Clinical Microbiology

JOURNAL OF CLINICAL MICROBIOLOGY, Dec. 1979, p. 934-936 0095-1 137/79/12-0934/03$02.00/0

Vol. 10, No. 6

Detection of Oral Anaerobic Spirochetes in Dental Plaque by the Indirect Fluorescent-Antibody Technique
E. JACOB,' A. L. ALLEN,2 AND R. K. NAUMAN`* Departments of Microbiology' and Periodontics,2 School of Dentistry, University of Maryland, Baltimore, Maryland 21201

Received forpublication 11 July 1979

The indirect fluorescent-antibody technique was found to be a rapid and sensitive tool for the detection of oral anaerobic spirochetes in dental plaque.

Anaerobic spirochetes are indigenous members of the oral flora of humans as well as other animal species (6). Cultural studies have shown that the appearance of spirochetes in the succession of microorganisms withindental plaque is closely correlated with the time of onset of experimental gingivitis (9). Light microscopic studies on subgingival plaque have revealed that there is a dramatic increase in the number of spirochetes from 0.6% in patients without periodontal disease to as much as 17% of the total microbial population in patients with chronic severe periodontitis (7). Although a specific role forspirochetes in periodontal disease has not yet been established, based on numerical evidence alone, there is reason to believe that they contribute to the periodontal disease state. The purpose of the present investigation was to determine the applicability of the indirect fluorescent-antibody (IFA) technique for the detection of immunologically distinct spirochetes within subgingival dental plaque. Ourlaboratory has been successful in isolating several strains of oral anaerobic spirochetes from subgingival plaque obtained from patients with chronic periodontitis. Three of these strains were designated W, 11, and 14, respectively. For the initial isolation, the membrane filter technique of Loesche and Socransky (5) was utilized, employing the media described by Socransky et al. (8) consisting ofPPLO broth (BBL Microbiology Systems) plus 5 ,ig of cocarboxylase per ml, 10% sterile pooled rabbit serum, and 0.7% ion agar. Subgingival plaque was obtained from the area of periodontal involvement and was immediately transferred to individual vials containing 0.2 ml of PPLO broth. The plaque was blended vigorously in a Vortex mixer for 1 min to disperse the organisms. One drop of this plaquewas carefully placed on top of a membrane filter with a diameter of 25 mm and a pore size of 0.45 ym (Schleicher and Schuell, Keene, N. H.) which had been layered on the surface of the PPLO-ion agar medium. All manipulations were carried out in a Coy anaerobic chamber

ment.

similar to that described by Aranki et al. (2). After 4 to 5 days of incubation at 37°C, the membrane filter was removedand typical spirochetal growth was observed within the agar and then confirmed by dark-field microscopy. A sterile Pasteur pipette was used to remove a plug of agar from the leading edge of spirochetal growth and streaked onto a separate plate of the same media composition for single clone isolation. Once individual clones were obtained, they were transferred to the medium, described above, whichlacked ion agar for liquid cultivation. Electron microscopic studies revealed that the three isolates were identical morphologically and possessed the "2-4-2" axial filament arrange-

Specific immune serum was prepared for each spirochetal isolate in New Zealand White rabbits by inoculating the marginal inner ear vein with 2.5 ml of a 6- to 7-day-old broth culture. The
immunization scheduleconsisted of a single inoculation per week for a total of 4 weeks. One week after the final inoculation, blood was collected by cardiac puncture and the serum was separated. Quantitation of specific spirochetal antibody was carried out by using the microscopic agglutination test as ordinarily used for the quantitation of leptospiral antibody (4). Microscopic agglutination titers obtained were...