(Porphyromonas) gingivalis Infection - Infection and Immunity

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... B. CHEN,'* LYNDA BETH DAVERN,1 ROBERT SCHIFFERLE,"2 AND JOSEPH J. ZAMBON" 2 ...... Mukkur, T. K. S., G. H. McDowell, B. A. D. Stoker, and A. K..

Vol. 58, No. 10

INFECTION AND IMMUNITY, OCt. 1990, p. 3394-3400 0019-9567/90/103394-07$02.00/0 Copyright X 1990, American Society for Microbiology

Protective Immunization against Experimental Bacteroides (Porphyromonas) gingivalis Infection PRISCILLA B.



Departments of Oral Biology' and Periodontology,2 School of Dental Medicine, State University of New York at Buffalo, Buffalo, New York 14214 Received 27 February 1990/Accepted 13 July 1990

The effects of immunization in modulating the pathogenesis of Bacteroides (Porphyromonas) gingivalis infection in a murine model system were examined. BALB/c mice were immunized by intraperitoneal injection with B. gingivalis ATCC 53977 (one injection per week for 3 weeks), or with a lithium diiodosalicylate (LIS) extract (one injection per week for 3 weeks), or with lipopolysaccharide (LPS; one intravenous or intraperitoneal injection) from this same strain. Two weeks after the final immunization, the mice were challenged by subcutaneous injection of B. gingivalis ATCC 53977. Mice immunized with bacteria had no secondary lesions and no septicemia, whereas mice immunized with LIS extract had few secondary lesions and no septicemia. Mice immunized with LPS and nonimmunized mice demonstrated secondary abdominal lesions and septicemia after challenge. Bacterial cells and LIS extract, but not LPS, induced serum antibody and antigen reactive lymphocytes, as measured by enzyme-linked immunosorbent assay, immunoblot, Western immunoblot transfer, and in vitro lymphoproliferative responses. The present study suggests that immunization with a LIS extract or whole cells may induce a protective response against experimental B. gingivalis infection.

Bacteroides (Porphyromonas) gingivalis is a gram-negative, anaerobic rod that has been associated with adult periodontitis and abscesses of oral origin. This species demonstrates significant heterogeneity as regards virulence in animal models. Studies in our laboratory (25) and other laboratories (8, 11, 30, 32) have demonstrated that B. gingivalis strains can be classified as either invasive or noninvasive depending on whether the strain migrates to a site distant from the injection site (invasive) or remains localized to the injection site (noninvasive). Immunization can alter the course of experimental B. gingivalis infections in a mouse model. Previously, we have shown that immunization with invasive B. gingivalis ATCC 53977 (A7A1-28) resulted in localization of a subsequent challenge infection with the homologous strain (4). However, mice immunized with a noninvasive strain of B. gingivalis or with Bacteroides intermedius or with Ringer solution (4) developed spreading infections after challenge with strain ATCC 53977. Similarly, Okuda and colleagues (26) found that there were slight reductions in the number of B. gingivalis 381 recovered from hamsters immunized with B. gingivalis 381 whole cells (a noninvasive strain) or its hemagglutinin when compared with sham-immunized controls. The present study sought to determine if bacterial extracts such as a membrane extract or lipopolysaccharide (LPS) could confer protective immunity against an experimental invasive B. gingivalis infection.

32). B. gingivalis ATCC 53977 was transferred from tryptic soy agar (Difco Laboratories, Detroit, Mich.) supplemented with 5% sheep blood (Crane Laboratories, Inc., Syracuse, N.Y.), 5 jig of hemin (Sigma Chemical Co., St. Louis, Mo.) per ml, 1.0 jig of menadione (Sigma) per ml, and 1% yeast extract (BBL Microbiology Systems, Cockeysville, Md.), henceforth called enriched tryptic soy agar (ETSA), to trypticase soy broth containing hemin (5 ,g/ml) and menadione (0.5 ,ug/ml). The microorganism was cultured in an anaerobic chamber containing an atmosphere of 85% N210% H2-5% CO2 (Forma Scientific, Marietta, Ohio) at 37°C to late logarithmic-early stationary phase. Bacterial purity was confirmed by phase-contrast microscopy, light microscopic examination of Gram stains, and monitoring of colony morphology after anaerobic culture. Cells were harvested by centrifugation, washed three times in phosphate-buffered saline (pH 7.2) and stored at -70°C until required. Antigen preparation and characterization. Two different preparations were prepared from whole cells of B. gingivalis ATCC 53977 for use in this study. (i) LIS extract. Bacterial cells were extracted with lithium diiodosalicylate (LIS; Eastman Kodak Co., Rochester, N.Y.) by stirring 2 x 1010 bacteria per ml in 0.3 M LIS (16) dissolved in Tris buffer (0.05 M, pH 7.2) for 1 h at room temperature and then overnight at 4°C. The extracted cells were removed by centrifugation at 12,000 x g. The supernatant was dialyzed against distilled water until the optical density at 323 nm (OD323) was

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