Scand. J. Dent. Res. 1977: 85: 106-113 (Key words: anfibodies; dental canes; dental plaque; periodcntal
disease}
Serum antibodies to plaque bacteria in subjects with dental caries and gingivitis DAG ORSTAVIK .AND PER BRANDTZAEG Department of Microbiology, Dental Faculty, University of Oslo, Oslo, Norway ABSTRACT — Correlations were sought between indices of gingival inflammation and dental caries experience and serum antibody titers to five species of oral bacteria. The material camprised 53 young adult males. .A statistically significant, negative correlation was observed between the antibody titer to a pool of Veillonella strains and dental caries experience. Multiple regression analyses failed to reveal significant associations between periodontal disease and serum antibody titers. However, the data suggested a combined association of the titers to the strains of Veillonella and a strain of Fusohacteriuni with the periodontal index. (Accepted for publication 25 April 1976)
Gingivitis in man results from accumulation of bacterial plaque at the gingiva! margin (LOE, THEIL.\DE &. JENSEN 1965).
The immunogenicity and mitogenicity of plaque bacteria are indicated by the appearance of lymphocytes with specificity for plaque antigens in peripheral blood (IvANYi & LEHNER 1970) and by popula-
tion of the gingival lesion with immunoglobulin (Ig)-producing cells (BRANDTZAEG & KRAUS 1965). Correlations have
been found between the severity of gingival inflammation and the stimulatory response of circulating lymphocytes {see LEHNER 1975). Less is known about the association of serum antibodies to plaque bacteria with periodontal disease (EVANS, SPAETH & MERGENHAGEN 1966, KRISTOFFERSEN & HOFSTAD 1970, GILMOUR & NiSENGARD 1974).
Serum antibodies tO' potentially cariogenic microorganisms have been found in subjects with and withotjt caries (KENNEDY, SHKLAIR, HAYAISHI & BAHN 1968),
and complex relationships between such antibodies and dental caries experience have been indicated (LEHNER, WILTON & WARD 1970, GHALLAGOMBE 1974).
In the present study an attempt was, made to detect associations between serum antibody titers to a number of plaque bacteria and the severity of gingiva! inflammation and dental caries experience. To reduce the likelihood of possible age-dependent, differential antibody responses, army recruits of near-uniform age were chosen for the study. The choice of antigens was based on the following considerations: Streptococcus mutans has been associated with dental caries; it is prevalent
SERUM ANTIBODIES TO PLAQUE BACTERIA in man and the serotype d is of frequent occurrence (BRATTHALL 1970, 1972); two strains of this serotype were included in the present study. Streptococcus salivarius is numerous in saliva and on the tongue dorsum (GIBBONS, KAPSIMALIS & SOGRANSKY 1964), but sparse in dental plaque (GARLSSON 1965,a); one strain was included as an indicator of the immunogenicity of bacteria other than the plaqueprevalent species. Streptococcus sanguis, on the other hand, is prevalent in dental plaque (CARLSSON 1965b); two strains of unknown antigenic relationship were used. Finally, Fusobacterium and Veillonella strains were included because sonicates of these species have been shown to induce transformation of lymphocytes from ,subjects with periodontal disease (IVANYI & LEHNER 1970),.
Material and methods SUBJECTS
Norwegian army recruits in good systemic health, aged 19 to 22 years, were studied. Their periodontal status was assessed by the periodontal index (PI) of RUSSELL (1956) and their dental caries experience by the DMF index of KLEIN, PALMER & KNUTSON (1938). Venous blood was
obtained and allowed to clot at room temperature for 1 h and at 4°C for approximately 4 h. The serum was then collected after centrifugation at 500 g for 10 min at 4°G. and stored at
BACTERIA
Streptococcus mutans OMZ 176E was a gift from Dr. J. E. NORTON. S. mutans 6715 and S. sanguis ATCG 10556 were obtained from Dr. H. J. SANDHAM, S. salivarius 9GS2 and S. sanguis 34 from Dr. R. J. GIBBONS, Veillonella antigenic types I to VII (ROGOSA 1965) from the National Institute of Dental Research (Bethesda, Maryland), and Fusobacterium strain F4 from Dr. T. KRISTOFFERSEN. The streptococci were grown anaerobically at 37°C in Trypticase Soy BrothTlI (BBL, Cockeysville, Maryland) for 24 h, harvested by centrifugation (10,000 g, 15 min, 4°C), washed three
107
times in PBS (0.01 M phosphate buffer, pH 7.2, 0.14 M NaCl), and resuspended to an absorbance (A) of 0.2 at 540 nm. Tbe Veillonella strains were grown anaerobically at 37 °C for 48 h in the V17 medium of ROGOSA (1964), harvested, washed, and resuspended to Ag^o = 0.2. Equal portions of the seven antigenic types were then pooled. The Fusobacterium strain F4 was received as a washed and frozen suspension in PBS; this suspension was thawed and adjusted to A540 = 0.2.
INDIRECT IMMUNOFLUORESCENCE
The bacterial suspensions were spotted in 10-[ll portions onto glass slides, air dried and fixed for 10 min in 96% ethanol at 4°C. The slides were then brought through decreasing concentrations of ethanol to PBS, dipped in demineralized w-ater, dried and incubated in a moist chamber with serial twofold dilutions (1:10 t° 1:640) of the human sera for 1 h at room temperature. One spot on each slide was incubated with PBS for control purposes. The slides were then washed twice in PBS for 7 min, dipped in demineralized water, dried, and incubated for 30 min with a fluorescent goat inimimogiobuiin (Ig) conjugate active against human IgG, IgA, and IgM (see below). After washing and dr>'ing, the preparations were mounted in a poly\'inyl alcohol medium (RODRIGUEZ & DEINHARDT I960). The fluorescence
microscope was a Leitz Ortholux microscope (E. Leitz, Inc., New York, N.Y.) equipped with a Ploem-type epi-illuminator. The smears "were examined twice by the same examiner, and the end-point titer was assigned to tlie last dilution which showed distinct fluorescence compared with the negligible background staining of PBSpreincubated control smears. When titers varied between the two readings, the slides were reexamined and a final titer determined. The examiner had no knowledge of which serum was being scored. For statistical analyses, end-point titers were transformed to an arithmetic scale as outlined in Table 1. .A multipotent goat antihuman Ig conjugate, which according to the manufacturer (Hyland, Los Angeles, Calif.) had a fiuorescein-to-protein ratio of 4.7 and a protein content of 21.7 mgfml, was used. In miero-double-diffusion it reacted with human IgG, IgA, and IgM at 1 mg/ml to dilutions of 1:8, 1:4, and 1:4, respectively. Before use, the conjugate was adsorbed for 2 h with mouse liver powder, and also with washed sediments of the bacterial suspensions.
108
ORSTAVIK AND BRANDTZAEG
By immunofluorescence performance testing a working dilution of 1:16 was established. Dilu- "" tions of the conjugate and of the human^ sera were made m 7.5 % bovine serum albumin m '
YD = rj. V H + ri. Vj + C + A ^jj,^^^^ AMUNDSEN & JANSEN 1974). Here, ^ „ ^ . ^ ^ j , ^he individual regression coefficients r . • •u^ ^ • . * .j A • ..-u for each variable, C is a constant, and A is the residual variation unexplained by the equation. A multiple correlation coefficient (R) was de™'"* f™'" *•= computations underlying this equation; its square denotes that part of the variability of VD which is statistically explained by the regression equation relative to the observed, total variability,
STATISTICAL METHODS The material consisted of 53 subjects and nine variables: PI, DMF, and the transformed titers to the seven bacterial preparations. Correlations were computed for all possible pairs of the nine variables. Thus, correlations of PI with DMF and with the individual serum antibacterial titers, correlations of DMF with bacterial titers. and correlations between pairs of titers were J
T.-
,1
•
1• I
Results
•
BACKGROUND INFORMATION
computed, r lnally, stepwise multiple regression analyses were performed to reveal possible linear relationships between one variable (VD; m ™™ PI or DMF) with two or more of the other variables (V;; in cam bacterial titers) according to the formula
Observed ranges, arithmetic means, and standard deviations of P I , DMF, and , ., . i .' transformed antibacterial Uters are shown in Table 2. Inspection of the frequency
Table 1 Transformation of titers to an arithmetic scale Dilution
1 :20
1:10
Titer
-
±
±
Assigned value
10
15 20
25 30
+
1 •AO +
+
35 40
1 :80
±
+
45 50
1: 160
1: 320
1: 640
±
±
±
+
55 60
+
65 70
75 80
Table 2 Observed ranges^ arithmetic means,, and standard deviations (s.d.) of PI, DMF, and transformed antibacterial titers n
Range
PI
53
0.68-2.00
DMF
53
0-28
16.6
Mean
s.d.
1.16
0.227 5.51
S. mutans OMZ 176E
53
10-75
44.6
10.91
S. mutans 6715
53 53
15-65 30-75
40.5 52.6
12.07
52
10-55
30.1
14.57
Titer to
S. salivarius 9GS2 S.sanguis 10556
-f-
12.51
S. sanguis 34
52
10-80
48.8
1 ] .44
VeiUonella types I-VII
53
49.8
14.12
Fusobacierium F4
53
20-80 20-75
43.2
13.08
SERUM ANTIBODIES TO PLAQUE BACTERIA
109
Table 3 Correlations between PI, DMF, and bacterial titers Coefficient of correlation
Probability of chance association
PI/DMF
T= +..276
P < 0.05
50
VljFusohacterium JF4
r = -.214
P = 0.13
48 48
Pair of comparison
Degrees of freedom
PllVeillonella
r = +,J20
P = 0.41
V>M-¥jFusobacterium F4
r = -.009
P = 0.55
47
J}MYIVeillonella
r=-.323
F
disease}
Serum antibodies to plaque bacteria in subjects with dental caries and gingivitis DAG ORSTAVIK .AND PER BRANDTZAEG Department of Microbiology, Dental Faculty, University of Oslo, Oslo, Norway ABSTRACT — Correlations were sought between indices of gingival inflammation and dental caries experience and serum antibody titers to five species of oral bacteria. The material camprised 53 young adult males. .A statistically significant, negative correlation was observed between the antibody titer to a pool of Veillonella strains and dental caries experience. Multiple regression analyses failed to reveal significant associations between periodontal disease and serum antibody titers. However, the data suggested a combined association of the titers to the strains of Veillonella and a strain of Fusohacteriuni with the periodontal index. (Accepted for publication 25 April 1976)
Gingivitis in man results from accumulation of bacterial plaque at the gingiva! margin (LOE, THEIL.\DE &. JENSEN 1965).
The immunogenicity and mitogenicity of plaque bacteria are indicated by the appearance of lymphocytes with specificity for plaque antigens in peripheral blood (IvANYi & LEHNER 1970) and by popula-
tion of the gingival lesion with immunoglobulin (Ig)-producing cells (BRANDTZAEG & KRAUS 1965). Correlations have
been found between the severity of gingival inflammation and the stimulatory response of circulating lymphocytes {see LEHNER 1975). Less is known about the association of serum antibodies to plaque bacteria with periodontal disease (EVANS, SPAETH & MERGENHAGEN 1966, KRISTOFFERSEN & HOFSTAD 1970, GILMOUR & NiSENGARD 1974).
Serum antibodies tO' potentially cariogenic microorganisms have been found in subjects with and withotjt caries (KENNEDY, SHKLAIR, HAYAISHI & BAHN 1968),
and complex relationships between such antibodies and dental caries experience have been indicated (LEHNER, WILTON & WARD 1970, GHALLAGOMBE 1974).
In the present study an attempt was, made to detect associations between serum antibody titers to a number of plaque bacteria and the severity of gingiva! inflammation and dental caries experience. To reduce the likelihood of possible age-dependent, differential antibody responses, army recruits of near-uniform age were chosen for the study. The choice of antigens was based on the following considerations: Streptococcus mutans has been associated with dental caries; it is prevalent
SERUM ANTIBODIES TO PLAQUE BACTERIA in man and the serotype d is of frequent occurrence (BRATTHALL 1970, 1972); two strains of this serotype were included in the present study. Streptococcus salivarius is numerous in saliva and on the tongue dorsum (GIBBONS, KAPSIMALIS & SOGRANSKY 1964), but sparse in dental plaque (GARLSSON 1965,a); one strain was included as an indicator of the immunogenicity of bacteria other than the plaqueprevalent species. Streptococcus sanguis, on the other hand, is prevalent in dental plaque (CARLSSON 1965b); two strains of unknown antigenic relationship were used. Finally, Fusobacterium and Veillonella strains were included because sonicates of these species have been shown to induce transformation of lymphocytes from ,subjects with periodontal disease (IVANYI & LEHNER 1970),.
Material and methods SUBJECTS
Norwegian army recruits in good systemic health, aged 19 to 22 years, were studied. Their periodontal status was assessed by the periodontal index (PI) of RUSSELL (1956) and their dental caries experience by the DMF index of KLEIN, PALMER & KNUTSON (1938). Venous blood was
obtained and allowed to clot at room temperature for 1 h and at 4°C for approximately 4 h. The serum was then collected after centrifugation at 500 g for 10 min at 4°G. and stored at
BACTERIA
Streptococcus mutans OMZ 176E was a gift from Dr. J. E. NORTON. S. mutans 6715 and S. sanguis ATCG 10556 were obtained from Dr. H. J. SANDHAM, S. salivarius 9GS2 and S. sanguis 34 from Dr. R. J. GIBBONS, Veillonella antigenic types I to VII (ROGOSA 1965) from the National Institute of Dental Research (Bethesda, Maryland), and Fusobacterium strain F4 from Dr. T. KRISTOFFERSEN. The streptococci were grown anaerobically at 37°C in Trypticase Soy BrothTlI (BBL, Cockeysville, Maryland) for 24 h, harvested by centrifugation (10,000 g, 15 min, 4°C), washed three
107
times in PBS (0.01 M phosphate buffer, pH 7.2, 0.14 M NaCl), and resuspended to an absorbance (A) of 0.2 at 540 nm. Tbe Veillonella strains were grown anaerobically at 37 °C for 48 h in the V17 medium of ROGOSA (1964), harvested, washed, and resuspended to Ag^o = 0.2. Equal portions of the seven antigenic types were then pooled. The Fusobacterium strain F4 was received as a washed and frozen suspension in PBS; this suspension was thawed and adjusted to A540 = 0.2.
INDIRECT IMMUNOFLUORESCENCE
The bacterial suspensions were spotted in 10-[ll portions onto glass slides, air dried and fixed for 10 min in 96% ethanol at 4°C. The slides were then brought through decreasing concentrations of ethanol to PBS, dipped in demineralized w-ater, dried and incubated in a moist chamber with serial twofold dilutions (1:10 t° 1:640) of the human sera for 1 h at room temperature. One spot on each slide was incubated with PBS for control purposes. The slides were then washed twice in PBS for 7 min, dipped in demineralized water, dried, and incubated for 30 min with a fluorescent goat inimimogiobuiin (Ig) conjugate active against human IgG, IgA, and IgM (see below). After washing and dr>'ing, the preparations were mounted in a poly\'inyl alcohol medium (RODRIGUEZ & DEINHARDT I960). The fluorescence
microscope was a Leitz Ortholux microscope (E. Leitz, Inc., New York, N.Y.) equipped with a Ploem-type epi-illuminator. The smears "were examined twice by the same examiner, and the end-point titer was assigned to tlie last dilution which showed distinct fluorescence compared with the negligible background staining of PBSpreincubated control smears. When titers varied between the two readings, the slides were reexamined and a final titer determined. The examiner had no knowledge of which serum was being scored. For statistical analyses, end-point titers were transformed to an arithmetic scale as outlined in Table 1. .A multipotent goat antihuman Ig conjugate, which according to the manufacturer (Hyland, Los Angeles, Calif.) had a fiuorescein-to-protein ratio of 4.7 and a protein content of 21.7 mgfml, was used. In miero-double-diffusion it reacted with human IgG, IgA, and IgM at 1 mg/ml to dilutions of 1:8, 1:4, and 1:4, respectively. Before use, the conjugate was adsorbed for 2 h with mouse liver powder, and also with washed sediments of the bacterial suspensions.
108
ORSTAVIK AND BRANDTZAEG
By immunofluorescence performance testing a working dilution of 1:16 was established. Dilu- "" tions of the conjugate and of the human^ sera were made m 7.5 % bovine serum albumin m '
YD = rj. V H + ri. Vj + C + A ^jj,^^^^ AMUNDSEN & JANSEN 1974). Here, ^ „ ^ . ^ ^ j , ^he individual regression coefficients r . • •u^ ^ • . * .j A • ..-u for each variable, C is a constant, and A is the residual variation unexplained by the equation. A multiple correlation coefficient (R) was de™'"* f™'" *•= computations underlying this equation; its square denotes that part of the variability of VD which is statistically explained by the regression equation relative to the observed, total variability,
STATISTICAL METHODS The material consisted of 53 subjects and nine variables: PI, DMF, and the transformed titers to the seven bacterial preparations. Correlations were computed for all possible pairs of the nine variables. Thus, correlations of PI with DMF and with the individual serum antibacterial titers, correlations of DMF with bacterial titers. and correlations between pairs of titers were J
T.-
,1
•
1• I
Results
•
BACKGROUND INFORMATION
computed, r lnally, stepwise multiple regression analyses were performed to reveal possible linear relationships between one variable (VD; m ™™ PI or DMF) with two or more of the other variables (V;; in cam bacterial titers) according to the formula
Observed ranges, arithmetic means, and standard deviations of P I , DMF, and , ., . i .' transformed antibacterial Uters are shown in Table 2. Inspection of the frequency
Table 1 Transformation of titers to an arithmetic scale Dilution
1 :20
1:10
Titer
-
±
±
Assigned value
10
15 20
25 30
+
1 •AO +
+
35 40
1 :80
±
+
45 50
1: 160
1: 320
1: 640
±
±
±
+
55 60
+
65 70
75 80
Table 2 Observed ranges^ arithmetic means,, and standard deviations (s.d.) of PI, DMF, and transformed antibacterial titers n
Range
PI
53
0.68-2.00
DMF
53
0-28
16.6
Mean
s.d.
1.16
0.227 5.51
S. mutans OMZ 176E
53
10-75
44.6
10.91
S. mutans 6715
53 53
15-65 30-75
40.5 52.6
12.07
52
10-55
30.1
14.57
Titer to
S. salivarius 9GS2 S.sanguis 10556
-f-
12.51
S. sanguis 34
52
10-80
48.8
1 ] .44
VeiUonella types I-VII
53
49.8
14.12
Fusobacierium F4
53
20-80 20-75
43.2
13.08
SERUM ANTIBODIES TO PLAQUE BACTERIA
109
Table 3 Correlations between PI, DMF, and bacterial titers Coefficient of correlation
Probability of chance association
PI/DMF
T= +..276
P < 0.05
50
VljFusohacterium JF4
r = -.214
P = 0.13
48 48
Pair of comparison
Degrees of freedom
PllVeillonella
r = +,J20
P = 0.41
V>M-¥jFusobacterium F4
r = -.009
P = 0.55
47
J}MYIVeillonella
r=-.323
F
