207
Intrafamilial Transmission of Actinobacillus actinomycetemcomitans Satu Alaluusua,
*
Sirkka Asikainen, f and
Chern-Hsiung Laif
Cross-sectional studies have shown that Actinobacillus actinomycetemcomitans is a frequent member of the oral flora in children with primary teeth. The purpose of the present study was to obtain information of the transmission of A. actinomycetemcomitans. Three types of families were studied for the prevalence and serotype distribution of A. actinomycetemcomitans. First, families whose periodontally healthy child member harbored A. actinomycetemcomitans; second, families of periodontally healthy children who did not harbor A. actinomycetemcomitans; third, families whose adult member harbored A actinomycetemcomitans and had been referred to treatment for severe Periodontitis. As a whole the study included 23 families. All the family members were invited for the examination. The final study population consisted of 38 children and 32 adults. The results showed that when a child was positive for A. actinomycetemcomitans, either the mother or the father was also positive with one exception, whereas when the adult member harbored A. actinomycetemcomitans, the children were infected in only 2 of the 9 families. Using immunodiffusion technique it was found that the child always harbored the same serotype of A. actinomycetemcomitans as the parent. In conclusion, the results suggest the intrafamilial transmittance of A actinomycetemcomitans. J Periodontol 1991; 62:207-10.
Key Words: Actinobacillus actinomycetemcomitans
occurrence; microbe
transmission;
periodontitis/etiology.
actinomycetemcomitans has been regarded putative pathogen in certain aggressive periodontal diseases.1-3 However, the organism can also be detected rather frequently in periodontally healthy teenagers and even in children with primary dentition.4'5 A actinomycetemcomitans may belong to the normal oral microflora.6 The occasional recovery of A actinomycetemcomitans in low proportions of the flora from healthy gingival sites and in periodontally healthy individuals supports the view of the opportunistic nature of infections associated with A actinomycetemcomitans.1·4>5>7 However, its possible exogenous origin has recently also been discussed.8'9 The evidence pointing to A. actinomycetemcomitans as an exogenous pathogen in man also points to the importance of understanding its transmission. Intrafamilial transmission of A actinomycetemcomitans in families with children suffering from juvenile Periodontitis has been suggested.10 However, little is known about the prevalence of A actinomycetemcomitans in families with periodontally healthy children. The aim of the present study is to report the prevalence and serotype distribution of A actinomycetemcomitans in families with A actinomycetemcomitans-positive and with
Actinobacillus as a
'Department
of Pedodontics and
Helsinki, Finland.
Orthodontics, University
'"Department of Periodontology. *University of Pennsylvania, School PA.
of Dental
of
Helsinki,
Medicine, Philadelphia,
A
actinomycetemcomitans-ntg&űs/t young children and in families with A actinomycetemcomitans-positive adult member(s) and further evaluate the possibility of intrafamilial transmission of A actinomycetemcomitans. MATERIALS AND METHODS
Subjects
selected on the following basis: First: child member harbored A actinomycetemcomitans. Second: a child member did not harbor A. actinomycetemcomitans. Third: an adult member harbored A actinomycetemcomitans and had been referred for treatment of severe Periodontitis. All the family members were invited for the examination. Finally, the records were obtained from 38 children and 32 adults. In the first type of families (N 7, 8 A actinomycetemcomitans-positive children, including one pair of siblings), the age of the children when A actinomycetemcomitans was first isolated varied from 4.9 to 10.4 years. The permanency of the infection was followed for 1 to 5 years (mean 2.7 years) and the number of sampling occasions varied from 2 to 7. The children were examined clinically and radiographically and the bacterial samples were taken at least once a year. For the present study, these 8 A actinomycetemcomitans-poúúve children and their 11 parents and 2 siblings were included. Three
types of families
were
a
=
208
J Periodontol March 1991
TRANSMISSION OF . ACTINOMYCETEMCOMITANS
The second type of families (N 7, 10 children, 8 had included children who parents) previously participated in our study of the detection frequency of A. actinomycetemcomitans in children.5 They were selected on the basis of being A actinomycetemcomitans-mga.tive and of the same age as the children in the first type of families. The third type of families (N 9) included 9 A. actinomycetemcomitans-positive parents, who suffered from severe Periodontitis, their 4 spouses, and 18 children. The age of the adults in the family groups ranged from 30 to 48. Alveolar bone loss was evaluated from orthopantomograms which were taken from all adults and from A. actinomycetemcomitans-posiüve children. The previous usage of antibiotics was recorded. =
=
Microbiological Sampling and Identification For the recovery of A. actinomycetemcomitans two pooled
plaque samples from four subgingival sites (mesial surfaces
of the first permanent molars or mesial surfaces of the second primary molars), and from dorsum of the tongue and/or stimulated saliva were examined (N 186). The plaque from the children taken with dental floss by were samples contact the floss the and movbringing through approximal in the sulcus. it and fro four times to buccolingually ing The plaque samples from the adults were taken with a sterile curet. The samples from the tongue were taken by scraping the mid-dorsum of the tongue with a sterile curet. The samples were placed in a 1/2-dram vial containing 1 ml sterilized 20% skim milk and glass beads.11 The vials, as well as about 1 ml of paraffin-stimulated saliva, were immediately transported to the microbiology laboratory of the Institute of Dentistry, and the cultures were set up at once. Bacteria were dispersed with Vortex mixing (20 strokes at maximum setting). For the selective culture of A. actinomycetemcomitans, aliquots of 100 µ of undiluted (plaque samples and samples from dorsum of the tongue) and 10-2 dilution samples (plaque samples) were inoculated on TSBVagar plates.12 For the saliva 10-3 dilution was used, thus giving the minimum detection limit of 104CFU/ml of saliva. The plates were incubated for 4 days at 37° C in a glove box§ containing 85% N2, 10% H2, and 5% C02. Small, circular, convex colonies adhering to agar and with a starlike inner structure were suspected to be A actinomycetemcomitans and subcultured. The isolates were confirmed as A. actinomycetemcomitans when they were Gram-negative, catalase-positive, coccobacilli which reduced nitrate. =
MA) and strain NCTC 9710 (serotype c, obtained from the National Collection of Type Cultures, London, England) were produced in rabbits with live whole cell suspensions in 0.05% formaline-saline solution at a concentration of 200 mg of nitrogen per ml as determined by the Nessler reaction.13 The rabbit was first injected in foot pad with 0.2 ml of mixture of bacterial suspension and complete Freund adjuvant. Subsequent ear vein injections with 0.2 ml of bacterial suspension without Freund adjuvant were performed a week after and once weekly for 2 weeks. Antisera were collected by intracardiac puncture beginning on the 5th week and again on alternate weeks as described previously.14 For the antigen extracts bacterial cells were grown in Todd-Hewitt broth" (10 ml) supplemented with 1% (wt/vol) yeast extract at 37°C for 3 days in candle jars. Antigen extracts were prepared by autoclaving the cells at 120°C for 15 minutes according to the method of Rantz and Randall.15 Immunodiffusion was performed in 1.2% (wt/vol) Noble agar11 in saline.16 ton,
RESULTS
Recovery of A. actinomycetemcomitans and Periodontal Status in Families With an A. actinomycetemcomitans-
Positive Child The study group included 8 children from 7 families who were positive for A actinomycetemcomitans (Table 1.) These children were studied longitudinally and from the total of 31 sampling occasions at least once a year (2 to 7 occasions/ child) A actinomycetemcomitans was detected in 27, indictating a constant colonization. On the basis of clinical and radiographical examination, all children except one (who had one
pathologically deepened pocket) were periodontally healthy.
Six children harbored serotype c strain and 2 children serotype b strain. If the child was positive for A actinomycetemcomitans either the father or the mother was positive, too, except in one family. In that family the father had a history of frequent usage of antibiotics. In 4 families A. actinomycetemcomitans was detected from the mother and in 2 families from the father. The child always harbored the same serotype as the parent. Only a single serotype was detected in all except one mother, who harbored serotypes a and c. Two siblings included for the present study were not positive for A actinomycetemcomitans. Six out of 11 parents did not have alveolar bone loss and 5 parents had bone loss around the cervical third of the root.
Recovery of A. actinomycetemcomitans and Periodontal Status in Families With an A. actinomycetemcomitansNegative Child
Immunological Methods Antisera against A. actinomycetemcomitans strain ATCC 29523 (serotype a, obtained from the American Type Culture Collection, Rockville, MD), strain FDC Y4 (serotype b, obtained from S.S. Socransky, Forsyth Dental Center, Bos-
actinomycetemcomitans.
§Forma Scientific Anaerobic System Model 1024, Marietta, OH.
'Difco
None of the
parents of the A. actinomycetemcomitans-ntg-
ative children
or
their
siblings
Laboratories, Detroit, MI.
were
positive
for A
Volume 62 Number 3
ALALUUSUA, ASIKAINEN,
Table 1. Recovery of A. actinomycetemcomitans in Families With A. actinomycetemcomitans-Positive Child Member Patient
Serotype and
Follow-up Age or Age
(years)
A.
Distribution of
actinomycetemcomitans
p00led
Plaque
Stimulated Saliva
Dorsum of
b
the
Tongue
Child (male) Mother Father
5.9-7.5 46 44
b * b
b
Child (female) Child (male) Mother Father
6.9-9.5 4.9-7.5 31 38
c
c
c
c
c
c
NN c
c
Child (male) Child (female) Mother
7.5-9.4 12 45
b b NN NN
b
Child (female) Mother
8.0-9.0 35
ce
Child (male) Mother Father
10.4-15.3 48 45
c
Child (male) Mother Father
9.6-13.5 45 45
c
Child (female) Child (male) Mother
7 33
*
=
t ND
not =
10.0-11.0
an
c
c
c
NDf
ND ND
ND c
ND
ND NN
c
c
NN c a,c
a
detected
not determined
All children
were
periodontally healthy. Seven of 8 parOnly one mother had
ents did not have alveolar bone loss.
bone loss around the cervial third of the root.
Recovery of A. actinomycetemcomitans and Periodontal Status in Families With an A. actinomycetemcomitansPositive Adult Member The A. actinomycetemcomitans-posiúve adults (N 9) selected for the study had been referred for treatment of severe Periodontitis. They all had alveolar bone loss extended to the apical third of the root length locally or generally in the dentition. From the 4 spouses included, 2 were positive for A. actinomycetemcomitans. Neither of them had bone loss. One of the two A actinomycetemcomitans-mgative spouses had bone loss extending to the middle third of the root length and the other did not have bone loss. Four adults (including a married couple) harbored serotype a strain. Three adults harbored serotype b strain, and 3 adults serotype c strain (including a married couple). One strain could not be categorized. In seven families A. actinomycetemcomitans was not isolated from the children, but in two families A. actinomycetemcomitans was isolated. In one family both parents and their 6 and 18 year-old children were infected with serotype c and in the other the mother and her 7 year-old son were infected with serotype a strain. The children did not have bone loss. =
Distribution of A.
actinomycetemcomitans
LAI
209
in the Oral
Cavity
A. actinomycetemcomitans was detected in 28 of the 70 individuals studied. Five individuals harbored serotype a, 7 serotype b, and 14 serotype c; one adult harbored both a and c and one strain was untypeable. If a subject was positive for A actinomycetemcomitans, the organism was most often isolated from plaque samples (86%), but also very frequently from samples taken from the dorsum of tongue (77%), or from stimulated saliva (68%). DISCUSSION We have looked at the distribution of A actinomycetemcomitans in families of periodontally healthy children and their parents with healthy periodontal tissues, or mild Periodontitis and in families where the adult member has severe Periodontitis and is infected with A actinomycetemcomitans. For the study it would have been interesting to include families whose child is periodontally diseased and harbors A actinomycetemcomitans. In Finland, however, prepupertal Periodontitis or juvenile Periodontitis in young permanent dentition is so rare that we could not gather such material. Based on the prevalence and serotype distribution of A actinomycetemcomitans in the families studied, we suggest that the transmission of A actinomycetemcomitans is intrafamilial. After detecting the organism in the child, it was also detected from either parent in all but one family. Furthermore it was shown that the child always harbored the same serotype strain as the parent. Serotype similarities in 5 mother/child pairs, 3 father/child pairs and in one whole family (both parents and 2 children) could be found. Comparable findings concerning mutans streptococci in children and their mothers have been reported.17 From 15 pairs of mothers and children, the discrepancies of the dominant serotypes oí mutans streptococci were observed only in 3 mother-child pairs. However, a similar distribution of mutans streptococci was not clearly seen in other relatives, including the father. Based on a determination of the serotypes and biotypes of A actinomycetemcomitans in families where a family member suffers from juvenile Periodontitis, Zambón et al. also suggest intrafamilial transmission of the organism.10 These investigators also pointed out that the organism does not readily colonize the tooth surface. In their material, over half of the family members studied were not infected. Our finding that children were infected in only 2 out of 9 families with an adult member suffering from severe Periodontitis and harboring A actinomycetemcomitans supports this statement.
actinomycetemcomitans is regarded as an exogenous pathogen the knowledge of the timing and route of the infection are important for its control. Little is known about the early colonization of A actinomycetemcomitans. A. actinomycetemcomitans has not been detected in study popIfA
ulations under the age of 2.5
years,18
but it is
frequently
210
detected in 5 to 6 year-old children with primary teeth.2 The present study demonstrated that the organism does not colonize transiently, but shows persistent oral colonization. The route of transmission may occur through direct person-to-person contact, by indirect contact through an inanimate object such as a shared toothbrush, or by droplets of saliva or other fluid which harbor the agent.8 When evaluating the role of saliva in transmission, it has been shown that, for example, mutans streptococci can be transmitted by saliva-contaminated spoons or other objects and that there is a correlation between the salivary count of mutans streptococci and the number of microorganisms transferred to the spoon.19 Furthermore it is more likely for infants to be infected with mutans streptococci if their mothers harbor dense salivary population of the organism than if the maternal salivary reservoirs are low or negligible.20 In our study population, if a person was positive for A. actinomycetemcomitans, the organism was detected in saliva in 68% of the subjects, indicating that saliva can act as direct or indirect vehicle in the transmission. Still, our method picked up only samples with an A. actinomycetemcomitans concentration of > 104 CFU/ml. Thus it is likely that the detection frequency of A. actinomycetemcomitans in saliva in persons infected with A. actinomycetemcomitans would be higher if a more sensitive method was used. For the immunodiffusion tests, crude autoclaved extracts of the clinical strains were used. This method was also used by Amano et al. for the serotyping of A. actinomycetemcomitans.21 Their preliminary results showed that the autoclaved extracts of serotype a, b, and c of A. actinomycetemcomitans strains formed strong precipitation lines only with the corresponding rabbit antiserum. In our study the method was found useful for serotyping the A. actinomycetemcomitans strains and all except three strains were easily characterized. Two strains (obtained from mother and her child) had strongest reaction with serotype c antiserum, but reacted also with antiserum of serotype a and b and one strain had poor reactivity with any of the antiserum used. In conclusion, based on the prevalence and serotype distribution of A. actinomycetemcomitans within families, intrafamilial transmittance is suggested. Because our data are cross-sectional we do not know the direction of infection. If the organism is transmitted from an adult to the child then parents are highly likely sources. However, the risk of the child getting the infection from an A. actinomycetemcomitans-positive parent is rather low. To prevent the infection, a more extensive survey must be performed to clarify the source and transmission route as well as the effect of the timing on the infection of A. actinomycetemcomitans.
Acknowledgment This
J Periodontol March 1991
TRANSMISSION OF . ACTINOMYCETEMCOMITANS
2. Asikainen S, Jousimies-Somer H, Kanervo A, Summanen P. Certain bacterial species and morphotype in localized juvenile Periodontitis and matched controls. / Periodontol 1987;58:224-230. 3. Slots J, Listgarten MA. Bacteroides gingivalis, Bacteroides intermedius and Actinobacillus actinomycetemcomitans in human periodontal diseases. / Clin Periodontol 1988;15:85-93. 4. Asikainen S, Alaluusua S, Kari , Kleemola-Kujala E. Subgingival microflora and periodontal conditions in healthy teenagers. / Periodontol 1986;57:505-509. 5. Alaluusua S, Asikainen S. Detection and distribution of Actinobacillus actinomycetemcomitans in the primary dentition. / Periodontol
1988;59:504-507. 6. Kilian M, Schiott CR. Haemophili and related bacteria in the human oral cavity. Arch Oral Biol 1975;20:791-796. 7. Kornman . Age, supragingival plaque, and steroid hormones as ecological determinants of the subgingival flora. In: Genco R, Mergenhagen S, eds. Host-Parasite Interactions in Periodontal Diseases. Washington DC: American Society of Microbiology, 1982:132-138. 8. Genco RJ, Zambón JJ, Christersson LA. The origin of periodontal infections. Adv Dent Res 1988;2:245-259. 9. Preus HR, Olsen 1. Possible transmittance of A. actinomycetemcomitans from a dog to a child with rapidly destructive Periodontitis. J Periodont Res 1988;23:68-71. 10. Zambón JJ, Christersson LA, Slots J. Actinobacillus actinomycetemcomitans in human periodontal disease. Prevalence in patient groups and distribution of biotypes and serotypes within families. J Periodontol 1983;54:707-711. 11. Sutter VL, Citron DM, Finegold SM. Wadsworth Anaerobic Bacteriology Manual. St. Louis: The CV Mosby Company; 1980. 12. Slots J. Selective medium for isolation of Actinobacillus actinomycetemcomitans. J Clin Microbiol 1982;15:606-609. 13. Campell DH, Garney JS, Cremer NE, Sussdorf DH. Nessler reaction. Methods in Immunology; A Laboratory Text for Instruction and Research. New York: Benjamin Wa, 1970;69-73. 14. Lai C- , Listgarten MA, Shirakawa M, Slots J. Bacteroides forsythus in adult gingivitis and Periodontitis. Oral Microbiol Immunol
1987;2:152-157. LA, Randall
15. Rantz
streptococci
for
1955;13:290-321.
E. Use of autoclaved extracts of
serological grouping. Stanford
hemolytic
Med Bull
16. Hamada S. Masuda N, Ooshima T. Sobue S. Kotani S. Epidemiologica! survey of Streptococcus mutans among Japanese children: Identification and serological typing of the isolated strains. Jpn J Microbiol 1976;20:33^14. 17. Masuda N, Shimamoto T, Kitamura K, Sobue S, Hamada S. Transmission of Streptococcus mutans in some selected families. Microbios
1985;44:223-232. KW, Higgins T, Palmer JM. The incidence of periodonto-
18. Frisken
pathic microorganisms
in young children. Oral Microbiol Immunol
1990;5:43^15. 19. Köhler , Bratthall D. Intrafamilial levels of Streptococcus mutans and some aspects of the bacterial transmission. Scand J Dent Res 1978;86:35^12. 20. Berkowitz RJ, Turner J, Green P. Primary oral infection of infants with Streptococcus mutans. Arch Oral Biol 1980;25:221-224. 21. Amano , Nishihara T, Shibuya , Noguchi , Koga T. Immunochemical and structural characterization of a serotype-specific Polysaccharide antigen from Actinobacillus actinomycetemcomitans Y4 (serotype b). Infect Immun 1989;57:2942-2946.
study has been supported by the Academy of Finland.
REFERENCES 1. Genco
RJ, Zambón JJ, Christersson LA. Use and interpretation of
microbiological munol
assays in
1986;1:73-79.
periodontal
diseases. Oral Microbiol Im-
Send reprint requests to: Dr. Satu Alaluusua, Department of Pedodontics and Orthodontics, Institute of Dentistry, University of Helsinki, Mannerheimintie 172, 00300 Helsinki, Finland. Accepted for publication August 29, 1990.
Intrafamilial Transmission of Actinobacillus actinomycetemcomitans Satu Alaluusua,
*
Sirkka Asikainen, f and
Chern-Hsiung Laif
Cross-sectional studies have shown that Actinobacillus actinomycetemcomitans is a frequent member of the oral flora in children with primary teeth. The purpose of the present study was to obtain information of the transmission of A. actinomycetemcomitans. Three types of families were studied for the prevalence and serotype distribution of A. actinomycetemcomitans. First, families whose periodontally healthy child member harbored A. actinomycetemcomitans; second, families of periodontally healthy children who did not harbor A. actinomycetemcomitans; third, families whose adult member harbored A actinomycetemcomitans and had been referred to treatment for severe Periodontitis. As a whole the study included 23 families. All the family members were invited for the examination. The final study population consisted of 38 children and 32 adults. The results showed that when a child was positive for A. actinomycetemcomitans, either the mother or the father was also positive with one exception, whereas when the adult member harbored A. actinomycetemcomitans, the children were infected in only 2 of the 9 families. Using immunodiffusion technique it was found that the child always harbored the same serotype of A. actinomycetemcomitans as the parent. In conclusion, the results suggest the intrafamilial transmittance of A actinomycetemcomitans. J Periodontol 1991; 62:207-10.
Key Words: Actinobacillus actinomycetemcomitans
occurrence; microbe
transmission;
periodontitis/etiology.
actinomycetemcomitans has been regarded putative pathogen in certain aggressive periodontal diseases.1-3 However, the organism can also be detected rather frequently in periodontally healthy teenagers and even in children with primary dentition.4'5 A actinomycetemcomitans may belong to the normal oral microflora.6 The occasional recovery of A actinomycetemcomitans in low proportions of the flora from healthy gingival sites and in periodontally healthy individuals supports the view of the opportunistic nature of infections associated with A actinomycetemcomitans.1·4>5>7 However, its possible exogenous origin has recently also been discussed.8'9 The evidence pointing to A. actinomycetemcomitans as an exogenous pathogen in man also points to the importance of understanding its transmission. Intrafamilial transmission of A actinomycetemcomitans in families with children suffering from juvenile Periodontitis has been suggested.10 However, little is known about the prevalence of A actinomycetemcomitans in families with periodontally healthy children. The aim of the present study is to report the prevalence and serotype distribution of A actinomycetemcomitans in families with A actinomycetemcomitans-positive and with
Actinobacillus as a
'Department
of Pedodontics and
Helsinki, Finland.
Orthodontics, University
'"Department of Periodontology. *University of Pennsylvania, School PA.
of Dental
of
Helsinki,
Medicine, Philadelphia,
A
actinomycetemcomitans-ntg&űs/t young children and in families with A actinomycetemcomitans-positive adult member(s) and further evaluate the possibility of intrafamilial transmission of A actinomycetemcomitans. MATERIALS AND METHODS
Subjects
selected on the following basis: First: child member harbored A actinomycetemcomitans. Second: a child member did not harbor A. actinomycetemcomitans. Third: an adult member harbored A actinomycetemcomitans and had been referred for treatment of severe Periodontitis. All the family members were invited for the examination. Finally, the records were obtained from 38 children and 32 adults. In the first type of families (N 7, 8 A actinomycetemcomitans-positive children, including one pair of siblings), the age of the children when A actinomycetemcomitans was first isolated varied from 4.9 to 10.4 years. The permanency of the infection was followed for 1 to 5 years (mean 2.7 years) and the number of sampling occasions varied from 2 to 7. The children were examined clinically and radiographically and the bacterial samples were taken at least once a year. For the present study, these 8 A actinomycetemcomitans-poúúve children and their 11 parents and 2 siblings were included. Three
types of families
were
a
=
208
J Periodontol March 1991
TRANSMISSION OF . ACTINOMYCETEMCOMITANS
The second type of families (N 7, 10 children, 8 had included children who parents) previously participated in our study of the detection frequency of A. actinomycetemcomitans in children.5 They were selected on the basis of being A actinomycetemcomitans-mga.tive and of the same age as the children in the first type of families. The third type of families (N 9) included 9 A. actinomycetemcomitans-positive parents, who suffered from severe Periodontitis, their 4 spouses, and 18 children. The age of the adults in the family groups ranged from 30 to 48. Alveolar bone loss was evaluated from orthopantomograms which were taken from all adults and from A. actinomycetemcomitans-posiüve children. The previous usage of antibiotics was recorded. =
=
Microbiological Sampling and Identification For the recovery of A. actinomycetemcomitans two pooled
plaque samples from four subgingival sites (mesial surfaces
of the first permanent molars or mesial surfaces of the second primary molars), and from dorsum of the tongue and/or stimulated saliva were examined (N 186). The plaque from the children taken with dental floss by were samples contact the floss the and movbringing through approximal in the sulcus. it and fro four times to buccolingually ing The plaque samples from the adults were taken with a sterile curet. The samples from the tongue were taken by scraping the mid-dorsum of the tongue with a sterile curet. The samples were placed in a 1/2-dram vial containing 1 ml sterilized 20% skim milk and glass beads.11 The vials, as well as about 1 ml of paraffin-stimulated saliva, were immediately transported to the microbiology laboratory of the Institute of Dentistry, and the cultures were set up at once. Bacteria were dispersed with Vortex mixing (20 strokes at maximum setting). For the selective culture of A. actinomycetemcomitans, aliquots of 100 µ of undiluted (plaque samples and samples from dorsum of the tongue) and 10-2 dilution samples (plaque samples) were inoculated on TSBVagar plates.12 For the saliva 10-3 dilution was used, thus giving the minimum detection limit of 104CFU/ml of saliva. The plates were incubated for 4 days at 37° C in a glove box§ containing 85% N2, 10% H2, and 5% C02. Small, circular, convex colonies adhering to agar and with a starlike inner structure were suspected to be A actinomycetemcomitans and subcultured. The isolates were confirmed as A. actinomycetemcomitans when they were Gram-negative, catalase-positive, coccobacilli which reduced nitrate. =
MA) and strain NCTC 9710 (serotype c, obtained from the National Collection of Type Cultures, London, England) were produced in rabbits with live whole cell suspensions in 0.05% formaline-saline solution at a concentration of 200 mg of nitrogen per ml as determined by the Nessler reaction.13 The rabbit was first injected in foot pad with 0.2 ml of mixture of bacterial suspension and complete Freund adjuvant. Subsequent ear vein injections with 0.2 ml of bacterial suspension without Freund adjuvant were performed a week after and once weekly for 2 weeks. Antisera were collected by intracardiac puncture beginning on the 5th week and again on alternate weeks as described previously.14 For the antigen extracts bacterial cells were grown in Todd-Hewitt broth" (10 ml) supplemented with 1% (wt/vol) yeast extract at 37°C for 3 days in candle jars. Antigen extracts were prepared by autoclaving the cells at 120°C for 15 minutes according to the method of Rantz and Randall.15 Immunodiffusion was performed in 1.2% (wt/vol) Noble agar11 in saline.16 ton,
RESULTS
Recovery of A. actinomycetemcomitans and Periodontal Status in Families With an A. actinomycetemcomitans-
Positive Child The study group included 8 children from 7 families who were positive for A actinomycetemcomitans (Table 1.) These children were studied longitudinally and from the total of 31 sampling occasions at least once a year (2 to 7 occasions/ child) A actinomycetemcomitans was detected in 27, indictating a constant colonization. On the basis of clinical and radiographical examination, all children except one (who had one
pathologically deepened pocket) were periodontally healthy.
Six children harbored serotype c strain and 2 children serotype b strain. If the child was positive for A actinomycetemcomitans either the father or the mother was positive, too, except in one family. In that family the father had a history of frequent usage of antibiotics. In 4 families A. actinomycetemcomitans was detected from the mother and in 2 families from the father. The child always harbored the same serotype as the parent. Only a single serotype was detected in all except one mother, who harbored serotypes a and c. Two siblings included for the present study were not positive for A actinomycetemcomitans. Six out of 11 parents did not have alveolar bone loss and 5 parents had bone loss around the cervical third of the root.
Recovery of A. actinomycetemcomitans and Periodontal Status in Families With an A. actinomycetemcomitansNegative Child
Immunological Methods Antisera against A. actinomycetemcomitans strain ATCC 29523 (serotype a, obtained from the American Type Culture Collection, Rockville, MD), strain FDC Y4 (serotype b, obtained from S.S. Socransky, Forsyth Dental Center, Bos-
actinomycetemcomitans.
§Forma Scientific Anaerobic System Model 1024, Marietta, OH.
'Difco
None of the
parents of the A. actinomycetemcomitans-ntg-
ative children
or
their
siblings
Laboratories, Detroit, MI.
were
positive
for A
Volume 62 Number 3
ALALUUSUA, ASIKAINEN,
Table 1. Recovery of A. actinomycetemcomitans in Families With A. actinomycetemcomitans-Positive Child Member Patient
Serotype and
Follow-up Age or Age
(years)
A.
Distribution of
actinomycetemcomitans
p00led
Plaque
Stimulated Saliva
Dorsum of
b
the
Tongue
Child (male) Mother Father
5.9-7.5 46 44
b * b
b
Child (female) Child (male) Mother Father
6.9-9.5 4.9-7.5 31 38
c
c
c
c
c
c
NN c
c
Child (male) Child (female) Mother
7.5-9.4 12 45
b b NN NN
b
Child (female) Mother
8.0-9.0 35
ce
Child (male) Mother Father
10.4-15.3 48 45
c
Child (male) Mother Father
9.6-13.5 45 45
c
Child (female) Child (male) Mother
7 33
*
=
t ND
not =
10.0-11.0
an
c
c
c
NDf
ND ND
ND c
ND
ND NN
c
c
NN c a,c
a
detected
not determined
All children
were
periodontally healthy. Seven of 8 parOnly one mother had
ents did not have alveolar bone loss.
bone loss around the cervial third of the root.
Recovery of A. actinomycetemcomitans and Periodontal Status in Families With an A. actinomycetemcomitansPositive Adult Member The A. actinomycetemcomitans-posiúve adults (N 9) selected for the study had been referred for treatment of severe Periodontitis. They all had alveolar bone loss extended to the apical third of the root length locally or generally in the dentition. From the 4 spouses included, 2 were positive for A. actinomycetemcomitans. Neither of them had bone loss. One of the two A actinomycetemcomitans-mgative spouses had bone loss extending to the middle third of the root length and the other did not have bone loss. Four adults (including a married couple) harbored serotype a strain. Three adults harbored serotype b strain, and 3 adults serotype c strain (including a married couple). One strain could not be categorized. In seven families A. actinomycetemcomitans was not isolated from the children, but in two families A. actinomycetemcomitans was isolated. In one family both parents and their 6 and 18 year-old children were infected with serotype c and in the other the mother and her 7 year-old son were infected with serotype a strain. The children did not have bone loss. =
Distribution of A.
actinomycetemcomitans
LAI
209
in the Oral
Cavity
A. actinomycetemcomitans was detected in 28 of the 70 individuals studied. Five individuals harbored serotype a, 7 serotype b, and 14 serotype c; one adult harbored both a and c and one strain was untypeable. If a subject was positive for A actinomycetemcomitans, the organism was most often isolated from plaque samples (86%), but also very frequently from samples taken from the dorsum of tongue (77%), or from stimulated saliva (68%). DISCUSSION We have looked at the distribution of A actinomycetemcomitans in families of periodontally healthy children and their parents with healthy periodontal tissues, or mild Periodontitis and in families where the adult member has severe Periodontitis and is infected with A actinomycetemcomitans. For the study it would have been interesting to include families whose child is periodontally diseased and harbors A actinomycetemcomitans. In Finland, however, prepupertal Periodontitis or juvenile Periodontitis in young permanent dentition is so rare that we could not gather such material. Based on the prevalence and serotype distribution of A actinomycetemcomitans in the families studied, we suggest that the transmission of A actinomycetemcomitans is intrafamilial. After detecting the organism in the child, it was also detected from either parent in all but one family. Furthermore it was shown that the child always harbored the same serotype strain as the parent. Serotype similarities in 5 mother/child pairs, 3 father/child pairs and in one whole family (both parents and 2 children) could be found. Comparable findings concerning mutans streptococci in children and their mothers have been reported.17 From 15 pairs of mothers and children, the discrepancies of the dominant serotypes oí mutans streptococci were observed only in 3 mother-child pairs. However, a similar distribution of mutans streptococci was not clearly seen in other relatives, including the father. Based on a determination of the serotypes and biotypes of A actinomycetemcomitans in families where a family member suffers from juvenile Periodontitis, Zambón et al. also suggest intrafamilial transmission of the organism.10 These investigators also pointed out that the organism does not readily colonize the tooth surface. In their material, over half of the family members studied were not infected. Our finding that children were infected in only 2 out of 9 families with an adult member suffering from severe Periodontitis and harboring A actinomycetemcomitans supports this statement.
actinomycetemcomitans is regarded as an exogenous pathogen the knowledge of the timing and route of the infection are important for its control. Little is known about the early colonization of A actinomycetemcomitans. A. actinomycetemcomitans has not been detected in study popIfA
ulations under the age of 2.5
years,18
but it is
frequently
210
detected in 5 to 6 year-old children with primary teeth.2 The present study demonstrated that the organism does not colonize transiently, but shows persistent oral colonization. The route of transmission may occur through direct person-to-person contact, by indirect contact through an inanimate object such as a shared toothbrush, or by droplets of saliva or other fluid which harbor the agent.8 When evaluating the role of saliva in transmission, it has been shown that, for example, mutans streptococci can be transmitted by saliva-contaminated spoons or other objects and that there is a correlation between the salivary count of mutans streptococci and the number of microorganisms transferred to the spoon.19 Furthermore it is more likely for infants to be infected with mutans streptococci if their mothers harbor dense salivary population of the organism than if the maternal salivary reservoirs are low or negligible.20 In our study population, if a person was positive for A. actinomycetemcomitans, the organism was detected in saliva in 68% of the subjects, indicating that saliva can act as direct or indirect vehicle in the transmission. Still, our method picked up only samples with an A. actinomycetemcomitans concentration of > 104 CFU/ml. Thus it is likely that the detection frequency of A. actinomycetemcomitans in saliva in persons infected with A. actinomycetemcomitans would be higher if a more sensitive method was used. For the immunodiffusion tests, crude autoclaved extracts of the clinical strains were used. This method was also used by Amano et al. for the serotyping of A. actinomycetemcomitans.21 Their preliminary results showed that the autoclaved extracts of serotype a, b, and c of A. actinomycetemcomitans strains formed strong precipitation lines only with the corresponding rabbit antiserum. In our study the method was found useful for serotyping the A. actinomycetemcomitans strains and all except three strains were easily characterized. Two strains (obtained from mother and her child) had strongest reaction with serotype c antiserum, but reacted also with antiserum of serotype a and b and one strain had poor reactivity with any of the antiserum used. In conclusion, based on the prevalence and serotype distribution of A. actinomycetemcomitans within families, intrafamilial transmittance is suggested. Because our data are cross-sectional we do not know the direction of infection. If the organism is transmitted from an adult to the child then parents are highly likely sources. However, the risk of the child getting the infection from an A. actinomycetemcomitans-positive parent is rather low. To prevent the infection, a more extensive survey must be performed to clarify the source and transmission route as well as the effect of the timing on the infection of A. actinomycetemcomitans.
Acknowledgment This
J Periodontol March 1991
TRANSMISSION OF . ACTINOMYCETEMCOMITANS
2. Asikainen S, Jousimies-Somer H, Kanervo A, Summanen P. Certain bacterial species and morphotype in localized juvenile Periodontitis and matched controls. / Periodontol 1987;58:224-230. 3. Slots J, Listgarten MA. Bacteroides gingivalis, Bacteroides intermedius and Actinobacillus actinomycetemcomitans in human periodontal diseases. / Clin Periodontol 1988;15:85-93. 4. Asikainen S, Alaluusua S, Kari , Kleemola-Kujala E. Subgingival microflora and periodontal conditions in healthy teenagers. / Periodontol 1986;57:505-509. 5. Alaluusua S, Asikainen S. Detection and distribution of Actinobacillus actinomycetemcomitans in the primary dentition. / Periodontol
1988;59:504-507. 6. Kilian M, Schiott CR. Haemophili and related bacteria in the human oral cavity. Arch Oral Biol 1975;20:791-796. 7. Kornman . Age, supragingival plaque, and steroid hormones as ecological determinants of the subgingival flora. In: Genco R, Mergenhagen S, eds. Host-Parasite Interactions in Periodontal Diseases. Washington DC: American Society of Microbiology, 1982:132-138. 8. Genco RJ, Zambón JJ, Christersson LA. The origin of periodontal infections. Adv Dent Res 1988;2:245-259. 9. Preus HR, Olsen 1. Possible transmittance of A. actinomycetemcomitans from a dog to a child with rapidly destructive Periodontitis. J Periodont Res 1988;23:68-71. 10. Zambón JJ, Christersson LA, Slots J. Actinobacillus actinomycetemcomitans in human periodontal disease. Prevalence in patient groups and distribution of biotypes and serotypes within families. J Periodontol 1983;54:707-711. 11. Sutter VL, Citron DM, Finegold SM. Wadsworth Anaerobic Bacteriology Manual. St. Louis: The CV Mosby Company; 1980. 12. Slots J. Selective medium for isolation of Actinobacillus actinomycetemcomitans. J Clin Microbiol 1982;15:606-609. 13. Campell DH, Garney JS, Cremer NE, Sussdorf DH. Nessler reaction. Methods in Immunology; A Laboratory Text for Instruction and Research. New York: Benjamin Wa, 1970;69-73. 14. Lai C- , Listgarten MA, Shirakawa M, Slots J. Bacteroides forsythus in adult gingivitis and Periodontitis. Oral Microbiol Immunol
1987;2:152-157. LA, Randall
15. Rantz
streptococci
for
1955;13:290-321.
E. Use of autoclaved extracts of
serological grouping. Stanford
hemolytic
Med Bull
16. Hamada S. Masuda N, Ooshima T. Sobue S. Kotani S. Epidemiologica! survey of Streptococcus mutans among Japanese children: Identification and serological typing of the isolated strains. Jpn J Microbiol 1976;20:33^14. 17. Masuda N, Shimamoto T, Kitamura K, Sobue S, Hamada S. Transmission of Streptococcus mutans in some selected families. Microbios
1985;44:223-232. KW, Higgins T, Palmer JM. The incidence of periodonto-
18. Frisken
pathic microorganisms
in young children. Oral Microbiol Immunol
1990;5:43^15. 19. Köhler , Bratthall D. Intrafamilial levels of Streptococcus mutans and some aspects of the bacterial transmission. Scand J Dent Res 1978;86:35^12. 20. Berkowitz RJ, Turner J, Green P. Primary oral infection of infants with Streptococcus mutans. Arch Oral Biol 1980;25:221-224. 21. Amano , Nishihara T, Shibuya , Noguchi , Koga T. Immunochemical and structural characterization of a serotype-specific Polysaccharide antigen from Actinobacillus actinomycetemcomitans Y4 (serotype b). Infect Immun 1989;57:2942-2946.
study has been supported by the Academy of Finland.
REFERENCES 1. Genco
RJ, Zambón JJ, Christersson LA. Use and interpretation of
microbiological munol
assays in
1986;1:73-79.
periodontal
diseases. Oral Microbiol Im-
Send reprint requests to: Dr. Satu Alaluusua, Department of Pedodontics and Orthodontics, Institute of Dentistry, University of Helsinki, Mannerheimintie 172, 00300 Helsinki, Finland. Accepted for publication August 29, 1990.
