Testosterone and estradiol modify the expression of adhesins and biofilm formation in Actinobacillus seminis.

Actinobacillus seminis is the causal agent of epididymitis and has other effects on the reproductive tracts of small ruminants and bovines. This bacterium causes infection when luteinizing (LH) or follicle-stimulating hormones increase, and hosts reach sexual maturity. LH induces female ovulation and male testosterone production, suggesting that these hormones affect A. seminis pathogenicity. In the present study, we evaluated the effect of testosterone (1-5 ng/ml) or estradiol (5-25 pg/ml) added to culture medium on the in vitro growth, biofilm production, and adhesin expression of A. seminis. Estradiol does not promote the growth of this bacterium, whereas testosterone increased A. seminis planktonic growth 2-fold. Both hormones induced the expression of the elongation factor thermo unstable (EF-Tu) and phosphoglycerate mutase (PGM), proteins that A. seminis uses as adhesins. Estradiol (5 or 10 pg/ml) decreased biofilm formation by 32%, whereas testosterone, even at 5 ng/ml, showed no effect. Both hormones modified the concentrations of carbohydrates and eDNA in biofilms by 50%. Amyloid proteins are characterized by their capacity to bind Congo red (CR) dye. Actinobacillus seminis binds CR dye, and this binding increases in the presence of 5-20 pg/ml estradiol or 4 ng/ml testosterone. The A. seminis EF-Tu protein was identified as amyloid-like protein (ALP). The effect of sexual hormones on the growth and expression of virulence factors of A. seminis seems to be relevant for its colonization and permanence in the host.

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)

Serovar-dependent differences in Hfq-regulated phenotypes in Actinobacillus pleuropneumoniae.

The RNA chaperone Hfq regulates diverse processes in numerous bacteria. In this study, we compared phenotypes (growth rate, adherence, response to different stress conditions and virulence in Galleria mellonella) of wild-type (WT) and isogenic hfq mutants of three serovars (1, 8 and 15) of the porcine pathogen Actinobacillus pleuropneumoniae. Similar growth in rich broth was seen for all strains except Ap1∆hfq, which showed slightly reduced growth throughout the 24 h time course, and the complemented Ap8∆hfqC mutant had a prolonged lag phase. Differences were seen between the three serovar WT strains regarding adherence, stress response and virulence in G. mellonella, and deletion of hfq affected some, but not all of these phenotypes, depending on serovar. Complementation by expression of cloned hfq from an endogenous promoter only restored some WT phenotypes, indicating that complex regulatory networks may be involved, and that levels of Hfq may be as important as presence/absence of the protein regarding its contribution to gene regulation. Our results support that Hfq is a pleiotropic global regulator in A. pleuropneumoniae, but serovar-related differences exist. These results highlight the importance of testing multiple strains/serovars within a given species when determining contributions of global regulators, such as Hfq, to expression of complex phenotypes.

Characterization of Actinobacillus seminis biofilm formation.

Actinobacillus seminis is an autochthonous gram-negative bacterium that affects reproductive organs, causing epididymitis, low fertility, and occasional abortions in ovine and goats. The virulence factors and the pathogenicity mechanisms of A. seminis have not been clearly elucidated yet. In this work, biofilm production by A. seminis in in vitro assays is described and characterized. After 48-h incubation at 37 °C in trypticase soy broth, A. seminis formed biofilms containing an extracellular matrix comprised mainly of fibrillar material. Microaerophilia or the presence of calcium diminished biofilm formation in approximately 50% and 70%, respectively, but low iron concentrations increased it 40%. Through enzymatic digestion, it was found that proteins were the main component of these biofilms. Structural observations through scanning electron microscopy indicated the presence of a high amount of fibrillar material in which bacteria were immersed. Antibodies against different bacterial surface proteins, such as anti-biofilm matrix and anti-adhesin, diminished biofilm formation in 70% and 25%, respectively; whereas furanone C-30 and LED-209, compounds described as quorum-sensing inhibitors, completely inhibited biofilm formation. In conclusion, environmental conditions can influence strongly biofilm formation in A. seminis, and this could be an advantageous strategy that allows bacteria to persist inside a host.

Antibodies against Actinobacillus pleuropneumoniae, Mycoplasma hyopneumoniae and influenza virus and their relationships with risk factors, clinical signs and lung lesions in pig farms with one-site production systems in Brazil.

A study was conducted on 21 pig herds using one-site production system in the southeast region of Brazil to assess the relationships among serological results for primary pathogens involved in respiratory diseases (Actinobacillus pleuropneumoniae, App; Mycoplasma hyopneumoniae, Mhyo; and swine influenza virus, SIV), cough index, pneumonia index, pleuritis and herd characteristics. The prevalence of antibodies against Mhyo and SIV increased throughout the raising phases, with the highest prevalence in slaughtered pigs (> 40%), while pigs in 65% (14/21) of nurseries demonstrated marked seroprevalence of App that decreased until the day of slaughter. Pleuritis and pulmonary consolidations were recorded in 9.0 and 72.4%, respectively, of the 908 evaluated lungs. Histopathological analysis of the lung lesions revealed suppurative bronchopneumonia in almost half of the lungs (48.9%). Regression analyses were conducted to identify risk factors associated with the cough index; pleuritis; pulmonary consolidation; and App, Mhyo and SIV serological results. All-in-all-out management in nursery buildings reduced the seroprevalence of Mhyo in herds. App seroprevalence was associated with pleuritis, and the presence of cough episodes in growing pigs was associated with SIV seropositivity in nursery pigs.

Microbiological, molecular, and histopathological findings in goats experimentally infected with Actinobacillus seminis.

The objective of this study was to experimentally evaluate the pathogenicity of an Actinobacillus seminis isolate named SAAS01 in goats. Animals were challenged with 2 mL of a suspension containing 1,5 × 108 CFU/mL of A. seminis (SAAS01 isolate) through the intrapreputial, epididymis tail, and conjunctival routes. Epididymis and testicular fragments were submitted to histopathological exam, and semen samples underwent microbiological and molecular diagnoses. Clinically, a unilateral increase in firm consistency was observed in the epididymis and testicles of two animals inoculated in epididymis tail and in one animal inoculated through conjunctival sac; this firmness continued until the day of euthanasia. Two goats inoculated through epididymis tail and conjunctival sac routes presented histopathological findings with macroscopically and microscopically significant changes. A. seminis was isolated from semen samples collected from goats inoculated through the epididymis tail and conjunctival sac routes. A. seminis DNA was amplified from six semen samples of three goats inoculated through the epididymis tail, two in conjunctival sac and one through intrapreputial route. The experimental infection model using goats confirmed the pathogenicity of the A. seminis isolate, demonstrating the predilection of the agent for the epididymis, with clinical signs, histopathological lesions, bacterial isolation, and a positive molecular diagnosis.

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.

New trends in innovative vaccine development against Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae is the etiological agent of porcine pleuropneumonia, a respiratory disease leading to severe economic losses in the swine industry. The most widely used commercial vaccines are bacterins comprising inactivated whole cells of A. pleuropneumoniae but these have only been partially effective in preventing disease. Innovative immuno-prophylactic preparations of A. pleuropneumoniae based on ApxI, ApxII, ApxIII, ApxIV toxins and outer membrane proteins, among others (i.e. RnhB, GalU, GalT, HflX, ComL, LolB, LppC), have high protective efficacy in mice and pigs. Some vaccine preparations have efficacy against homologous and heterologous A. pleuropneumoniae serovars, which constitute an important advance to control porcine pleuropneumonia. In this arena, subunit vaccines based on toxins are one of the most advanced and promising developments. Many research groups have focussed on the development of live attenuated vaccines comprising strains with inactivated Apx toxins and/or other virulence factors, their protective efficacy being determined in mouse and/or swine models. Other innovative approaches such as bacteria, yeast and plants as production and oral delivery platforms have been explored in animal models and the definitive host with encouraging results. In addition, further research into A. pleuropneumoniae-based DNA and nano-vaccines, as well as bioencapsulation of antigens in plants, is envisaged. Here, the recent findings and future trends in innovative vaccine development against A. pleuropneumoniae are reviewed and placed in perspective.

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.

Characterization of Actinobacillus pleuropneumoniae field strains antigenically related to the 3-6-8-15 group from diseased pigs in Japan and 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.

Differential cellular immune response of Galleria mellonella to Actinobacillus pleuropneumoniae.

In the present work, we have investigate the cellular immune response of Galleria mellonella larvae against three strains of the gram-negative bacterium Actinobacillus pleuropneumoniae: low-virulence (780), high-virulence (1022) and the serotype 8 reference strain (R8). Prohemocytes, plasmatocytes, granulocytes, oenocytoids and spherulocytes were distinguished according to their size and morphology, their molecular markers and dye-staining properties and their role in the immune response. Total hemocyte count, differential hemocyte count, lysosome activity, autophagic response, cell viability and caspase-3 activation were determined in circulating hemocytes of naive and infected larvae. The presence of the autophagosome protein LC3 A/B within the circulating hemocytes of G. mellonella was dependent on and related to the infecting A. pleuropneumoniae strain and duration of infection. Hemocytes treated with the high-virulence strain expressed higher levels of LC3 A/B, whereas treatment with the low-virulence strain induced lower expression levels of this protein in the cells. Moreover, our results showed that apoptosis in circulating hemocytes of G. mellonella larvae after exposure to virulent bacterial strains occurred simultaneously with excessive cell death response induced by stress and subsequent caspase-3 activation.

Galleria mellonella is an effective model to study Actinobacillus pleuropneumoniae infection.

Actinobacillus pleuropneumoniae is responsible for swine pleuropneumonia, a respiratory disease that causes significant global economic loss. Its virulence depends on many factors, such as capsular polysaccharides, RTX toxins and iron-acquisition systems. Analysis of virulence may require easy-to-use models that approximate mammalian infection and avoid ethical issues. Here, we investigate the potential use of the wax moth Galleria mellonella as an informative model for A. pleuropneumoniae infection. Genotypically distinct A. pleuropneumoniae clinical isolates were able to kill larvae at 37 °C but had different LD50 values, ranging from 10(4) to 10(7) c.f.u. per larva. The most virulent isolate (1022) was able to persist and replicate within the insect, while the least virulent (780) was rapidly cleared. We observed a decrease in haemocyte concentration, aggregation and DNA damage post-infection with isolate 1022. Melanization points around bacterial cells were observed in the fat body and pericardial tissues of infected G. mellonella, indicating vigorous cell and humoral immune responses close to the larval dorsal vessel. As found in pigs, an A. pleuropneumoniae hfq mutant was significantly attenuated for infection in the G. mellonella model. Additionally, the model could be used to assess the effectiveness of several antimicrobial agents against A. pleuropneumoniae in vivo. G. mellonella is a suitable inexpensive alternative infection model that can be used to study the virulence of A. pleuropneumoniae, as well as assess the effectiveness of antimicrobial agents against this pathogen.

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.

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

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

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

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

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

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

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.

Effect of bovine apo-lactoferrin on the growth and virulence of Actinobacillus pleuropneumoniae.

Actinobacillus pleuropneumoniae (App) is a Gram-negative bacterium that causes porcine pleuropneumonia, leading to economic losses in the swine industry. Due to bacterial resistance to antibiotics, new treatments for this disease are currently being sought. Lactoferrin (Lf) is an innate immune system glycoprotein of mammals that is microbiostatic and microbicidal and affects several bacterial virulence factors. The aim of this study was to investigate whether bovine iron-free Lf (BapoLf) has an effect on the growth and virulence of App. Two serotype 1 strains (reference strain S4074 and the isolate BC52) and a serotype 7 reference strain (WF83) were analyzed. First, the ability of App to grow in iron-charged BLf was discarded because in vivo, BapoLf sequesters iron and could be a potential source of this element favoring the infection. The minimum inhibitory concentration of BapoLf was 14.62, 11.78 and 10.56 µM for the strain BC52, S4074 and WF83, respectively. A subinhibitory concentration (0.8 µM) was tested by assessing App adhesion to porcine buccal epithelial cells, biofilm production, and the secretion and function of toxins and proteases. Decrease in adhesion (24-42 %) was found in the serotype 1 strains. Biofilm production decreased (27 %) for only the strain 4074 of serotype 1. Interestingly, biofilm was decreased (60-70 %) in the three strains by BholoLf. Hemolysis of erythrocytes and toxicity towards HeLa cells were not affected by BapoLf. In contrast, proteolytic activity in all strains was suppressed in the presence of BapoLf. Finally, oxytetracycline produced synergistic effect with BapoLf against App. Our results suggest that BapoLf affects the growth and several of the virulence factors in App.

A BOX-SCAR fragment for the identification of Actinobacillus pleuropneumoniae.

Bacterial respiratory diseases are responsible for considerable mortality, morbidity and economic losses in the swine industry. Actinobacillus pleuropneumoniae, the causative agent of porcine pleuropneumonia, is one of the most important disease agents, but its identification and surveillance can be impaired by the existence of many other related bacteria in normal swine microbiota. In this work, we have evaluated a BOX-A1R-based repetitive extragenic palindromic-PCR (BOX-PCR) sequence characterised amplified region (SCAR) marker for the specific identification of A. pleuropneumoniae and its use in a multiplex PCR to detect additionally Haemophilus parasuis and Pasteurella multocida, two other major respiratory pathogens of pigs that are members of the family Pasteurellaceae. PCRs based on the BOX-SCAR fragment developed were rapid, sensitive and differentiated A. pleuropneumoniae from all swine-related members of the Pasteurellaceae family tested. Single and multiplex BOX-SCAR fragment-based PCRs can be used to identify A. pleuropneumoniae from other bacterial swine pathogens and will be useful in surveillance and epidemiological studies.

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.(AU)

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.