Antibody response of swine to outer membrane components of Haemophilus pleuropneumoniae during infection

Abstract

Sera from pigs infected with Haemophilus (Actinobacilus) pkuropneumoniae were tested for antibodies to outer membrane proteins (OMPs) of the organism by immunoblotting. Convalescent sera were produced in naturally born, colostrum-fed pigs and in cesarean-derived, colostrum-deprived pigs given H. pleuropneumoniae serotype 5 intranasally twice at 5-week intervals. Sera, collected at weekly intervals, were reacted with Sarkosyl-insoluble, OMP-enriched preparations of H. pleuropneumonwe which had been separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to nitrocellulose. Antibodies were detected to OMPs with an apparent molecular weight of 16,500 (16.5K OMP); to 29K, 38.5K, 43.5K, 45K, 49.5K, and 66.5K OMPs; and to several high-molecular-weight (.94,000) OMPs, but not to the major 42K OMP. Antibodies to the heat-modifiable OMP (29K/43.5K) and the 38.5K OMP were detected in sera from noninfected pigs. Antibodies were also detected to two broad 54,000and 95,000-molecular-weight bands which did not stain with Coomassie blue, stained with silver nitrate, resisted proteinase K digestion, and were eliminated by oxidation with sodium metaperiodate. This indicates that the 54,000and 95,000-molecularweight bands represent polysaccharide, possibly capsular or lipopolysaccharide immunogens. Adsorption of sera with cells from the homologous serotype 5 strain removed antibodies to the 45K, 49.5K, 66.5K, and .94K OMPs and to the two polysaccharide bands, indicating that these antibodies were directed primarily to surface-exposed epitopes. When tested with OMP preparations from other serotype 5 strains, heterogeneity was apparent, both in the reactions with OMPs and with the polysaccharide bands. Silver staining of proteinase K-treated, whole-cell lysates from serotype 5 strains also indicated variable expression of the polysaccharide bands. Sera also reacted with OMPs from H. pkuropneumoniae serotypes 1 and 7; however, several OMPs and the lipopolysaccharide or polysaccharide determinants of these serotypes appeared to be type specific. Haemophilus (Actinobacillus) pleuropneumoniae is a leading cause of pleuropneumonia in swine throughout the world (36). Specific immunity to infection can be acquired following exposure to the organism (37) or vaccination (17, 24, 38, 51), and antibodies to H. pleuropneumoniae are routinely assayed by several techniques (31, 34, 35, 37). The complement fixation (CF) test, in particular, has proven to be useful for monitoring disease prevalence and for disease control (34, 52

Outer membrane proteins of Actinobacillus actinomycetemcomitans and Haemophilus aphrophilus studied by SDS‐PAGE and immunoblotting

“…Our present results were also close to those achieved with SDS-PAGE and itnmunoblotting of OMP frotn Haemophilus pleuropneumoniae (12,25). Pohl et al (19) and Maclnnes & Rosendahl (12) found that H. pleuropneumoniae shared greater homology with members of genus Aetinobaeillus based on phenotypic and deoxyribonucleie acid relatedness than with metnbers of genus Haemophilus.…”

Characterization of antigens recognized by new monoclonal antibodies raised against culture filtrate proteins of Mycobacterium bovis bacillus Calmette-Guérin

“…In addition, treatment of CF with NaIO R , known to oxidize sugar residues to aldehydes, did not abrogate recognition by the mAb, showing that no glycidic substituent was involved in mAb-antigen binding [17]. This treatment only abolished recognition by mAb WB8A11, indicating that at least one of the antigens, the 82-kDa protein, is glycosylated (Fig.…”

“…Samples of BCG CF (15 Wg) were diluted in 100 Wl PBS containing 1 mg ml 3I trypsin or 1 mg ml 3I papain and incubated at 37³C for 2 h. To determine if the epitopes reacting with the mAbs were glycidic, 15 Wg of BCG CF were diluted in 100 Wl of 100 mM Na-acetate pH 4.5, 10 mM NaIO R and incubated at 37³C for 2 h. They were then subjected to SDS-PAGE and abrogation of recognition by the mAbs was evaluated by Western blotting [17].…”

Identification and characterization of a porcine‐specific transferrin receptor in Actinobacillus pleuropneumoniae

“…1986a,b). Analysis of the serological response to outer-membrane antigens during A. pieuropneumoniae infection in pigs has identified a number of outer-membrane proteins that are only reactive with convalescent serum (Rapp and Ross, 1986), including 76kD and 95 kD iron-repressible proteins (Deneer and Potter, 1989). Reactivity of the sera with outer-membrane proteins from other serotypes and removal of reactive antibody by adsorption with intact cells implicate the existence of conserved surface epitopes.…”