative controls in PCR with all of which were prepared by routing phenol-chloroform method. A multiplex PCR assay, using three sets of primers specific to 16S rDNA genes of the three anaerobes, was developed to detect the specimens. The target amplification fragments from 3 cases of PCR products positive for all the three anaerobes were sequenced after T-A cloning. Chi-square test was applied to analyze the correlation between different coinfection of the three anaerobes and severity of the disease.</p><p><b>RESULTS</b>The established 16S rDNA multiplex PCR assay was able to detect P. gingivalis, A. actinomycetemcomitans and T. denticola at a minimum of 10, 20 and 20 cells, respectively. In comparison with the reported corresponding sequences, similarities of the nucleotide sequences from the three anaerobes 16S rDNA amplification fragments were as high as 99.45%, 97.08% and 96.59%, respectively. Of the 30 periodontally healthy gingival sulcus specimens, only one (3.3%) positive for P. gingivalis and two (6.7%) for A. actinomycetemcomitans could be identified but all of the rest were negative. In the 152 CP periodontal pocket specimens, 147 cases (96.7%) were positive for P. gingivalis, A. actinomycetemcomitans and/or T. denticola, respectively, and 5 cases (3.3%) were negative for all the three anaerobes. The positive rate of P. gingivalis detection (91.5%, 139/152) was significantly higher than those of A. actinomycetemcomitans (72.4%, 110/152) and T. denticola (80.9%, 123/152) (chi(2) = 7.07, 18.67; P < 0.01). 89.8% of the specimens from patients showed coinfections with two (26.5%) or three anaerobes (63.3%), and the coinfection rates in the specimens from moderate and advanced CP were remarkably higher than that from mild CP (chi(2) = 10.43, P < 0.01).</p><p><b>CONCLUSION</b>The 16S rDNA multiplex PCR established in this study showed high sensitivity and specificity, which could be used to detect P. gingivalis, A. actinomycetemcomitans and T. denticola in clinical specimens. CP was an disease caused by multiple pathogenic microbes while the synergistic pathopoiesis of the three microbes was closely related to the severity of the disease.</p>