Serotype specificity and immunogenicity of the capsular polymer of Haemophilus pleuropneumoniae serotype 5

Abstract

Serotyping of Haemophilus pleuropneumoniae and serologic assays for detection of serotype-specific antibody are problematic due to the potential cross-reactivity of the crude antigens used for raising immune serum or for serology. The capsular polymer (CP) of H. pleuropneumoniae serotype 5 was investigated for serotypespecific activity with antiserum to whole cells or with antiserum made monospecific to CP by adsorption with a capsule-deficient mutant. When antiserum to whole cells or monospecific antiserum to CP was tested against purified CP from serotypes 1 to 7 by immunodiffusion or enzyme-linked immunosorbent assay, only capsules of serotype 5 were reactive. In addition, only encapsulated serotype 5 cells reacted with serum monospecific to CP in an indirect immunofluorescent-antibody assay. Serotype-specific antibody was completely inhibited in each assay by preincubation of purified CP with the serum. Antiserum to whole cells of H. pleuropneumoniae serotype 5 contained antibodies to proteins and lipopolysaccharide; these antibodies cross-reacted with antigens of heterologous serotypes by dot-blot enzyme-linked immunosorbent assay and immunoblotting. The antigenic activity of CP was stable after heating for at least 30 min at 100°C. High titers of antibody to CP were present in the sera of rabbits immunized intravenously with whole log-phase cells or in the convalescent sera of pigs experimentally infected with H. pleuropneumoniae serotype 5. However, the purified CP was poorly immunogenic in rabbits and swine. Our results indicate that the capsule is the serotype-specific antigen of H. pleuropneumoniae and that a monospecific antiserum to capsule or purified capsule should be used for serotyping or serologic assays, respectively.

Safety, stability, and efficacy of noncapsulated mutants of Actinobacillus pleuropneumoniae for use in live vaccines

“…The bacterial strains used in this study were A. pleuropneumoniae J45 serotype 5, A. pleuropneumoniae 4074 serotype 1, and the noncapsulated mutants of these strains. The source of the parent strains and growth conditions have been described previously (9). Bacteria were treated with ethyl methanesulfonic acid by modification of a procedure described by Murchison et al (15).…”

“…Serum for specific antibody determination was obtained prior to immunization and again immediately prior to challenge. Antibody titers were determined for noncapsulated whole cells by ELISA (9), for Apx toxins I and II by indirect ELISA (13), and for capsule by ELISA (9) or radioimmunoassay (7). Pigs were necropsied as soon as possible after death or immediately after euthanasia with sodium pentobarbital.…”

Oral immunization of pigs with viable or inactivated Actinobacillus pleuropneumoniae serotype 9 induces pulmonary and systemic antibodies and protects against homologous aerosol challenge

“…The EU data were analyzed by the positive reference standard method (5), which enables us to compare our results with those for experimentally infected convalescent pigs. Several authors have presented evidence that parenteral immunization with bacterial components or virulence factors like lipopolysaccharides, hemolysins, transferrin-binding receptor, and outer membrane proteins increases the concentrations of specific antibodies in sera or lung-washing fluids and is correlated positively with protection against clinical pleuropneumonia (2,6,7,9,10,16,27). However, the mucosal immune response induced in our FIG.…”

Efficacy of a cell extract from Actinobacillus (Haemophilus) pleuropneumoniae serotype 1 against disease in swine

“…Additionally, a portion of the free carbohydrate may be attributed to precipitation of capsular material that is released from the cells during growth and processing. The capsule may not play a major role in protection, since capsule alone is a poor immunogen (18) and acapsular mutants appear to be as virulent as the parent strain (17). This theory may also be extended to include endotoxin, on the basis of previous work by Fenwick and co-workers (10)(11)(12).…”

“…Identification of specific virulence factors and their role in stimulating protective immunity has been the focus of current research (17, 18, 22, 25, 26, 31, 36, 40, 41; J. Ma, J. Todd, and T. Inzana identified for A. pleuropneumoniae include hemolysins, endotoxins, exotoxins, capsular polysaccharides, lipopolysaccharides, permeability factor, and outer membrane proteins, including iron-regulated proteins (6, 7, 10-15, 18, 20, 22, 25-29, 31,36,41). Capsular polysaccharides are serotype specific and poorly immunogenic (18). Protection with partially purified outer membrane proteins (OMPs) and lipopolysaccharides was comparable with that of current commercial products (10)(11)(12)33), and partial protection was reported for a capsular polymer (17).…”