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.

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.

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.