Outer membrane protein profiles of Haemophilus pleuropneumoniae were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cells were disrupted by sonication, and outer membrane-enriched fractions were prepared by differential centrifugation and selective solubilization of the inner membrane with sodium N-lauroyl sarcosinate. Colony type, growth medium, time of harvest, and in vitro or in vivo passage had no appreciable effect on the protein profiles of the strains examined. Seven patterns were distinguished among the reference strains of the nine capsular serotypes. These patterns were based on the mobility of the major outer membrane proteins migrating in the 39,000to 44,000-molecular-weight region of the gel, a 16K to 16.5K protein, and a heat-modifiable 29K protein. Strains of serotypes 1 and 9 had identical outer membrane protein profiles, as did strains of serotypes 2 and 6. The reference strains of the remaining five serotypes each had a distinct pattern. The outer membrane protein proffles of 95 field isolates belonging to serotypes 1, 5, 7, and 9 from swine in the midwestern United States were determined and compared with the reference patterns. The results indicate that the population of H. pleuropneumoniae is clonal, with three predominant clones distinguished by both serotype and outer membrane protein profile responsible for the majority of H. pleuropneumoniae disease occurring in swine in the United States.

Genetic diversity among 135 isolates of nine serotypes of Haemophilus pleuropneumoniae recovered from pigs with pleuropneumonia or other invasive diseases in 14 countries was estimated by multilocus enzyme electrophoresis, which detects allelic variation in structural genes. Thirty-two multilocus genotypes (electrophoretic types [ETs]) were distinguished on the basis of allele profiles at 15 enzyme loci, and 36 distinctive combinations of ET and serotype were identified. The recovery of isolates with identical properties in widely separated geographic regions and over a 20-year period indicated that the population structure of H. pleuropneumoniae is clonal. Isolates of the same ET generally shared the same serotype and electrophoretic pattern of the outer membrane proteins, but some ETs were represented by isolates of several different serotypes, outer membrane protein patterns, or both. On average, the genetic diversity among ETs of the same serotype was 56% of the total genetic diversity in the species. Isolates of serotype 1, which are unusually pathogenic, belong to a distinctive group of clones that are closely related to clones marked by serotype 9.

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

Of 30 sows from a herd believed to be free of Haemophilus pleuropneumoniae infection, 2 had complementfixing antibodies to H. pleuropneumoniae serotype 5. Necropsy and microbiological examination of the two sows revealed no evidence of H. pleuropneumoniae infection; however,)haemophilus taxon "minor group" and a urease-negative, indole-positive Haemophilus sp. were isolated from numerous respiratory tract sites in both sows. Isolation of these Haemophilus spp. was facilitated by serially diluting specimens in two broth media. Pigs from a closed, respiratory disease-free herd were inoculated with four strains of Haemophilus taxon "minor group" to determine whether the organism induces antibodies which cross-react with H. pleuropneumoniae in the complement fixation test. Antigenic heterogeneity among the taxon "minor group" strains was apparent; however, antibodies cross-reacting between these strains and H. pleuropneumoniae serotypes 1 through 5 and 7 were not detected.

SDS-PAGE has proven to be a powerful technique for the separation and identification of OMPs. Data indicate that OMR profiles are stable, genetic markers for strains of Haemophilus influenzae type b (12), and E. coli (1). As such, OMP profiles have proven useful as epidemiologic markers (12), and in the characterization of strains by serotyping (5, 10) and subtyping (12, 32). Expression of OMPs may reflect differences in morphologic and virulence attributes as has been demonstrated with pathogenic strains of Neisseria spp. (26, 62) and Bordetella spp. (7). External to the OM of most gram-negative bacteria is a glycocalyx which generally takes the form of a well-defined capsule (6). Bacterial capsules are biochemically and physically heterogeneous. They may be rigid, flexible, integral, or peripheral, and are generally composed of a fibrous anionic polysaccharide matrix (6). Direct examination of bacteria in nature and disease indicates the almost universal presence of an extensive glycocalyx, and it is presumed to be a structure essential to in vivo survival in the presence of antibacterial factors (6). Expression of the glycocalyx, however, is often reduced or lost in vitro, exposing other cellular components with different properties of adhesion, antibiotic sensitivity, and antigenicity (6). Antigenic determinants of cell surface components which are accessible to antibody in situ are candidate protective antigens. Therefore, it is not unexpected that with encapsulated organisms, investigations often focus on the potential immunogenicity of capsular material. The demonstration that immunity to meningococcal disease was 19 SECTION I: SEROTYPING OF HAEMOPHILUS PLEUROPNEUMONIAE