Forms Of Early-onset Periodontitis Research Articles

Evidence for the role of highly leukotoxic Actinobacillus actinomycetemcomitans in the pathogenesis of localized juvenile and other forms of early-onset periodontitis.

Actinobacillus actinomycetemcomitans leukotoxin is thought to be an important virulence factor in the pathogenesis of localized juvenile and other forms of early-onset periodontitis. Some highly leukotoxic A. actinomycetemcomitans strains produce 10 to 20 times more leukotoxin than other minimally leukotoxic strains. The distribution, clonality, and intrafamilial transmission of highly leukotoxic A. actinomycetemcomitans were examined in order to determine the importance of leukotoxin in the pathogenesis of periodontitis. The polymerase chain reaction (PCR) was used to differentiate highly leukotoxic from minimally leukotoxic strains in examining 1,023 fresh A. actinomycetemcomitans isolates and strains from our culture collection. These were obtained from 146 subjects including 71 with localized juvenile periodontitis (LJP), 4 with early-onset periodontitis, 11 with post-localized juvenile periodontitis, 41 with adult periodontitis, and 19 periodontally normal subjects. The arbitrarily primed polymerase chain reaction (AP-PCR) analysis of 30 oral isolates from each of 25 subjects was used to determine the intraoral distribution of A. actinomycetemcomitans clones. AP-PCR was also used to examine the transmission of A. actinomycetemcomitans in 30 members of 6 families. The clonality of 41 highly leukotoxic A. actinomycetemcomitans strains was evaluated by both AP-PCR and ribotyping. Highly leukotoxic A. actinomycetemcomitans was found only in subjects with localized juvenile and early-onset periodontitis. Fifty-five percent of the LJP subjects harbored highly leukotoxic A. actinomycetemcomitans isolates. Seventy-three percent of the A. actinomycetemcomitans isolates in these subjects were highly leukotoxic. Highly leukotoxic A. actinomycetemcomitans infected younger subjects (mean age 13.95 years, range 5 to 28 years) than minimally leukotoxic (mean age 35.47 years, range 6 to 65 years). Most subjects were infected with only one A. actinomycetemcomitans genotype. However, PCR of whole dental plaques and subsequent analysis of up to 130 individual oral isolates suggested a possible shift in A. actinomycetemcomitans over time in that a few subjects harbored both highly leukotoxic and minimally leukotoxic strains. AP-PCR analysis was consistent with intrafamilial A. actinomycetemcomitans transmission. Ribotyping and AP-PCR analysis confirmed a previous report that highly leukotoxic A. actinomycetemcomitans consists of a single clonal type. This study suggests that localized juvenile and other forms of Actinobacillus-associated periodontitis are primarily associated with the highly leukotoxic clone of A. actinomycetemcomitans.

Localized adhesion molecule expression and circulating LFA-3 levels in adult and early onset forms of periodontitis

Because of their importance in mediating cellular interactions in chronic inflammatory diseases, this study has examined the expression of a number of adhesion molecules in adult (n=11), generalized early onset (n=5) and localized early onset (n=2) forms of periodontitis. In comparison with immunostaining profiles of cryostat sections of healthy gingival tissue (n=7), the beta 1 integrins VLA-1, VLA-2 and VLA-4 were found to be up-regulated in periodontitis, with VLA-6 being markedly elevated. Although only small differences were observed in ICAM-1 and LFA-3 expression in the gingival epithelium, there was particularly notable up-regulation of these adhesion molecules within the inflammatory infiltrates of the diseased tissues. However, there were no statistically significant differences between the serum levels of a soluble form of LFA-3 in periodontitis patients (n=47) compared with healthy control subjects (n=40), although the generalized early onset and adult periodontitis groups exhibited wider ranges of circulating LFA-3. These findings show that there is localized modulation of adhesion molecule expression in the chronic inflammatory periodontal diseases studied, but that the levels of LFA-3 in the circulation nevertheless remain unaffected.

Neutrophil migration, oxidative metabolism and adhesion in early onset periodontitis.

The aim of this study was to evaluate neutrophil function in patients suffering from the generalized form of early onset periodontitis (EOP). We investigated neutrophil migration in vivo and neutrophil superoxide production and adhesion in response to a variety of compounds; neutrophils were isolated both from blood and a skin experimental exudate of 15 patients with EOP and of 15 sex- and age-matched normal control subjects. No difference was found in neutrophil migration in vivo (71.2+/-16.4x10(6) and 68.8+/-10.7x10(6) PMN/cm2/24 h in patients affected by early onset periodontitis and normal subjects respectively) and in adhesion. The superoxide production in response to STZ and PMA was similar between the 2 groups, while superoxide production in response to fMLP was markedly lower in patients than in control subjects both in circulating neutrophils (5.6+/-2.2 versus 10.4+/-2.3 nmoles O2-/10(6) cells, p<0.0001) and in exudate neutrophils (16.3+/-4.3 versus 22.3+/-4.7 nmoles O2-/10(6) cells, p<0.005). In general, neutrophil function in patients suffering from early onset periodontitis does not differ from control subjects, suggesting that the overall defence function of these cells is normal. The only parameter that we have found to be different between the 2 groups is the low superoxide production after fMLP stimulation. The stimulus- and function-specificity of this defect in neutrophils from patients indicates the existence of a dysregulation of the signal transduction pathway distal to fMLP receptor and proximal to NADPH oxidase activation.

No association with HLA-DQB1 in European Caucasians with early-onset periodontitis.

Periodontitis is a chronic inflammatory disease of the supporting tissues of the teeth that may ultimately result in tooth loss. The human leukocyte antigen (HLA) complex plays an important role in immune responsiveness and may be involved in antigen recognition of periodontal pathogens. A recent report of a Japanese population found an association between an atypical BamHI site in the HLA-DQB1 gene and a severe form of early-onset periodontitis (EOP). The aim of the present study was to test for the existence of the site in a European Caucasian EOP population using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. No statistically significant differences were found between the patients and controls with regard to the presence of the BamHI site. It was concluded that this lack of association could reflect racial genetic variation in HLA allelic frequencies.

Actinobacillus actinomycetemcomitans serotype e--biotypes, genetic diversity and distribution in relation to periodontal status.

Actinobacillus actinomycetemcomitans isolates from 356 individuals were screened for identification of serotype e in order to investigate its distribution in relation to periodontal status. From subjects with serotype e, 1-6 isolates per subject (n = 61) were genotyped using arbitrarily primed-polymerase chain reaction (AP-PCR) and apaH gene polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) analysis to determine the genetic heterogeneity within the serotype. Furthermore, one serotype e strain per subject was tested for fermentation of 8 carbohydrates for biotyping. Among patients with adult periodontitis (n = 219), localized juvenile periodontitis (n = 55) and other forms of early-onset periodontitis (n = 18) serotypes b, a and c, respectively, were the most frequently detected serotypes. Non-periodontitis subjects (n = 64) were predominantly colonized with serotype c. Serotype e was found in 30 (14%) adult periodontitis patients, 2 (11%) early-onset periodontitis patients and in 5 (8%) non-periodontitis individuals, but in none of the 55 localized juvenile periodontitis patients. AP-PCR distinguished 3 and apaH gene PCR-RFLP analysis 2 genotypes among the 61 A. actinomycetemcomitans serotype e isolates, one genotype per subject. The AP-PCR genotypes 1 and 3 represented the apaH genotype 1 and the AP-PCR genotype 2 the apaH genotype 2. On the basis of variable fermentation of galactose and xylose, 3 biotypes among A. actinomycetemcomitans serotype e were established. Contrary to the absence of A. actinomycetemcomitans serotype e in localized juvenile periodontitis patients, its detection frequency was comparable among other forms of periodontitis and periodontal health. Clinical serotype e isolates form at least 2 genetic types and 3 biotypes.

Smoking and patterns of bone loss in early-onset periodontitis.

The aims of this study were to investigate the extent and distribution of bone loss in subjects with early-onset periodontitis (EOP) referred for periodontal care and to study the relationship between smoking and EOP. A total of 71 consecutive referrals (21 male, 50 female) under 35 years old, who were otherwise healthy, with a clinical diagnosis of severe periodontitis were recruited for the study. Bone loss was measured from available radiographs using a Schei ruler to identify 2 patterns of destruction: localized (LEOP) in 41 (58%) and generalized early-onset periodontitis (GEOP) in 30 (42%) subjects. The study population had a mean of 25.0 (SD 2.4) teeth, excluding third molars, and mean bone loss of 28.7% (SD 13.0). Bone loss was more severe in the maxilla, 30.9% (SD 13.8) compared with 26.6% (SD 14.0) in the lower arch. More than one-third (36%) of the teeth examined had at least 30% bone loss. Mean smoking experience was 9.2 pack years (SD 5.6), and 39 (55%) of the EOP subjects smoked. Smokers had significantly more maxillary bone loss than non-smokers. A much higher proportion of GEOP (70%) currently smoked compared with 44% of LEOP, P = 0.029. It is concluded that young adults with early-onset forms of periodontitis often have advanced periodontal destruction before they are referred for specialist care. In addition, there was a relationship between smoking and severe bone destruction in subjects with EOP, particularly those with generalized disease.

Structures of the antigenic O-polysaccharides of lipopolysaccharides produced by Actinobacillus actinomycetemcomitans serotypes a, c, d and e.

Actinobacillus actinomycetemcomitans is a gram-negative capnophilic coccobacillus that has been implicated in the etiology of certain forms of early-onset periodontitis as well as non-oral infections, mostly bacterial endocarditis. Five distinct serotypes of A. actinomycetemcomitans have been described. Although the O-polysaccharide (O-PS) of lipopolysaccharide (LPS) has been shown to define the serologic specificity for this species, only the structure of the O-PS of serotype b has been characterized. The focus of the current study was to define the structures of the O-PS of A. actinomycetemcomitans serotypes a, c, d and e. Structure determination was accomplished through the use of methylation, periodate oxidation, and one-dimensional and two-dimensional NMR methods. The O-PS of A. actinomycetemcomitans (OMZ-542) serotype d had [alpha]D +15 degrees and was composed of D-glucose, D-mannose and L-rhamnose (L-Rha) in a molar ratio of 1:2:1. Methylation, periodate oxidation, and two-dimensional 1H-NMR and 13C-NMR studies showed that the antigenic O-PS was a high-molecular-mass polymer composed of repeating tetrasaccharide units with the structure: [formula: see text] The O-PS of the LPS produced by A. actinomycetemcomitans (ATCC 29523) serotype a had [alpha]D +150 degrees and was found to contain 6-deoxy-D-talose (6dTalp) and O-acetyl (2:1) and was a high-molecular-mass polymer composed of O-acetyl-substituted repeating disaccharide units with the structure: [formula: see text] The O-PS of the LPS of A. actinomycetemcomitans (SUNY 67) serotype c had [alpha]D -170 degrees and was composed of 6-deoxy-L-talose and O-acetyl (2:1). Structural analysis showed that the O-PS was a high-molecular-mass polymer of repeating disaccharide units with the structure: [formula: see text] The O-PS of the LPS of A. actinomycetemcomitans (OMZ 534) serotype e had [alpha]D +57 degrees and was composed of 2-acetamido-2-deoxy-D-glucose and L-rhamnose (1:1) and by chemical and NMR analysis was found to be a polymer of repeating disaccharide units with the structure: -->4)-alpha-D-GlcpNAc-(1-->3)-alpha-L-Rhap-(1-->.

Association between HLA antigens and early onset periodontitis

HLA-A, B, C and DR antigen frequencies were determined in a group of patients with juvenile periodontitis and rapidly progressive periodontitis. In juvenile periodontitis patients, HLA-A24 and DR4 were found at a significantly higher level than in the control group, and in rapidly progressive periodontitis patients, A9 and DR4 were found at a significantly higher level than the control group. The presence of these antigens gives evidence as to the susceptibility of various forms of early onset periodontitis.

Molecular Genetics and the Analysis of Leukotoxin in A. actinomycetemcomitans.

The primary pathogen involved in certain forms of early-onset periodontitis is A. actinomycetemcomitans. Among its numerous potential virulence factors is a leukotoxin that kills certain host defense cells. In order to analyze the regulation and in vivo function of leukotoxin and other virulence factors in A. actinomycetemcomitans, molecular genetic approaches are being established. Although there has been significant progress in developing plasmids and bacteriophage as E. colilA. actinomycetemcomitans shuttle vectors, more work needs to be done. Tn5-based transposon mutagenesis has been shown to work in this organism. Targeted mutagenesis is now possible in A. actinomycetemcomitans; exogenously introduced DNA recombines efficiently with the homologous chromosomal locus. These techniques have been applied to studies of leukotoxin. Targeted mutagenesis has been used to construct leukotoxin negative mutants that are otherwise isogenic with their leukotoxin-producing parent strain. These mutants can be tested in animal models to ascertain the in vivo role of leukotoxin in A. actinomycetemcomitans pathogenesis. Gene targeting has also been used to make strains in which the leukotoxin promoter is regulating the synthesis of a β-galactosidase reporter gene. Such strains have been used to show that leukotoxin synthesis increases in cells grown anaerobically, but that several other environmental changes had little effect on leukotoxin synthesis. Finally, plasmid shuttle vectors with leukotoxin promoters from various strains of A. actinomycetemcomitans fused to reporter genes have been used in cis/trans tests to show that leukotoxin promoter sequences and strain-specific trans-acting factors are important in determining why different strains of A. actinomycetemcomitans produce different levels of leukotoxin. J Periodontol 1996;67:309-316.

HLA A9 and B15 are associated with the generalized form, but not the localized form, of early-onset periodontal diseases.

HLA proteins are genetically determined, and account in part for individual immune response. Several studies have been performed seeking an association between HLA antigens and various forms of periodontitis with no conclusive results. The aim of the present study was to determine the frequency of HLA antigens of patients suffering from the localized (LJP) and the generalized (SGP) forms of early-onset periodontitis (EOP). Twenty-six EOP patients from the same ethnic group were studied in comparison to 113 race-matched controls. The EOP group included 11 LJP and 15 SGP patients. HLA-A9 and B15 antigens were found to be significantly elevated in the patient group. These differences were found to be due to the high frequency of A9 and B15 antigens in the SGP patients, with the LJP patient group showing no significant difference from the control group. The results are in agreement with previous studies in which A9 and B15 were found to be associated with EOP. However, previous studies did not differentiate between the localized and the generalized form of EOP. These results support the hypothesis that the generalized and the localized forms of EOP are under different genetic control.

Reevaluation of the chromosome 4q candidate region for early onset periodontitis.

Evidence of linkage (lod = 3.1, theta = 0.05) was reported previously in one large kindred (the Brandywine genetic isolate) for an autosomal dominant form of early onset periodontitis (EOP) with a protein polymorphism in the vitamin D binding protein (GC) located on chromosome 4q12-q13. To evaluate the generality of this finding, 19 unrelated families (228 individuals), each with two or more EOP affected individuals, were ascertained and sampled. A restriction fragment length polymorphism (RFLP) at the GC locus and eight other polymorphic DNA markers and two red blood cell antigens located on proximal chromosome 4q in the vicinity of the GC locus were typed. Twelve genetic models of EOP were evaluated, which varied in diagnostic classification, penetrance, and mode of disease transmission. Results for all models strongly exclude linkage between an EOP susceptibility gene and this chromosomal region assuming locus homogeneity. Our data statistically exclude (lod < or = -2.0) the possibility that more than 40% of our families are linked to this candidate region for one model tested. Linkage under heterogeneity was excluded less strongly for other models, but no significant evidence in support of linkage was obtained for any model. Our results indicate that either the previous report of linkage was a false positive, or that there are two or more unlinked forms of EOP, with the form located in 4q12-q13 being less common.

Host response tests for diagnosing periodontal diseases.

Extensive data collected over the past decade demonstrate clearly that disease-active and disease-inactive periodontal pockets exist, disease progression is infrequent and episodic, and most progression occurs in a small proportion of highly susceptible individuals. Furthermore, traditionally used diagnostic procedures do not identify susceptible individuals nor distinguish between disease-active and disease-inactive periodontal sites. New diagnostic tests based on host response factors that will aid in resolving these problems appear to be possible. Sources of material for use in such tests include gingival crevicular fluid (GCF), blood cells, and blood serum. Of these, components in GCF are most promising, at least in the immediate future. Although more than 40 GCF components have been studied, efforts that attempt to relate the presence and amount of a given component to an independent measure of active disease are very few in number. As a consequence, we do not yet know the potential for most GCF components as the basis of diagnostic tests. Those components that have been documented to associate with active disease as measured by attachment loss of 2 mm or greater include alkaline phosphatase, beta-glucuronidase, prostaglandin-E2, aspartate aminotransferase, and IgG4 antibody subclass. Even in these cases, the data base is small and additional clinical studies are needed to document claims. At the present time, tests based on beta-glucuronidase, nonspecific neutral proteases, and aspartate aminotransferase are being commercialized. One test has received FDA approval. Tests based on blood cells have limited application for patients with adult periodontitis, but are useful for patients with early-onset forms of periodontitis. An abnormality in the leukocyte adherence molecules on the surfaces of neutrophils is diagnostic for generalized prepubertal periodontitis, and defects in chemotactic receptor numbers and in a surface molecule designated as GP110 are found on the neutrophils of most but not all localized juvenile periodontitis patients. Recent data indicate that enhanced unstimulated or stimulated release of PGE2 and Interleukin-1 by peripheral blood monocytes may be an indicator of susceptibility to severe periodontitis. Assessment of the humoral immune response as reflected by serum antibodies to antigens of periodontopathic bacteria shows little promise as the basis for tests diagnostic of site-specific disease activity. However, the capacity of an individual to mount an IgG2 subclass response to carbohydrate antigens may have potential as an indicator of disease susceptibility.

Early Onset Periodontitis in the United States of America

A National Survey of the Oral Health of U.S. children aged 5 to 17 was conducted by the National Institute of Dental Research during the 1986-87 school year. Eleven thousand and seven adolescents aged 14 to 17 years received a periodontal assessment. Their patterns of loss of periodontal attachment as assessed by probing at mesial sites were used to classify adolescents as cases of early onset periodontitis. Approximately 0.53% of adolescents nation-wide were estimated to have localized juvenile periodontitis (LJP), 0.13% to have generalized juvenile periodontitis (GJP), and 1.61% to have incidental loss of attachment (LA) (greater than or equal to 3 mm on 1 or more teeth). The total number of adolescents affected were not trivial. Close to 70,000 adolescents in the U.S. were estimated to have LJP in 1986-87. More destructive GJP affected an estimated 17,000 adolescents. Another 212,000 adolescents were estimated to have incidental LA. Blacks were at much greater risk for all forms of early onset periodontitis than whites. Males were clearly more likely (4.3 to 1) to have GJP than females when other variables were statistically controlled. Gender associations were more complicated for LJP because gender interacted with race. Black males were 2.9 times as likely to have LJP as black females. In contrast, white females were more likely than white males to have the disease by about the same odds. When interactions among demographic variables exist, caution must be taken in comparing results from different studies.

Clinical and laboratory characterization of early onset periodontitis.

Clinical and laboratory data were compared in 72 patients with localized periodontitis (LP) and 103 patients with generalized periodontitis (GP). Significantly more LP than GP cases had decreased neutrophil chemotaxis (CTX), and were seropositive for Actinobacillus actinomycetemcomitans (Aa). Significantly, more GP cases were seropositive for Bacteroides gingivalis (Bg). All clinical indices were similar on affected teeth in LP and GP, but the attachment loss was greater on clinically unaffected teeth in GP when compared with LP. LP cases with CTX defects had a significantly lower mean age, were more often seropositive for Aa antibodies, and were more often female than LP patients with normal CTX. Significantly more GP cases with CTX defects were seropositive for Aa antibody. GP patients with normal CTX had a higher plaque index on both affected and unaffected teeth than did GP patients with a CTX defect. Our data suggest that chemotaxis and/or specific bacteria may be contributory, but not always necessary, factors in these disorders. The overlap in clinical and laboratory profiles of LP and GP continues to cloud the distinction of these early onset forms of periodontitis.

Neutrophil chemotactic behaviour in patients with early-onset forms of periodontitis (II). Assessment using the under agarose technique.

The locomotory behaviour of peripheral blood neutrophils (PMNs) from patients with juvenile (JP) and rapidly progressive (RPP) forms of early-onset periodontal disease was studied using the under agarose technique and n-formyl-methionyl-leucyl-phenylalanine (FMLP) as the chemotractant. PMNs from experimental patients showed normal random, chemotactic and chemokinetic locomotory behaviour when compared with control subjects. Further investigation of single-cell movements using time-lapse video analysis also failed to show any significant differences in locomotory behaviour between the PMNs of experimental and control individuals. We conclude that differences in technique may account for much of the variation which exists in the literature with respect to PMN locomotion in periodontal disease. In the final analysis, it is difficult to dispute direct observation of moving cells, and using this approach, we have been unable to confirm the presence of any PMN locomotory defect in our series of patients with early-onset periodontal disease.

Neutrophil chemotactic behaviour in patients with early-onset forms of periodontitis (I). Leading front analysis in Boyden chambers.

Despite some reports to the contrary, it is generally assumed that early-onset forms of periodontal disease (including both juvenile and rapidly progressive periodontitis) are associated with a defect in neutrophil (PMN) chemotactic behaviour. Using the Boyden chamber technique and N-formyl-methionyl-leucylphenylalanine (FMLP) to assess locomotion by the leading front method, we have failed to show any evidence for such depressed PMN locomotion. Indeed, when PMN chemotaxis and chemokinesis are considered in response to a range of chemoattractant doses our results indicate significant enhancement of all but random locomotion. When taken together with other studies, our results suggest that PMNs from patients with early-onset periodontitis may show abnormal locomotory behaviour which can either be enhanced or reduced in nature. The extent to which these results reflect in vitro methodology in uncertain and, furthermore, their in vivo significance is unclear.

Abnormalities of leukocyte chemotaxis in patients with various forms of periodontitis.

Prior studies have documented that a majority of patients with juvenile periodontitis have suppressed polymorphonuclear leukocyte (PMN) chemotaxis. However, in other forms of earlyonset periodontitis, PMN migration has been studied only to a limited extent or not at all, and monocyte (MN) chemotaxis has not been extensively studied in patients with any of the forms of periodontitis. Accordingly, PMN and MN chemotaxis was studied in 7 patients with prepubertal, 37 with juvenile, and 35 with rapidly progressive periodontitis. In addition, PMN chemotaxis was studied in 8 patients with adult periodontitis. In the prepubertal group, 5 patients had abnormal PMN chemotaxis, 5 had depressed MN chemotaxis, and one had reduced serum chemotactic activity. All patients in this group had at least one form of leukocyte chemotaxis abnormality. In the juvenile periodontitis group, 17 patients had abnormal PMN responses, 16 of these were depressed while 1 was enhanced. MN chemotaxis was depressed in 4 of these patients and elevated in 2. Five patients had reduced serum chemotactic activity and one manifested a serum chemotactic inhibitor. In total, 65% of juvenile patients had some form of abnormality. In the rapidly progressive group, 15 had abnormal PMN chemotaxis, 7 had aberrant MN chemotaxis, 4 had reduced serum chemotactic activity, and 8 had a serum inhibitor of chemotaxis. No abnormalities were found in the PMNs or sera of patients with adult periodontitis. Overall, 66% of the early‐onset patients manifested some form of cell or serum‐related leukocyte chemotactic abnormality. Thus, abnormalities of leukocyte chemotaxis were present in all patients with prepubertal periodontitis and in a majority of those with juvenile and rapidly progressive disease, while no abnormalities were found in 8 patients with adult periodontitis.

Assessment of serum antibody patterns and analysis of subgingival microflora of members of a family with a high prevalence of early-onset periodontitis.

In a study of members of a large family with a high prevalence of early-onset periodontitis, we sampled the subgingival microflora and characterized 40 isolates from each sample. We surveyed serum samples by enzyme-linked immunosorbent assay for antibodies reacting with any of a panel of 21 periodontal bacteria. The mother and 7 of her 13 children had early-onset periodontitis. Bacteroides gingivalis was not detected in the subgingival flora of any affected or unaffected family member, and Actinobacillus actinomycetemcomitans was isolated from only one affected child. Capnocytophaga ochracea was isolated from five of seven affected children and from none of their normal siblings. We found no significant differences among the floras from family members who had rapidly progressive, juvenile, and prepubertal forms of periodontitis. Elevated levels of serum antibody reacting with one or more of the bacteria tested were found in all family members with disease, but in only one periodontally normal family member. Both children with prepubertal periodontitis had antibodies reacting with C. sputigena, a species not found in their subgingival floras, but with none of the other bacteria tested. All remaining affected family members had antibodies to one or more serotypes of A. actinomycetemcomitans, and four had antibodies reacting with additional bacteria, including C. sputigena, Eikenella corrodens, Fusobacterium nucleatum, and Haemophilus aphrophilus. Sera from patients contained antibodies specific for putative periodontal pathogens not found in their pocket flora, and conversely, putative periodontal pathogens for which no serum antibodies were found frequently comprised a large proportion (10% or more) of the pocket flora. In no case were both the bacterium and its antibody found. These observations are suggestive of sequential infection in the early-onset forms of periodontitis and of induction of protective immunity against reinfection by the same microorganism.