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.

Pulmonary lesions with asteroid bodies in a pig experimentally infected with Actinobacillus pleuropneumoniae serovar 15.

Five pigs experimentally infected with Actinobacillus pleuropneumoniae serovar 15 isolated in our previous study were pathologically examined. One pig died at 2 days post inoculation (dpi) and four pigs were euthanized at 7 dpi. Autopsy revealed fibrinohemorrhagic pleuropneumonia in all pigs. Histopathologically, the lesions were characterized by extensive hemorrhage and necrosis, fibrin deposition, and multifocal abscesses composed of numerous neutrophils including oat cells and numerous Gram-negative bacilli. In one survived pig, asteroid body formation was confirmed in the lung. The bacteria within the abscesses and asteroid bodies were immunohistochemically positive for antiserum raised against A. pleuropneumoniae serovar 15. This is the first report describing porcine pleuropneumonia with asteroid bodies in a pig experimentally infected with A. pleuropneumoniae serovar 15.

Characterization of nontypeable Actinobacillus pleuropneumoniae isolates.

Two Actinobacillus pleuropneumoniae isolates from clinical cases of porcine pleuropneumonia in Japan were positive in the capsular serovar 15-specific PCR assay, but nontypeable (NT) in the agar gel precipitation (AGP) test. Nucleotide sequence analysis of gene clusters involved in the biosynthesis of capsular polysaccharide (CPS) and lipopolysaccharide O-polysaccharide (O-PS) revealed that both clusters contained transposable element ISApl1 of A. pleuropneumoniae belonging to the IS30 family. Immunoblot analysis revealed that these 2 isolates could not produce O-PS. We conclude that the ISApl1 of A. pleuropneumoniae can interfere in the biosynthesis of both CPS and O-PS.

Isolation and characterization of atypical Actinobacillus pleuropneumoniae serovar 15 lacking the apxIICA genes in Japan.

Six atypical Actinobacillus pleuropneumoniae serovar 15 strains were isolated from pneumonic lesions of naturally infected dead pigs from the same farm in Japan. Genetic analyses of apx genes revealed that the atypical isolates contained the toxin-associated genes apxIBD, apxIIICA, apxIIIBD, and apxIVA, but not apxIICA. Coinciding with the result of the atypical gene profile, analyses of toxin protein production revealed that these atypical isolates expressed only ApxIII but not ApxII. A mouse pathogenicity test showed that the atypical isolate tested seemed to be less virulent than the typical isolates. This is the first report describing the emergence of atypical A. pleuropneumoniae serovar 15, which does not produce ApxII due to the absence of apxIICA genes, in Japan.

Complete Genome Sequence of Mycoplasma bovis Strain KG4397, Isolated from Cattle in Japan.

Mycoplasma bovis is a major bacterial pathogen that causes pneumonia, mastitis, and arthritis in cattle. In this study, we performed whole-genome sequencing of an M. bovis strain isolated in Japan for the first time and announce the complete genome sequence of strain KG4397, which caused respiratory diseases in cattle in 2012.

Application of an enzyme-linked immunosorbent assay for detection of antibodies to Actinobacillus pleuropneumoniae serovar 15 in pig sera.

An indirect enzyme-linked immunosorbent assay (ELISA) using lipopolysaccharide extract as antigen was evaluated for detection of antibodies to Actinobacillus pleuropneumoniae serovar 15. The serovar 15 ELISA had a higher sensitivity and specificity than latex agglutination test for 63 and 80 sera from pigs experimentally infected and not infected with A. pleuropneumoniae, respectively. When the serovar 15 ELISA was applied to 454 field sera, high rates of seropositivity were found in pigs from farms infected with A. pleuropneumoniae serovar 15, but not in those from farms free of A. pleuropneumoniae serovar 15. The results suggest that the serovar 15 ELISA may be useful for the serological surveillance of infection with A. pleuropneumoniae serovar 15.

Molecular, biological, and antigenic characterization of a Border disease virus isolated from a pig during classical swine fever surveillance in Japan.

In the current study, molecular, biological, and antigenic analyses were performed to characterize Border disease virus (BDV) strain FNK2012-1 isolated from a pig in 2012 in Japan. The complete genome comprises 12,327 nucleotides (nt), including a large open reading frame of 11,685 nt. Phylogenetic analysis revealed that FNK2012-1 was clustered into BDV genotype 1 with ovine strains. FNK2012-1 grew in porcine, bovine, and ovine primary cells and cell lines, but grew better in bovine and ovine cells than in porcine cells. Specific pathogen-free pigs inoculated with FNK2012-1 did not show any clinical signs. Noninoculated contact control pigs also did not show clinical signs and did not seroconvert. The results suggest that FNK2012-1 may be of ruminant origin and is poorly adapted to pigs. Such observations can provide important insights into evidence for infection and transmission of BDV, which may be of ruminant origin, among pigs.

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.