MATERIALS and METHODS

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2211
Temporal Changes in Porphyromonas gingivalis
Clonal Types During a Macaca fascicularis
Ligature-Induced Periodontitis Vaccine Study P.
H. Braham, R. P. Darveau, T. J. Sims, F. A.
Roberts, G. R. Persson, L. S. Houston, and R. C.
Page University of Washington, Seattle, WA 98195
ABSTRACT In a previous Porphyromonas gingivalis
(Pg) vaccine study, protection of alveolar bone
loss in a Macaca fascicularis (Mf) ligature
induced periodontitis model was reported.
Although Pg was suppressed, it was not eliminated
in vaccinated animals. Objective To determine
if the observed persistence of Pg might be
related to the acquisition of different Pg clonal
types (CTs) following vaccination. Method
Heteroduplex analysis (HA) of the Pg 16S/23S
ribosomal intergenic spacer region (ISR) has
successfully been used to detect multiple Pg CTs
in the oral cavities of humans and Mf. This
method was chosen to initially examine 2
experimental and 3 control animals for changes in
Pg CTs in another Pg-based vaccine study of 20
animals. Pooled subgingival plaque and tongue
scrapings were taken at baseline, week 16 (prior
to ligature placement), and week 32. DNA was
extracted and nested PCR was performed to amplify
the ISR. HA was used to detect differences in
the ISR fragments and therefore differences in Pg
CTs. Results Preliminary results showed no
change in Pg CTs in 1 experimental and 2 control
animals. However, a second clonal type was
acquired after ligation in 1 experimental animal,
and 1 of 2 clonal types was apparently lost in 1
control animal by week 16. Conclusion
Heteroduplex analysis revealed that Porphyromonas
gingivalis clonal types may be both lost and
acquired during a longitudinal vaccine study
using the Macaca fascicularis ligature-induced
periodontitis model. Patterns of acquisition and
loss of Pg CTs, and any correlations to
vaccination will be determined upon analysis of
all 20 animals. Supported by NIH/NIDCR R01
DE12939.

Fig.2

• INTRODUCTION
• Periodontitis is a chronic inflammatory disease
  that is a major cause of tooth loss.
  Porphyromonas gingivalis (Pg) is strongly
  implicated in the pathogenesis of the disease.
• Previously, we have employed a ligature-induced
  periodontitis model in the nonhuman primate,
  Macaca fascicularis (Mf), to examine a whole-cell
  based Pg vaccine, which was subsequently shown to
  be protective against alveolar bone loss.
• It has been reported that in the oral cavity of
  Mf harboring Pg, the number of Pg clonal types
  that may be detected ranges from 1 to 3 using
  the method of Heteroduplex Analysis (HA) of the
  Pg 16S/23S ribosomal intergenic spacer region (2
  ) as originally described by Leys et al. in their
  studies on differentiation and detection of
  multiple Pg clonal types in humans (3 ).
• In our previous study, immunized animals that
  obtained high serum antibody titers to the
  vaccine strain of Pg (Pg 5083 originally isolated
  from Mf ) demonstrated suppression, but not
  elimination of Pg (1).
• In contrast to suppression effected by
  vaccination, the placement of ligatures in the
  animal model enhances the numbers of total
  bacteria including Pg.
• Therefore we asked if Pg clonal types were
  affected by conditions which altered Pg growth in
  vivo (vaccine suppression or ligature
  enhancement).

• MATERIALS and METHODS
• HA was employed to detect changes in Pg clonal
  types in a ligature-induced periodontitis
  vaccine study by comparing pre-vaccination
  numbers of Pg clonal types at baseline with
  samples collected post-vaccination at week 16 and
  post-ligation at week 32.
• HA PROTOCOL - Method established by Leys et al.
  (3) was employed. The procedure is briefly
  described below and exceptions to the referenced
  protocol are noted.
• Principle of HA the ISR is predominately a
  non-coding region between the highly conserved
  16S and 23S ribosomal genes. Lack of selective
  pressure results in accumulation of mutations and
  greater sequence diversity thus allowing for
  discrimination between strains of a species. When
  PCR amplified ISR DNA is melted and slowly
  cooled, double-stranded DNA is formed.
  Identical strands reanneal to form homoduplexes
  and mismatched strands form heteroduplexes that
  migrate more slowly than homoduplexes during
  polyacrylamide gel electrophoresis.
• IMMUNOGEN A Pg-derived peptide-based immunogen,
  Rgp-Kgp, was used in this study.
• RELEVANT TIME POINTS to SAMPLING

S sample collection for HA V vaccination L
ligature placement

• SAMPLE COLLECTION paperpoint subgingival plaque
  samples from all teeth tongue
  scrapings
• DNA ISOLATION and PURIFICATION
• AMPLIFICATION of ISR using NESTED POLYMERASE
  CHAIN REACTION (PCR)