Actinomyces viscosus T14V-Jl and its fimbria-deficient mutant strain possessing type 1 fimbriae strongly aggregated with latex beads treated with acidic proline-rich protein 1, basic proline-rich proteins, and proline-rich glycoprotein and its deglycosylated derivative. These type 1+ strains did not aggregate with latex beads treated with other proteins, such as salivary amylase, salivary histidine-rich polypeptides, laminin, type 1 collagen, fibronectin, or Clq. The type 1+ strains also adsorbed well to experimental pellicles formed with acidic proline-rich protein 1, basic proline-rich proteins, and proline-rich glycoprotein and its deglycosylated derivative on hydroxyapatite (HA) surfaces. These interactions were inhibited with immunoglobulins and Fabs specific for type 1 fimbriae. Type 1-actinomyces exhibited feeble adsorption to latex beads or HA treated with any of the aforementioned proteins. Collectively, these data indicate that actinomyces type 1 fimbriae may specifically interact with several proline-rich salivary molecules, forming experimental pellicles on HA or polystyrene surfaces. PRPs (bPRPs) were localized in the included fractions from the Sephadex G-200 after samples were monitored by amino acid analysis (22). Acidic PRP-1 isolated as previously described (15) was kindly provided by D. I. Hay (Forsyth Dental Center, Boston, Mass.). Commercial human salivary amylase (Sigma Chemical Co., St. Louis, Mo.) was purified further by gel filtration on columns (1.5 by 110 cm) of Bio-Gel P60 (

The objective of this study was to determine whether the squirrel monkey (Saimiri scuireus) is indigenously colonized with black-pigmented bacteroides (BPB) resembling human Bacteroides gingivalis and Bacteroides intermedius (suspected periodontal pathogens) and to determine the usefulness of the squirrel monkey as an in vivo model for studying colonization by putative pathogens. We assayed the subgingival plaques of 138 monkeys of various ages and in four different colonies for the presence of anaerobic BPB microorganisms. We also tested half the animals for the presence ofActinobacillus actinomycetemcomitans. Clinical indices and levels of serum antibody to B. gingivalis were recorded. We detected BPB in 50% of the animals and A. actinomycetemcomitans in 69% of the animals. The presence of BPB was generally associated with increased age, increased gingival index, presence of calculus, and increased levels of serum antibody. These data indicate that the squirrel monkey may be a good model for studying the parameters of natural infection of the gingival crevice with suspected periodontopathogenic BPB microorganisms.

Hemolysin production was measured in strains of Prevotella intermedia. Zones of beta-hemolysis were detected on agar plates supplemented with either sheep, rabbit or human erythrocytes. A standard tube assay was performed on cell suspensions of the organism to measure hemolytic activity, which was found to be dose dependent, eliminated by heat treatment, and saturable with increasing concentrations of blood. Growth-phase experiments suggested that hemolysin production was increased during logarithmic growth and was reduced during stationary phase. Cell fractionation, performed on several strains of P. intermedia, localized the activity in the outer membrane and in cell vesicles. The biological implication of this study is that P. intermedia, by virtue of its hemolytic activity, is capable of liberating the hemoglobin from erythrocytes, thereby acquiring an essential nutrient, iron, for its metabolism.

The squirrel monkey (Saimiri sciureus) has been proposed as an in vivo model for the study of subgingival colonization by suspected periodontopathogens, such as black-pigmented porphyromonads and prevotellas (BP/P). However, the indigenous microbiota of the squirrel monkey has not been well described. Therefore, in order to more fully characterize the oral microbiota of these animals, we studied two groups of squirrel monkeys from widely different sources. Group I consisted of 50 breeding colony monkeys ranging in age from 9 months to over 6 years which had been raised in captivity; group II consisted of 16 young sexually mature monkeys recently captured in the wild in Guyana. Group I animals in captivity had developed moderate to severe gingivitis, with a mean gingival index (GI) of 2.6; 52% of the sites bled, 26% had detectable calculus, and 83% had detectable BP/P. A group I subset (six animals), for which predominant cultivable microbiota was described, had a mean GI of 2.4. Colony morphology enumeration revealed that five of the six subset animals were detectably colonized with BP/P (range, 0 to 16.9%) and Actinobacillus actinomycetemcomitans (range, 0 to 3.9%); all subset animals were colonized with Fusobacterium species (range, 0.8 to 3.6%), Actinomyces species (range, 2.3 to 11%), and gram-positive cocci (range, 1.4 to 21.4%). Predominant cultivable microbiota results revealed the presence of many bacterial species commonly found in the human gingival sulcus. At baseline, group II animals were clinically healthy and had a mean GI of 1.4; 67% of the sites bled and 2.1% had calculus, and none of the animals had detectable BP/P. Neisseriae were very common in noninflamed sites. Subsequently, when inflamed sites were compared with noninflamed sites in group II animals after they had been maintained in captivity for 6 months, inflamed sites exhibited a more complex microbiota and increased proportions of gram-negative rods and asaccharolytic bacteria.

A gene bank was created from Prevotella intermedia strain 27 chromosomal DNA, and a clone was isolated that conferred the expression of two separate modes of hemolytic activity in recombinant Escherichia coli. The original recombinant hemolytic strain (EB34) contained plasmid, pEB34, with a 5.6-kb insert from Sau 3 AI-digested P. intermedia strain 27 chromosomal DNA cloned into the Bam HI site of pUC18. EB34 and deletion subclones were tested for expression of hemolytic activity in a standard tube assay, measuring lysis of erythrocytes spectrophotometrically as a function of hemoglobin release. Cell suspensions of EB34 demonstrated a dose-dependent hemolytic activity, inhibitable by proteases, and heat treatment but not dependent on calcium ions, and not inhibitable by osmoprotectants. Cell-free lysates also demonstrated a heat inhibitable, dose dependent hemolytic activity. Sub-cloning experiments localized the hemolytic region of the insert to a 3.9-kb fragment under direction of the lac promoter. Sequence analysis of the entire insert revealed the presence of multiple open reading frames (1 to 3) in this region which correlated to different forms of hemolytic expression, such that subclones containing all open reading frames 1 to 3 demonstrated strong hemolytic phenotype on blood plates and in the tube assay. Subclones containing only ORF1 demonstrated hemolysis on plates, but not in the tube assay. Subclones containing only open reading frames 2 and 3, but not ORF1 demonstrated hemolysis in the tube assay but not on plates. Homology searches of DNA and protein databases have not revealed significant homologies with reported hemolysins or proteins in any of the open reading frames.