ANTIGENIC DIFFERENCES WITHIN ACTINOBACILLUS-PLEUROPNEUMONIAE SEROTYPE-1

Two distinct antigenic subtypes of Actinobacillus pleuropneumoniae serotype 1 were identified via coagglutination (Co-A) and designated as 1A and 1B. The reference strains, ATCC 27088 (1A) and ISU 158 (1B), were used to prepare hyperimmune rabbit sera for Co-A reagents. Of 75 serotype 1 field isolates tested by Co-A, 35 isolates typed as 1A, 12 as 1B and 28 as 1A/1B. Significant crossreactivity between the 2 subtypes was found in the Co-A and was eliminated in 20/28 1A/1B strains by using Co-A reagents prepared with rabbit sera absorbed with the heterologous reference strain. However, twelve isolates (5 1A and 7 1A/1B; 16%) showed no reaction with Co-A reagents prepared with absorbed sera. Immunoblots of outer membranes (OM) prepared from APP 1A or 1B reference strains and field isolates indicated that antigenic differences between subtypes 1A and 1B were located within the high molecular weight (MW) region of the gels (40-100 kDa). Hyperimmune rabbit sera against 1A or 1B and sera from pigs vaccinated with whole-cell, formalin inactivated 1A or 1B bacterins reacted with the high MW region only in strains of the homologous subtype. In contrast, 4 of 5 sera from 1B infected pigs and 2 of 5 sera from 1A infected pigs reacted with all APP serotype 1 strains regardless of subtype. Apparently, infection exposed cross-reactive antigenic determinants that were not exposed by immunization with killed bacteria preparations. SDS-PAGE gels with LPS purified from APP 1A, 1B, 9 and 11 showed that 1A, 9 and 11 LPS O antigens had an identical smooth ladder pattern, while 1B LPS was distinctly different. In immunoblots with OM or LPS and in dot-immunobinding assays with LPS, rabbit antiserum against APP 1A reacted with 1A, 9 and 11. In contrast, rabbit antiserum against APP 1B only reacted with APP 1B and weakly with APP 9 in the OM immunoblot and with LPS from APP 1B, 9 and 11 in the LPS immunoblot and dot-immunobinding assay. We conclude that 2 subtypes of APP serotype 1 can be distinguished based on their antigenic differences. These differences are located, at least in part, within the LPS O-antigens. LPS O-antigens from APP 1B appear more antigenically similar to APP 9 LPS than to either APP 1A or APP 1 1 LPS. There may also be antigenic differences in the capsular polysaccharides of APP 1A and 1B strains. The antigenic differences between APP subtypes 1A and 1B may be sufficient so that vaccination with one subtype would not elicit a protective immune response against the heterologous subtype.