INFECTION AND IMMUNITY, Dec. 1979, p. 1137-1145 Vol. 26, No.

3
0019-9567/79/12-1 137/09$02.00/0

Relationship Between Host Age and Susceptibility to Oral

Colonization by Actinomyces viscosus in Sprague-Dawley Rats
STEPHEN M. BRECHER"2* AND JOHANNES VAN HOUTE'
Department of Oral Microbiology, Forsyth Dental Center, Boston, Massachusetts 02115' and Department of
Biology, Northeastern University, Boston, Massachusetts 021152
Received for publication 29 August 1979

The colonization of Actinomyces viscosus strain Ny-1R on the molar teeth of

conventional and ex-germfree rats of various ages fed either a high-sucrose diet,
a high-glucose diet, or laboratory chow was studied. Conventional rats directly
after weaning and up to 30 days of age are less susceptible to experimental
infection by strain Ny-1R than are older rats regardless of the test diet. The
relationship between host age and susceptibility to infection is also demonstrable
in ex-germfree rats fed a high-sucrose diet. Host factors responsible for the
differences in susceptibility were investigated. The results from these studies do

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not implicate host antibodies, host indigenous flora, or host saliva. In other
studies, it was demonstrated that within the mouths of rats, strain Ny-iR
preferentially colonizes in the pits and fissures of the molar teeth rather than on
the dorsum of the tongue or on the vestibular mucosa. In short-term experiments,
it was found that strain Ny-1R attaches to the first molars of 40-day-old conven-
tional rats to a greater extent than it attaches to the first molars of 20-day-old
rats. The differences in attachment and subsequent colonization of strain Ny-iR
in 20- and 40-day-old rats may be related to the varying amounts of the reduced
enamel epithelium and connective tissue present in the fissures of the molar
teeth.
Actinomyces spp. are among the predominant until weaning of the pups at 17 to 21 days of age. All
organisms in human dental plaque (1, 18, 26) rats were individually housed in screen-bottomed
and have been implicated in the etiology of stainless steel cages without bedding and were pro-
certain types of periodontal disease (20, 21, 26, vided with experimental diet and drinking water ad
29, 30), root surface caries (19, 31, 32), and fissure libitum. The experimental diet consisted of 34.7% vi-
tamin-free casein, 5% salt mixture W, and 3.5% brew-
caries (16). However, little is known about the ers' yeast extract (all from Nutritional Biochemicals
factors that govern their establishment in the Corp., Cleveland, Ohio); this was supplemented with
mouth. Information about the mechanisms in- either 56% sucrose or 56% glucose. In some experi-
volved in the colonization of Actinomyces spp. ments, pelleted laboratory chow (Ralston-Purina, St.
on oral surfaces or the susceptibility of a host to Louis, Mo.) was used. The laboratory chow consisted
infection by these organisms may aid in the of 23.0% protein, 4.5% crude fat, 6.0% crude fiber, 8.0%
prevention and eradication of oral infections by ash, 2.5% minerals, 49.8% carbohydrate (mostly starch
Actinomyces spp. with less than 2.0% lactose and sucrose), and 6.2%
Recent studies suggest that Sprague-Dawley moisture. The test diets were provided at least 24 h
before inoculation with the test strain and for the
rats up to the age of about 3 months are more duration of the experimental periods. Before arrival
susceptible to infection by strains of Streptococ- from the supplier, the rats had been fed stock diet 4-
cus mutans than are older rats (36). These ob- RF (Agway, Inc., Syracuse, N.Y.).
servations with S. mutans, presently considered Germfree male and female Sprague-Dawley rats
as an important causative agent in dental caries raised and maintained at the Forsyth Dental Center
(15, 34), prompted similar investigation of the (12) were conventionalized before inoculation with the
role of host age in the susceptibility of rats to test strain by removing them from the germfree iso-
infection by Actinomyces viscosus. lators. These rats were also caged individually and fed
the sucrose diet.
Bacterial strains and inoculum preparation. A.
MATERIALS AND METHODS viscosus strain Ny-1 was kindly provided by B. Gug-
Animals and diet. Conventional male Sprague- genheim, Zurich, Switzerland. It was originally isolated
Dawley rats of various ages were obtained from from glucose-fed rats (33). Streptomycin-resistant mu-
Charles River Breeding Laboratories, Wilmington, tants were selected by serial transfer of the test strain
Mass. Mothers and male pups were caged together in Actinomyces broth (BBL Microbiology Systems,
1137
1138 BRECHER AND VAN HOUTE
Cockeysville, Md.) with increasing concentrations (up
to 5,000 jg/ml) of streptomycin sulfate (ICN Biochem-
icals, Cleveland, Ohio). For experimental purposes,
fresh isolates of strain Ny-lR (suffix R indicates strep-
tomycin-resistance) were obtained from plaque sam-
ples of previously infected conventional Sprague-Daw-
ley rats fed a diet with 56% sucrose. Strain Ny-1R was
cultivated in Actinomyces broth in candle jars for 48
h at 370C. Cells for animal inoculation were washed
once in quarter-strength Ringer solution (35) and, if
required, appropriately diluted or concentrated in the
same solution.
S. mutans strain 6715 (resistant to streptomycin),
obtained from the culture collection at the Forsyth
Dental Center, was cultivated in Trypticase broth (11)
with 0.1% glucose in Brewer Anaerobic Jars (Becton,
Dickinson & Co.) with an atmosphere of 90% N2, 5%
H2, and 5% CO2 for 48 h at 370C. S. mutans cells for
animal inoculation were washed once in quarter-
strength Ringer solution (35) and diluted in the same
solution before use.
Animal inoculation and determination of bac-
terial establishment. Rats were orally inoculated
once via a 1.0-ml pipette with 0.2 ml of the desired cell
inoculum, unless noted otherwise. The number of col-
ony-forming units (CFU) per 0.2 ml of inoculum was
determined by culturing duplicate samples of appro-
priate dilutions on agar plates containing 5% sheep
blood (Scott Laboratories, Fiskville, R.I.), which were
incubated in candle jars for 48 h at 37°C. The inocula
used in the different experiments varied from 3.0 x 103
up to about 109 CFU; the inocula for the experiments
are given in the tables. To determine whether strain
Ny-lR became implanted in the mouth, the molar
teeth were sampled with Calgiswabs (Wilson Diagnos-
tic, Inc., Glenwood, Ill.) within 1 week after inocula-
tion. The swabs were streaked directly onto agar plates
containing 5% sheep blood and 200 yg of streptomycin
sulfate per ml. Plates were incubated in candle jars for
48 h at 37°C. At the end of the experiments, rats were
sacrificed by chloroform inhalation, and some or all of
the 12 molar teeth were carefully extracted with a
sterile forceps. The teeth were ground in glass tissue
grinders containing 3.0 ml of quarter-strength Ringer
solution for 60 s. The resulting suspensions were dis-
persed with a Vortex mixer (Scientific Products, Ev-
anston, Ill.) at setting 6 for 30 s. Samples of undiluted
and appropriately 10-fold-diluted suspensions were
cultured in duplicate on streptomycin-containing
blood agar plates. All plates were incubated in candle
jars for 48 h at 37°C. Plates with about 30 to 300
colonies were counted.
Relationship between rat age and colonization
of strain Ny-iR on teeth. Conventional rats ranging
in age from 20 to 180 days were used. Groups consisting
of 5 or 10 rats of the same age were fed the sucrose
diet before being inoculated with strain Ny-lR (var-
ious cell sizes) inocula. In other experiments, groups
of five 21- or 50-day-old ex-germfree rats were fed the
sucrose diet and inoculated 6 h thereafter. The pres-
ence of the test strain on the teeth of all rats was
determined within 1 week after inoculation. The pop-
ulations of strain Ny-IR present on the teeth of all
animals were determined at the end of experimental
periods of different lengths.
INFECT. IMMUN.
Relationship between rat age, rat diet, and
colonization of strain Ny-1R on teeth. Groups of
five conventional rats either 21 or 40 days old were
used. These rats were fed the sucrose diet, the glucose
diet, or the laboratory chow diet. The populations of
strain Ny-1R on the teeth of all rats were determined
at 30 or 50 days after inoculation.
Colonization of strain Ny-1R at various sites
in the mouth. Ten conventional rats 30 days old were
used. These rats were fed the sucrose diet. Rats were
inoculated on each of 7 days with high cell inocula (108
to 109 CFU) of strain Ny-IR to ensure infection of all
animals. All rats were sacrificed 75 days after inocula-
tion. Samples from the dorsum of the tongue and
vestibular mucosa were taken by thorough swabbing
with calgiswabs. Six molars were extracted from one
maxillary and one mandibular quadrant of the denti-
tion and finely ground in tissue grinders. The calgiswab
samples were placed in quarter-strength Ringer solu-
tion. The suspensions of all three types of samples
were dispersed with a Vortex mixer. Appropriate 10-
fold dilutions of all samples were cultured in duplicate
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on blood agar plates with streptomycin for the enu-
meration of strain Ny-iR and on blood agar plates
without streptomycin for the enumeration of all bac-
teria. All plates were incubated anaerobically in
Brewer Anaerobic Jars for 48 to 72 h at 370C. The
proportions of strain Ny-1R in the samples were ex-
pressed as percentages of the total anaerobically cul-
tivated flora.
Four conventional rats were fed the glucose diet
and inoculated with 108 CFU of strain Ny-1R. Rats
were sacrificed 60 days after inoculation, and all four
molar quadrants, each consisting of three molars with
surrounding oral tissues and alveolar bone, were care-
fully dissected. The quadrants were placed in Karnov-
sky fixative (23) for 24 h, postfixed in 2% osmium
tetroxide in S-collidine buffer, dehydrated through
increasing concentrations of ethyl alcohol, passed
through amyl acetate, and critical point dried with
CO2. Specimens were mounted on aluminum stubs
with silver paint, coated with palladium-gold, and
examined with a model JEOL U3 scanning electron
microscope (Japan Electronics and Optical Laborato-
ries, Tokyo, Japan). The entire surface of each molar
was inspected for the presence of cells of the test
organism.
Relationship between rat age and colonization
of the first molar teeth by strain Ny-1R. Groups
of conventional rats 18 and 38 days old were fed the
sucrose diet. Rats of both age groups were inoculated
with approximately 106 CFU (close to the minimum
infective dose) of strain Ny-IR and were sacrificed 4
or 24 h thereafter. All four first molar teeth were
extracted, and the populations of strain Ny-1R on
these teeth were determined.
Colonization of A. viscosus strain Ny-iR and S.
mutans strain 6715 on the first molars of sucrose-
fed rats. Groups of 10 conventional rats either 18 or
38 days old were fed the sucrose diet and were inocu-
lated with a suspension containing a mixture of 2.0 x
107 CFU of A. viscosus strain Ny-LR and 3.0 x 107
CFU of S. mutans strain 6715. All rats were sacrificed
24 h after inoculation to determine the populations of
both test strains on four extracted first molars.
VOL. 26, 1979 AGE OF RATS AND COLONIZATION OF A. VISCOSUS
Survival of strain Ny-1R in saliva. Pilocarpine
nitrate-stimulated saliva was collected from sedated
20- and 40-day-old conventional rats (2). Before use
the saliva was placed in a 60'C water bath for 30 min
to inactivate salivary enzymes and was then clarified
by centrifugation at 12,000 x g for 10 min (2, 5). Strain
Ny-1R was cultivated for 48 h in Actinomyces broth.
Cells were harvested by centrifugation and washed
twice in Ringer solution, and 3.5 x 105 CFU was then
added to 2 ml of saliva. Duplicate samples (0.1 ml)
were taken periodically during aerobic, stationary in-
cubation of the saliva-bacteria mixtures at 370C and,
after being blended with a Vortex mixer for 15 s at
setting 6, were cultured on blood agar plates with
streptomycin to determine the number of viable or-
ganisms present.
Adherence of strain Ny-1R to HA beads. The
adherence of [3H]thymidine-labeled cells of strain Ny-
1R to uncoated and saliva-coated spheroidal hydrox-
yapatite (HA) beads was determined by the method
of Clark et al. (4), which was modified and has been
described previously (2). In brief, saliva was obtained
from conventional rats 20, 40, and 90 days old as
described above and elsewhere (2). Cells of strain Ny-
1R were cultivated in Trypticase broth (11) with 5 tCi
of [3H]thymidine (New England Nuclear Corp., Bos-
ton, Mass.) per ml and 0.2% glucose. A 15-mg amount
of spheroidal HA beads (BDH, Poole, England) was
placed in 500-ml polypropylene micro-test tubes (Dy-
ana Laboratories, Rochester, N.Y.) and washed three
times in 0.01 M phosphate-buffered saline pH 6.0.
Tubes were filled with approximately 0.5 ml of saliva
or phosphate-buffered saline, tightly stoppered, and
placed at room temperature in a turning apparatus
which continuously inverted them 10 times per min
for 16 to 18 h. Unabsorbed saliva was removed by
washing twice in phosphate-buffered saline. Tubes
with phosphate-buffered saline-treated HA beads or
saliva-coated beads were then filled with 0.5 ml of
[3H]thymidine-labeled cell suspensions of strain Ny-
1R containing approximately 2.2 x 107 cells per ml,
with a specific activity of 8 x 104 to 12 x 104 cpm. The
tubes were continuously turned at room temperature
for 2 h. The beads were allowed to settle for 1 min,
and the supernatant, which contained the unabsorbed
organisms, was removed. The cells in the supernatant
were collected on a 0.45-,.m membrane filter (Millipore
Corp., Bedford, Mass.), and the activity on the filters
was counted in a Packard Tri-Carb scintillation spec-
trometer. Cell adherence to HA was determined after
washing the beads twice with phosphate-buffered sa-
line to remove the few remaining unabsorbed cells and
then transferring them to scintillation vials for count-
ing. Control bacteria suspensions (0.5 ml) were incu-
bated without HA beads and counted similarly to
correct for cell loss due to adsorption to the tubes. The
counts per minute of cells adhering to the HA beads
were expressed as percentages of the counts per min-
ute of cells in the control suspensions. All assays were
carried out in duplicate.
RESULTS
Relationship between rat age and colo-
nization of strain Ny-1R on teeth. Conven-
1139
tional rats 20 days old were less susceptible to
infection by strain Ny-iR than were rats 40 days
old or older (Table 1). Oral swabbing 6 days
after inoculation with about 107 CFU (Table 1,
experiment 1) revealed that only a few of the
younger rats but all of the older rats were in-
fected. The level of infection in the younger rats
(+) was lower than that in the older rats (+ to
4+). At the end of the experiment, only one-half
of the younger rats but nearly all of the older
rats were infected. The populations of strain Ny-
1R on the molar teeth of infected rats of both
age groups were in the order of 108 CFU.
The demonstration of the effect of host age on
the implantation of strain Ny-1R appeared to
depend on the inoculum size (Table 1, experi-
ment 2). Thus, with an inoculum of 5.0 X 108
CFU, little difference was evident between 20-
and 40-day-old rats. With an inoculum of 5.0 x
107 CFU, a clear difference was observed.
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Other experiments with 20-, 25-, 30-, and 35-
day-old rats (Table 2) indicated that the lower
susceptibility of young rats to infection by strain
Ny-1R lasted until 25 to 30 days of age.
The effect of age on susceptibility to infection
by strain Ny-1R was also observed with ex-germ-
free rats (Table 3). The minimum infective dose
for these rats was on the order of 104 CFU and
was over 100-fold lower than that for conven-
tional rats.
Relationship between rat age, rat diet,
and colonization of strain Ny-iR on teeth.
The results of implantation of strain Ny-1R in
21- and 40-day-old conventional rats fed differ-
ent diets are shown in Table 4. The effect of rat
age on the implantation of strain Ny-iR was less
marked in the case of glucose-fed rats than in
the case of rats fed the sucrose diet or laboratory
chow. Glucose appeared to favor somewhat the
implantation of strain Ny-1R, as judged by the
number of rats that became infected. The pop-
ulations of strain Ny-1R on the teeth of sucrose-
and glucose-fed rats were similar. Its populations
on the teeth of rats fed laboratory chow in the
experiment shown (Table 4), as well as in other
experiments, were always significantly lower (P
< 0.01; Student's t test) than in the case of rats
fed a sucrose or glucose diet.
Colonization of strain Ny-1R in various
sites of the mouth. Strain Ny-1R was found to
preferentially colonize the teeth, where it com-
prised 81% of the total cultivable flora (Table 5);
in contrast, its proportions on the vestibular
mucosa and the dorsum of the tongue were
significantly lower (P < 0.01; Student's t test).
Examination of molar teeth of conventional rats
infected with strain Ny-lR by scanning electron
microscopy revealed the presence of large plaque
masses in the fissures and to a lesser extent in
1140 BRECHER AND VAN HOUTE INFECT. IMMUN.
TABLE 1. Establishment of A. viscosus strain Ny-JR on the teeth of conventional rats of various ages fed
high-sucrose diet
ExptInoculum Days No. of rats infected/no. of rats inoculated when age at time of inoculation was:
Expt (CFU) after in-
oculation 20 days 40 days 60 days 180 days
1 2.3 x 107 6 2/10a (+)" 10/10 (+ to 4+) 10/10 (+ to 4+) 10/10 (+ to 4+)
30 5/10 [1.4 x 105f' 10/10 [1.2 x 108] 10/10 [9.4 x 10'] 10/10 [9.0 x 10']
2 5.0 x 108 6 9/10 (+) 10/10 (+ to 4+)
45 10/10 [1.6 x 108] 10/10 [2.2 x 108]
5.0 x 10' 6 3/10 (+) 10/10 (+ to 4+)
45 3/10 [1.7 x 108] 10/10 [1.7 x 108]
"Symbols in parentheses indicate recoveries from oral swabs obtained from infected rats and streaked on
streptomycin blood agar plates: +, 1 to 25 CFU; 2+, 26 to 100 CFU; 3+, 101 to 200 CFU; 4+, >200 CFU.
b
Numbers in brackets are arithmetic means of total recoveries (colony-forming units) from 12 molar teeth of
infected rats at sacrifice.

TABLE 2. Establishment of A. viscosus strain Ny-
JR on the teeth of conventional rats 20 to 35 days
old fed high-sucrose diet
No. of rats infected/no. of rats inoculated
were lower in the younger animals. However, 24
h after inoculation the typical age effect was
evident, and the mean recoveries of strain Ny-
1R from the first molars of the younger rats

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Inoculum when age at time of inoculation was: were significantly lower (P < 0.05; Student's t
(CFU) test).
20 days 25 days 30 days 35 days Colonization of A. viscosus strain Ny-iR
1.0 x 108 2/5" 4/5 5/5 5/5 and S. mutans strain 6715 on the first mo-
1.0 x 107 1/5 2/5 4/5 4/5 lars of sucrose-fed rats. S. mutans strain 6715
1.0 x 106 0/5 1/5 2/5 3/5 has been shown previously to colonize the mo-
a Determinations were made at sacrifice (45 days lars of rats 17 days to about 3 months of age
after inoculation) by oral swabbing of molar teeth. with about equal efficiency; in older rats, how-
ever, its colonization was found to be impaired
TABLE 3. Establishment of A. viscosus strain Ny- (36). A difference between 20- and 40-day-old
1R on the teeth of ex-germfree rats 21 and 50 days rats with respect to their susceptibility to strains
old fed high-sucrose diet Ny-1R and 6715 was shown in a short-term
No. of rats infected/no. of rats inoculated experiment in which rats were inoculated with
Inoculum when age at time of inoculation was: a mixture of the two organisms (Table 7). At 24
(CFU) h after inoculation, the typical age effect was
21 days 50 days observed with strain Ny-1R; in contrast, strain
3.0 X 104 3/5 (2.8 X 108)a 5/5 (2.3 X 108) 6715 became established in all rats of both age
3.0 X 103 1/5 (1.6 X 108) 3/5 (4.0 X 108) groups. The mean recovery of strain Ny-1R from
" Numbers in parentheses are arithmetic means of the first molars of the older rats was higher than
total recoveries (colony-forming units) from 12 molar that from the younger rats (P < 0.05; Student's
teeth of infected rats. Determinations were made after t test). No difference in this respect was observed
sacrifice of the rats at 65 days after inoculation. with strain 6715. Also, although the proportions
of cells of the two organisms in the inoculation
the interproximal areas (Fig. IA). Plaque was mixture were about equal, about 100-fold-higher
virtually absent on the smooth tooth surfaces numbers of strain 6715 than of strain Ny-IR
(Fig. 1B). Higher magnification of the plaques were present on the first molars of 20-day-old
revealed the presence of rod-shaped organisms rats 24 h after inoculation (P < 0.01; Student's
that clearly resembled cells of strain Ny- iR (Fig. t test).
1C). Survival of strain Ny-1R in saliva. Strain
Relationship between rat age and colo- Ny-1R survived equally well in saliva obtained
nization of first molars by strain Ny-lR. from conventional rats 20 and 40 days old (Table
The relationship between rat age and suscepti- 8). At the end of the 24-h experimental period,
bility to strain Ny-1R was also demonstrable in the recoveries of strain Ny-iR were higher than
short-term experiments in which the recoveries those at the start of the experiment.
of the test strain were determined from first Adherence of strain Ny-1R to HA beads.
molars only (Table 6). At 4 h after inoculation, Cells of strain Ny-1R adhered equally well to
strain Ny-iR was recovered from first molars in uncoated HA beads and to HA beads coated
all 20- and 40-day-old rats. The mean recoveries with saliva obtained from conventional rats 20,
VOL. 26, 1979 AGE OF RATS AND COLONIZATION OF A. VISCOSUS
TABLE 4. Establishment of A. viscosus strain Ny-JR on the teeth of conventional rats 21 and 40 days old
fed sucrose, glucose, or laboratory chow
Expt Inoculum (CFU) Dietary carbohydrate
1 2.3 X 107 Sucrose
Glucose
2 2.8 X 106 Sucrose
Glucose
Laboratory chow
a
Numbers in parentheses are arithmetic means of total recoveries (colony-forming units) from 12 molar teeth
of infected rats. Determinations were made after sacrifice of the rats at 30 and 50 days after inoculation in
experiments 1 and 2, respectively.
TABLE 5. Proportional distribution of A. viscosus
strain Ny-JR in the mouths of conventional rats fed
high-sucrose diet
Oral site Proportion of strain
Ny-lRa
Vestibular mucosa 33 ± 24
Teeth 81 ± 14
Dorsum of the tongue 10 ± 8
a Proportions of strain Ny-1R are expressed as per-
centages of the total anaerobically cultivable flora of
10 rats. Strain Ny-1R was implanted in all rats. Mean
± standard deviation.
40, and 90 days of age (Table 9). In all of these
experiments, more than 90 percent of the labeled
cells adhered to the HA beads.
DISCUSSION
This study demonstrates that the molars of
Sprague-Dawley rats immediately after weaning
and up to about 30 days of age are less suscep-
tible to infection by A. viscosus strain Ny-1R
than are the molars of older rats. This effect of
age was demonstrable with conventional rats fed
a high-sucrose diet, a high-glucose diet, or labo-
ratory chow, as well as with ex-germfree rats fed
a high-sucrose diet. In long-term experiments
(usually 30 to 50 days), the effect of age was
manifest only as a difference in the proportions
of rats that became infected. The numbers of
strain Ny-iR on the teeth of infected rats were
usually on the order of 107 to 108 CFU, regardless
of rat age. In short-term studies in which the
implantation of the test organism was deter-
mined within 24 h after inoculation, differences
in the numbers of the test organism on the teeth,
as well as in the proportions of rats that became
infected, were evident.
The age-associated difference in the suscepti-
bilities of rats to infection by strain Ny-1R ap-
parently involves the colonization of the oral
cavity rather than the colonization of other parts
of the body. Studies of the intraoral distribution
1141
No. of rats infected/no. of rats inoculated when age at
time of inoculation was:
21 days 40 days
2/5 (1.1 X 108)a 5/5 (1.4 X 108)
4/5 (1.2 X 108) 5/5 (1.0 X 108)
0/5 (0) 2/5 (2.5 X 108)
3/5 (1.3 X 108) 5/5 (1.2 X 108)
0/5 (0) 3/5 (2.3 X 107)
of our strain of Ny-iR showed that this organism
is particularly adept at colonizing the pits and
fissures of the molar teeth and to a lesser extent
the approximal tooth surfaces as compared with
the oral epithelial surfaces. Recent work by El-
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len (8) suggests a similar distribution for A.
viscosus in humans. In other experiments (data
not shown), cultivation of feces of 40-day-old
rats infected a few days previously with strain
Ny-1R revealed that the intestinal concentra-
tions of the test strain were approximately 102
CFU/100 mg (wet weight) of feces; this concen-
tration is far below the minimum infective dose.
It was also found that Ny-1R cells could not be
detected in feces until at least 12 to 16 h after
oral inoculation. Since the age effect was clearly
evident within 24 h after oral inoculation, it
appears that recycling of Ny-1R cells from intes-
tinal reservoirs to the mouth via copraphogy
was not of major importance with respect to the
colonization of molar teeth by strain Ny-IR.
Results from the present study may provide
some clues about the mechanisms that underlie
the differences in host susceptibilities to infec-
tion by strain Ny-1R. First, they do not readily
suggest that antibody is involved. The relative
resistance to infection of conventional rats early
after weaning but not later in life seems to argue
against this. Furthermore, similar patterns of
susceptibility were observed with the ex-germ-
free rats; these rats would not be expected to
possess effective concentrations of homologous
or cross-reacting antibody directed against Ac-
tinomyces or related organisms acquired from
the outer environment only a few hours after
conventionalization. An effect of cross-reacting
antibody induced by germfree diet components,
although unlikely, cannot be ruled out. Second,
the differences in the inocula required for infec-
tion of ex-germfree and conventional rats as
observed in the present study indicate that the
oral indigenous flora may influence the implan-
tation of strain Ny-1R on the molar teeth. How-
1142 BRECHER AND VAN HOUTE INFECT. IMMUN.
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FIG. 1. Localization of A. viscosus strain Ny-JR on the molar teeth of conventional rats. (A) Presence of
bacterial plaque in the fissures (>p) and in the interproximal area (ip). x50. (B) Presence of bacterial plaque
in the fissures (fp) and absence of bacterial plaque on smooth surface areas (s). x50. (C) Higher magnification
of fissure plaque. The bacterial cells resemble strain Ny- IR. x 7,000.
ever, it does not seem likely that the 20- and 50- The relationship between host age and sus-
day-old germfree rats conventionalized for only ceptibility to infection by strain Ny-iR could
a few hours acquired a different flora which also be due to changes in the composition of
subsequently influenced the implantation of saliva. Salivary components engage in a variety
strain Ny-1R. of interactions with oral bacteria (5, 9, 13, 14, 17,
VOL. 26, 1979 AGE OF RATS AND COLONIZATION OF A. VISCOSUS 1143
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TABLE 6. Establishment of A. viscosus strain Ny- TABLE 7. Establishment of A. viscosus strain Ny-
IR on the first molars of conventional rats fed high- IR and S. mutans strain 6715 on the first molars of
sucrose diet conventional rats 24 h after inoculation with a
No. infected/no. inoculated mixture containing both test strains
Inoculum Time after when age at time of inocula-
Age at inoc- No. of rats in- Total recovery (CFU) of:.
(CFU) inoculation tion was: fected with:'
~~(h) ulation
20 days 40 days (days) Ny-IR 6715 Ny-1R 6715
7.6 X 10" 4 7/7a (75)b 7/7 (140) 20 6 10 4.5 X 102 3.6 X 104
5.0 X 106 24 4/15 (17) 11/15 (370) 40 10 10 5.0 X 101 1.5 X 104
a Number of rats with infected first molars/number
" Numbers of rats infected with each test strain
of rats inoculated. after inoculation of 10 rats with a mixture containing
'Numbers in parentheses are arithmetic means of 2.0 x 10' CFU of strain Ny-1R and 3.0 X 10' CFU of
total recoveries (colony-forming units) from all four strain 6715.
first molar teeth of sacrificed rats. h Arithmetic means of total recoveries of each strain
from all four first molar teeth of rats sacrificed 24 h
after inoculation.
34, 37) and can probably significantly influence
the adherence-colonization processes. For ex-
ample, it has been shown that the adsorption of in which the saliva used was obtained from rats
S. mutans cells to HA disks is influenced by 20 or 40 days of age.
pretreatment of the disks with saliva for various Highly specific adherent interactions between
periods of time (5). Changes in salivary compo- bacteria and host tissue may determine the re-
sition or flow rate occur after weaning of animals sistance or susceptibility of the host to bacterial
(3, 7, 28) and can be induced by the administra- infection (10, 14, 34). Kalberer and co-workers
tion of sex hormones (6, 25, 27, 28). However, in have shown that rat teeth, immediately after
the present studies no differences were observed eruption, are covered by the reduced enamel
in the adherence to HA, survival, or aggregation epithelium and connective tissue (22). These
(data not shown) of strain Ny-iR in experiments tissues gradually degenerate within 1 to 2 weeks
1144 BRECHER AND VAN HOUTE
TABLE 8. Recovery ofA viscosus strain Ny-IR from
pilocarpine-stimulated rat saliva
Recovery (CFU/ml of saliva)
Time (h)
20 days' 40 days
0 3.5 X 105 3.5 X 105
0.5 3.7 X 105 3.6 X 105
1.0 4.0 X 105 3.6 X 105
4.0 4.6 X 105 3.8 X 105
8.0 4.9 X 105 4.6 X 105
24.0 3.8 X 106 3.6 X 106
a Age of rats at the time that saliva was collected.
TABLE 9. Adherence of [3Hjthymidine-labeled cells
of A. viscosus strain Ny-JR to uncoated and saliva-
coated HA beads
% Adherence % Adherence to saliva-coated HA beads at
to uncoated age.a
HA beads 20 days6 40 days6 90 days6
3d 3d
91 ± 5c 92 ± 94 ± 94 ±
a Four different experiments.
bThree different experiments.
'
Age of rats at the time saliva was collected.
d
Mean ± standard deviation.
after molar eruption (22). The presence of sur-
face integuments in the fissures of molar teeth
could directly affect the adherence of Ny-1R to
the teeth; they could also have an indirect effect
by altering the acquired salivary pellicle which
forms on top of these tissues and to which Ny-
1R bacteria must adhere in order to initiate
plaque formation. The first, second, and third
molars of Sprague-Dawley rats erupt at ages 13
to 20 days, 17 to 22 days, and 33 to 41 days,
respectively (24). Based on the data of Kalberer
et al., it is likely that remnants of these tissues
are still present in the first molars of 20-day-old
rats. The first molars of rats 40 days old should
be fully erupted and virtually devoid of these
tissues. Thus, the difference in susceptibility
observed between 20- and 40-day-old rats could
be due to the varying amounts of these tissues
present in the fissure spaces after molar erup-
tion. This is supported by the significant differ-
ences in the mean recoveries of strain Ny-iR
from the first molars of 20- and 40-day-old rats
in the short-term experiments. This view is also
supported by the similarity of the survival of the
organism in the saliva obtained from rats of the
two age groups and by the fact that its adherence
to HA coated with the two salivas was similar.
Ellen (8) recently reported that the coloniza-
tion of organisms resembling catalase-positive
A. viscosus in human mouths does not occur
until after tooth eruption. The frequency of iso-
lation of these bacteria slowly increases with
host age, and by age 7 years, A. viscous is
INFECT. IMMUN.
detectable in over 50% of the subjects. Thus,
there is some analogy between rats and humans
with respect to the oral acquisition of A. visco-
sus. However, it does not appear that the mech-
anisms responsible for the age effect are similar
for the two hosts. With humans, a role of the
reduced enamel epithelium seems diminished
because this tissue is lost soon after tooth erup-
tion, whereas the delay in the oral colonization
of A. viscosus persists after the eruption of the
deciduous dentition and its replacement by per-
manent teeth. Furthermore, in the present study
changes in saliva of rats during their aging which
could be responsible for a change in the effi-
ciency with which A. viscosus strain Ny-1R ad-
heres to teeth could not be detected. In contrast,
recent work by Qureshi and Gibbons involving
an adherence assay system similar to that em-
ployed by us suggests that changes in saliva may
3d occur during aging of human subjects, which
Downloaded from iai.asm.org by on May 14, 2010
favorably influence the adherence ofA. viscosus
to teeth (V. Qureshi and R. J. Gibbons, Abstr.
Int. Assoc. Dent. Res. 1979, abstr. no. 996).
It is of interest to note that in the present
study the colonization of S. mutans strain 6715,
in contrast to that of strain Ny-lR, was not
different in rats 20 and 40 days old. Further
studies of the affinities of these two strains for
the developmental integuments and salivary ma-
terials present on the teeth at various times after
tooth eruption may help to clarify the difference
in the susceptibilities of rats of various ages to
infection by these organisms.
ACKNOWLEDGMENTS
This investigation was supported by Public Health Service
grant DE-02847 from the National Institute of Dental Re-
search.
The technical assistance of S. Edelstein and M. Berk is
gratefully acknowleged. Special thanks are due to Z. Skobe
and D. Stern for assistance with electron microscopy and to
0. Marsh for typing assistance.
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