I. N. Rocas, J. F. Siqueira Jr, A. F. B. Andrade &M. Uzeda
Department of Endodontics, Faculty of Dentistry, Estacio de Sa University
Department of Microbiology, State University of Rio de Janeiro, and Institute of Microbiology, Federal University of Rio de Janeiro, Rio de Janeiro, RJ, Brazil.

Introduction.
The association of spirochaetes with oral diseases has been known for many years. Amongst the oral spirochaetes, nine species have been cultured and named. Four species have been cultured widely and reliably by many laboratories: T. denticola, T. pectinovorum, T. socranskii andT. vincentii (Chan & McLaughlin 2000). Recently, 16S rRNA-based analyses have allowed the description and characterization of additional cultivable oral treponemes: T. malthophilum (Wyss et al. 1996), T. medium (Umemoto et al. 1997), T. amylovorum (Wyss et al. 1997), T. lecithinolyticum (Wyss et al. 1999) and T. parvum (Wyss et al. 2001).
Oral treponemes, particularly the species T. denticola, T. socranskii, T. pectinovorum and T. vincentii, have been reported to be associatedwithgingivitis, adult periodontitis, acute necrotizing ulcerative gingivitis, localized juvenile periodontitis, refractory periodontitis and HIV associated periodontal diseases (Socransky & Haffajee 1997, Moter et al. 1998,Willis et al. 1999, Dewhirst et al. 2000). It has been revealed that the periodontal pocket harbours a highly diverse treponeme population (Choi et al.1994, Dewhirst et al. 2000).Whilst treponemes are usually detected in periodontally diseased sites, some species are only infrequently found at periodontally healthy sites (Moter et al.1998,Willis et al.1999,Dewhirst et al.2000).This indicates a possible causal relationship.
Miller (1894) was the first to report the occurrence of spirochaetes in endodontic infections. Brown &Rudolph (1957) observed spirochaetes in 14% of their pulp samples using dark field and phase-contrast microscopy. Also using darkfield microscopy, Thilo et al. (1986) reported 6% of spirochaetes amongst the root canal microbiota. With the use of light and transmission electron microscopy, Nair (1987) revealed that spirochaetes seemed to form a significant component of the root canal microbiota. In a dark field and scanning electron microscopic study, Dahle et al. (1993) described a spirochaete isolated from a root canal that was much longer (140 mm in length) and thicker (2 mm in diameter) than usual for oral treponemes. van Winkelhoff et al. (1985) reported the presence of spirochaetes in pus samples from acute periradicular abscesses. Darkfield examination of the exudate from periodontal and endodontic abscesses found that in periodontal abscesses the spirochaete count ranged from 30 to 60%, whereas in endodontic abscesses the range was 0-10%(Trope et al.1984).I n situ scanning electron microscopic investigations have also disclosed spirochaetes in infected root canals (Molven et al.1991, Siqueira et al. 2002).
Although spirochaetes had already been observed in infected root canals by microscopy, they have only recently been identified. Dahle et al. (1996) reported that four spirochaete strains isolated from endodontic infections were found to have similarities to T. pectinovorum, but probably represented two previously unidentified newspecies. Smallwood et al. (1998) reported the occurrence of spirochaetes in all samples collected from infected canals using PCR, but did not characterize which species of spirochaetes were involved. Recently, the occurrence of T. denticola in cases of endodontic infections by means of molecular methods has been reported (Siqueira et al. 2000a,b, 2001a, Rocas et al. 2001). The checkerboard DNA-DNA hybridization method has been used to investigate the prevalences of 42 bacterial species in infections of endodontic origin, and T. denticola was found in17.9% of the cases (Siqueira et al.2000b). When the more sensitive single PCR method was used, this species was found in 34.5% of the canals associated with asymptomatic chronic periradicular lesions, 53.3% of the cases of acute apical periodontitis and in 50%of the teethwithacute periradicular abscess. In general, T. denticola was found in 42.6% of the cases (Siqueira et al.2001a).Roc¸as et al. (2001) found relatively high prevalence of T. denticola in endodontic infections, sometimes forming a complex with Bacteroides forsythus and Porphyromonas gingivalis. Taken together, these reports suggested that T. denticola can participate in the pathogenesis of periradicular diseases. Dot-blot hybridization with species-specific oligonucleotide probes and amplified bacterial DNA has recently allowed the detection of T. maltophilum and T. socranskii in root canal infections (Jung et al. 2001). T. socranskii was also recently detected in pus samples from acute periradicular abscesses by checkerboard DNA-DNA hybridization (Siqueira et al. 2001b).
Studies have revealed that PCR methods are more sensitive, rapid and accurate than the culture method for the detection of anaerobic bacteria (including spirochaetes) in saliva and subgingival plaque samples (Ashimoto et al. 1996, Sakamoto et al. 2001). Therefore, the purpose of this study was to investigate the prevalence of four Treponema species, T. denticola, T. socranskii, T. vincentii and T. pectinovorum, in root canal infections using a nested PCR assay.

Materials and methods.

Specimen sampling.
Thirty-two single-rooted teeth from adult patients, all of them having carious lesions, necrotic pulps and radiographic evidence of periradicular disease, were included in this study. Of these, 22 cases had chronic asymptomatic periradicular lesions and 10 cases were diagnosed as acute apical periodontitis (six cases showing both spontaneous symptoms and tenderness to percussion and four cases showing only tenderness to percussion). The teeth had no periodontal pockets over 4 mm deep. The ages of the patients ranged from 18 to 60 years.
Samples were collected using strict asepsis. The tooth was cleansed with pumice and isolated with a rubber dam. The tooth and the surrounding field were then cleansed with 3% hydrogen peroxide and decontaminated with a 2.5% sodium hypochlorite solution. Access cavities were prepared using sterile burs without water spray. The operative field, including the pulp chamber, was then swabbed with 2.5% sodium hypochlorite. This solution was inactivated with sterile 5%sodiumthiosulphate. If the root canal was dry, a small amount of sterile saline solution was introduced into the canal. Samples were initially collected by means of a size 15 K-type file (Dentsply Maillefer, Ballaigues, Switzerland) with the handle cut off. The file was introduced to a level, approximately1mmshort of the tooth apex, based on diagnostic radiographs and a discrete filing motion was applied. Afterwards, two sequential paper points were placed to the same level and used to soak up the fluid in the canal. Each paper point was retained in position for 1min. The cut file and the two paper points were then transferred to cryotubes containing 1mL of 5% dimethyl sulphoxide in trypticase-soy broth (Difco, Detroit, MI, USA) (TSB-DMSO). Samples were immediately frozen at _20 8C.

DNA extraction.
The samples in TSB-DMSO were thawed to 37 8C for 10 min and vortexed for 30 s. Microbial suspensions were washed three times with100 mL of double-distilled water by centrifugation for 2 min at 2500 g. Pellets were then resuspended in 100 mL of double-distilled water, boiled for10 min and chilled on ice. After centrifugation to remove cell debris for10 s at 9000 g at 4 8C, the supernatant was collected and used as the template for PCR amplification.
Reference DNA from T. denticola B1 (Forsyth Dental Center),T. pectinovorum ATCC 33768,T. socranskii S1 (Forsyth Dental Center) and T. vincentii ATCC 35580was also extracted to serve as positive control for the primers used.

Oligonucleotide primers.
Primer sequences are shown in Table 1. Universal primers anneal at conserved regions near the 50- and 30- ends of 16S rDNA, generating a practically full-length 16S product. Sequences for T. denticola and T. socranskii consisted of specific forward and reverse primers as described by Ashimoto et al. (1996) and Willis et al. (1999), respectively. Primers for T. pectinovorum and T. vincentii utilized an universal forward sequence (positions 8-27 of E. coli16S rDNA) with a species-specific reverse sequence as described by Willis et al. (1999). Primers were purchased from Oligos Etc. Inc. (Wilsonville, OR, USA).

Table 1. PCR primer pairs used for detection of four treponemal species in infected root canals.

Nested PCR.
Five microlitres of the supernatant from clinical samples was used as target in the PCR reaction using universal 16S rDNA primers. One microlitre of the universal reaction was then used as template for each nested specific reaction. PCR amplification was performed in 25 mL of reaction mixture containing 0.2 mm concentration of forward and reverse primers, 2.5 mL of10X PCR buffer, 1.5 mm MgCl2, 1.25 U of Taq DNA polymerase (Gibco BRL, Gaithersburg, Md, USA) and 25 mm concentrations of each deoxyribonucleoside triphosphate (dATP, dCTP, dGTP and dTTP) (Gibco BRL). PCR reactions were performed in 25-well microtitre plates.
Preparations were amplified in a DNA thermocycler (Primus 25/96, MWG-Biotech, Ebersberg, Germany). The PCR temperature profile included an initial denaturation step at 97 8C for1min, followed by 26 cycles of a denaturation step at 97 8C for45 s, a primer annealing step at 55 8C (for the universal16S rDNA primer),60 8C (for T. denticola primer), 53 8C (for T. pectinovorum or T. socranskii) or 56 8C (for T. vincentii) for 45 s, an extension step at 72 8C for 1min and a final step of 72 8C for 4 min.
Eight microlitres of each PCR product were electrophoresed on a 1.5% agarose gel at 4 Vcm_1 in Tris- borate-EDTA buffer. The gel was stained for 15 min with 0.5 mg mL_1ethidiumbromide and photographed under ultraviolet light. Positive reactions were determined by the presence of bands of the appropriate sizes (Table 1). Both 100 bp and 1 kb DNA ladder digests (Gibco BRL) were used to size markers.