We purified and characterized CP from several strains of Hps, and determined that each CP contained similar sugars, but in different amounts and structures. We also discovered that the production of CP is variable. In other words, environmental conditions control whether the CP is present on the cell or not. For instance, when the bacteria are grown on agar medium CP was not found, but when the bacteria were grown in liquid culture the CP was present. In addition, the presence of sodium bicarbonate, a molecule found in increased amounts in the bloodstream during episodes of stress (such as weaning, transport, and prior infection) enhanced CP production. Increased bicarbonate levels in swine before Hps infection may be an indicator the animals are more susceptible to this bacterial infection.
We generated antibodies against purified CP and used the antibodies to confirm that CP can induce an immune response, and is the serotype-specific antigen (there are 15 known serotypes). These antibodies also proved to be useful in research assays to further characterize the role of CP in protection against Hps. For example, we determined that bacteria that lack CP are able to be killed by normal host serum, but that bacteria expressing CP were resistant to normal serum unless CP antibodies were also present. We also hypothesized that a CP-protein complex could generate a robust antibody response to the CP in piglets, but the level of protection against Hps infection that these antibodies could provide is still being evaluated.
Finally, we incorporated antibodies to the CP into a rapid diagnostic test to determine if disease in swine is due to Hps, and to assist with proper typing of various Hps clinical isolates. The diagnostic test involved permanently binding CP antibodies to latex beads, which agglutinate, or clump together, in the presence of CP as the antibodies react to CP found on whole cells or is shed into the infection site. The beads agglutinate within a minute of mixing with a positive sample of the identical serotype, and significantly less or not at all with a different serotype. Furthermore, the antibody-sensitized beads agglutinated when mixed with lysed bacteria grown on agar medium, the current industry standard for the preparation of the bacteria for clinical serotyping, but not with unlysed bacteria. These results indicates that CP is present within the bacteria grown on agar medium, but appears not to be expressed on the cell surface unless triggered by the proper environmental stimuli.
Our results have identified the CP as the molecule responsible for Hps serotype specificity, as a virulence factor, and though still preliminary, as an immuno-protective antigen. The results of our work will lead to safer, more efficient and more rapid diagnostic assays and prevention of Hps infection in swine herds without the use of potentially toxic whole cell bacterins. While more research is necessary to finalize these products for commercial use, the identification of CP as a factor in Hps infection and diagnosis will positively influence the future directions of swine health research.
Corresponding author: Phone: 540-231-5188, Fax: 540-231-5553, Email: [email protected]
