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

Abstract

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.

Application of an enzyme-linked immunosorbent assay for detection of antibodies to <i>Actinobacillus pleuropneumoniae</i> serovar 15 in pig sera

“…In Japan, serovar 2 is the most predominant, followed by serovars 1, 5 and 15. Disease due to A. pleuropneumoniae serovar 15 has been found in Aichi (unpublished data), Chiba, Ehime, Fukuoka, Hokkaido and Kagawa prefectures of Japan [ 8 , 13 , 16 , 21 , 24 ]. Thus, the development of more effective vaccines against the infection by serovar 15, and of rapid and specific serological tests for detection of antibodies to serovar 15 are highly desired.…”

“…LPS was extracted from A. pleuropneumoniae serovar 15 reference strain HS143 using the lysozyme-phenol-water method as previously described [ 11 , 24 ]. Briefly, cell pellet was suspended in 50 mM sodium phosphate buffer, pH 7.0 containing 5 mM EDTA and 0.05% sodium azide, treated with lysozyme and then stirred overnight at 4°C.…”

“…Those studies provided suggestive evidence that the antibodies to A. suis reacted with the LPS core-lipid A of serovar 15 but not with CPS and other LPS antigens of serovar 15. A recent study from our laboratory [ 24 ] shows that the low molecular weight region, corresponding to the LPS core-lipid A, was not observed in the LPS preparation obtained by the lysozyme-phenol-water method. The antigen containing mainly O-chain LPS of serovar 15 seems to be, in our hand, the antigen that retains the best specificity while keeping a good sensitivity for detection of antibodies to serovar 15 by the ELISA.…”

Characterization of nontypeable Actinobacillus pleuropneumoniae isolates

“…agar gel precipitation (AGP) test as described previously. 17 All 3 earlier field isolates of serovar 15 and the reference serovar 15 strain, but not isolates IB19-1 and IB19-2, were serotyped as serovar 15 by the AGP test, based on the observation of serovar 15-specific precipitation lines (Fig. 1).…”

“…Serotyping of the 2 field isolates in our study, as well as the serovar 15 reference strain and the 3 earlier field isolates of serovar 15, was carried out with serovar-specific rabbit antisera prepared against serovar reference strains by using an agar gel precipitation (AGP) test as described previously. 17 All 3 earlier field isolates of serovar 15 and the reference serovar 15 strain, but not isolates IB19-1 and IB19-2, were serotyped as serovar 15 by the AGP test, based on the observation of serovar 15–specific precipitation lines (Fig. 1).…”