Human isolates of Streptococcus pneumoniae tested by traditional immunochemical methods produce a protease that cleaves human immunoglobulin A1 (IgA1) into Fab and Fc fragments. The protease may be an important virulence factor, but studies of its pathogenetic significance have been hampered by lack of a suitable animal model. Since S. pneumoniae is a respiratory pathogen for several species of animals, we sought to determine whether isolates of this organism from animals with pneumococcal infection, including fatal diplococcal pneumonia, produced an IgA protease. Isolates from six animal species including the mouse, rat, dog, guinea pig, rhesus monkey, and chimpanzee were tested for protease activity against IgA preparations from the mouse, rat, dog, guinea pig, rabbit, rhesus and cynomolgus monkeys, gorilla, and human. Cleavage of IgA was demonstrated by the appearance of Fc fragments in Western blots (immunoblots) treated with specific antisera. All these isolates except that from the guinea pig produced a protease that cleaved IgA of human, rhesus monkey, and gorilla origin. Cleavage was inhibited by 5 mM EDTA. IgA cleavage from the other species could not be demonstrated. Although S. pneumoniae can colonize the respiratory tracts of several animal species, it is a significant pathogen principally of humans and some other primates. Our data suggest that some species of nonhuman primates including the rhesus monkey could be suitable for experimental studies on the significance of IgA protease in the pathogenesis of pneumococcal disease.
Whole cells and lipopolysaccharides (LPS) of 10 isolates of Pasteurella multocida from laboratory rabbits were subjected to chemical and serological analysis. LPS of most of these isolates possessed pyrogenic potency comparable to LPS from Salmonella minnesota 9700, although their average ketodeoxyoctonate content was only 18% of that of salmonella. A gel diffusion precipitin test for somatic antigens extracted in a formal-saline solution demonstrated several isolates with three to four somatic antigens, with some variation in the major somatic type from one test to another. Conversely, the use of LPS as antigen in the gel diffusion precipitin test (i) eliminated cross-reactivity with reference antisera and (ii) often resulted in the organism being typed as serotype 12 even when the type 12 antigen was a minor antigen in the formal-saline extracts. Antisera from specific pathogen-free rabbits immunized with either whole cells or LPS of two isolates were tested against whole cells or LPS of the 10 isolates by enzyme immunoassay and indirect hemagglutination. Both whole cells and LPS of one of the isolates (isolate 2) were serologically specific, whereas those of the other isolate (isolate 1) were moderately to strongly cross-reactive with other isolates. The data indicate that although LPS is the major antigen responsible for typing based on the gel diffusion precipitin test, substances other than LPS (probably capsular polysaccharide) are responsible for the type specificity that forms the basis for the A, B, D, or E classification of this organism.
The lipopolysaccharides (LPSs) of five isolates of Pasteurella multocida from rabbits were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblots, and enzyme-linked immunosorbent assay. Silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles of purified unaggregated LPSs resembled those of semirough strains of gram-negative enterobacteria and consisted of one or two bands that migrated within an interval just ahead or slightly behind the migration of the Ra chemotype of "Salmonella minnesota," which has a molecular size of 4.3 kilodaltons. Polyclonal rabbit antisera to P. multocida whole cells used in Western blots and enzyme-linked immunosorbent assays of unabsorbed and LPS-absorbed antisera revealed that the LPS of these isolates of P. multocida contained at least two types of antigens: a nonserospecific antigen and a serospecific antigen. The LPSs of four isolates each had a different serospecific antigen. The nonserospecific antigen was expressed in two isolates and was the only demonstrable LPS antigen in one other isolate.
The immunoglobulin G (IgG) response to whole-cell and lipooligosaccharide (LOS) antigens of Pasteurella pneumotropica was evaluated in mice with latent pasteurellosis by enzyme-linked immunosorbent assay (ELISA) and immunoblots. Antibodies to cell wall proteins of P. pneumotropica also reacted with several protein antigens from isolates of Actinobacillus spp. and other pasteurellae. Conversely, antibodies to LOS antigens of P. pneumotropica demonstrated no cross-reactivity with LOSs of other Pasteurella or Actinobacillus species. IgG to cell wall proteins was detected initially by ELISA 4 weeks after experimental oronasal inoculation of specific-pathogen-free mice; antibody to LOSs was first detected 7 weeks after infection and at that time exceeded titers to other cell wall antigens. Naturally infected conventional mice from a colony with endemic latent pasteurellosis had high IgG titers to P. pneumotropica antigens at 8 to 10 weeks of age, and, as in the experimentally infected mice, antibody to LOSs predominated. Thus, LOSs of P. pneumotropica can be used as an ELISA or immunoblot antigen to detect serospecific antibodies in laboratory mice with latent pasteurellosis.
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