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Infection and Immunity, November 1999, p. 6098-6103, Vol. 67, No. 11
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
A New Rat Model of Otitis Media Caused by
Streptococcus pneumoniae: Conditions and Application in
Immunization Protocols
Leo T. M.
van der
Ven,1,*
Germie P. J. M.
van den Dobbelsteen,2
Bhawani
Nagarajah,1
Harry
van Dijken,2
Paul M.
Dortant,1
Josephus G.
Vos,1 and
Paul J. M.
Roholl1
Laboratory of Pathology and
Immunobiology1 and Laboratory of Vaccine
Development and Immune Mechanisms,2 National
Institute of Public Health and the Environment, Bilthoven, The
Netherlands
Received 26 April 1999/Returned for modification 26 May
1999/Accepted 25 August 1999
 |
ABSTRACT |
Streptococcus pneumoniae (pneumococcus [Pn]) can be
cultured from up to 50% of acute otitis media (AOM) effusions, and
these bacteria are the most common cause of AOM-related complications. With the recent advent of antibiotic-resistant Pn strains, treatment of
Pn infections may meet with serious difficulties. Prevention through
vaccination, notably for the four most common occurring Pn serotypes in
humans (i.e., Pn 6B, Pn 14, Pn 19F, and Pn 23F), is a helpful
alternative. Testing of vaccine efficacy should occur in an appropriate
animal AOM model, which is presented here. The four involved Pn
serotypes are not pathogenic to the rat, which was chosen as the
experimental animal for practical reasons. To induce a natural
infection (i.e., ascending through the eustachian tube), the
mucociliary clearance of the eustachian tube was impaired by infusing
histamine into the tympanic cavity on 2 consecutive days before
intranasal inoculation of the bacteria. With this simple protocol, high
and reproducible infection rates, as determined with bacterial
cultures, of Pn-induced AOM (approximately 70%) with the two major Pn
serotypes 14 and 19F (Pn 14 and Pn 19F) were obtained, whereas lower
infection rates (25 to 50%) with Pn 6B and Pn 23F were obtained. In
this model, intranasal priming with pneumococci, as well as
subcutaneous vaccination with Pn 14 tetanus toxoid-conjugated
polysaccharide, induced a protective effect against the induction of
otitis media with these bacteria. This shows that immunity to Pn 14 AOM
can be induced by both mucosal and systemic presentations of antigen.
In conclusion, we have developed an animal model for Pn-induced AOM,
which is suitable for the evaluation of the protecting effect of immunization.
| |
INTRODUCTION |
Acute otitis media (AOM) is
characterized by acute inflammation of the middle ear and is associated
with a concurrent or subsequent suppurative process in the middle ear
(9). AOM is a common disease in preschool children. Review
of epidemiological surveys on otitis media shows that 50 to 70% of
children have experienced their first episode of otitis media before 2 or 3 years of age (11). Most cases of AOM resolve
spontaneously in less than 3 or 4 weeks, but a selected population will
develop recurrent and severe disease (2, 9). Possible
complications include meningitis, lateral sinus thrombosis, chronic
suppurative otitis media, and bacteremia (22, 23).
Streptococcus pneumoniae (pneumococcus [Pn]) can be
isolated from up to 50% of AOM effusions (2) and is the
most common cause of complications (23). Antibacterial
treatment has shown good activity against pneumococcal AOM, but with
the recent advent of antibiotic-resistant pneumococcal strains there is
an increasing risk for serious and fatal infections (16, 23,
27). Preventive immunization against pneumococcal disease,
especially in individuals at risk, may be preferred to treatment of
existing infections. However, current licensed vaccines based on
streptococcal capsular polysaccharides are poorly immunogenic in
children under 2 years of age and in immunocompromised individuals
(3). Therefore, new vaccines with increased immunogenicity,
such as the tetanus toxoid-conjugated pneumococcal polysaccharide
vaccine (24), are being constructed. There is a general need
for an animal model that can test the efficacy of such vaccines and
that can help to develop correlates of protection for Pn-induced
diseases, particularly AOM. This animal model should meet the following
conditions. (i) Infection should occur through a natural route, i.e.,
ascending from the nasopharynx through the eustachian tube to the
middle ear, to enable determination of the effect of mucosal immunity in the eustachian tube. (ii) Minimal manipulation of the laboratory animal should facilitate induction of otitis media with the
pneumococcal serotypes covering approximately 60% of Pn-induced middle
ear infections in young children, i.e., Pn serotype 6B (Pn 6B), Pn 14, Pn 19F, and Pn 23F (4, 6). (iii) The laboratory animal of
choice should be widely available and well characterized with respect
to genetic, microbiologic, and immunologic determinants. Established
clearance models for infectious otitis media employ direct instillation
of bacteria into the tympanic cavity in rats or chinchillas (10,
15), thus avoiding the eustachian tube route. Other models
combine infection with a respiratory virus (influenza virus A or
adenovirus) and intranasal inoculation of bacteria (13, 21).
These models do not suit our purpose since they were established in
chinchillas, a species which doesn't meet the conditions mentioned above.
We developed a new animal model for Pn-induced otitis media in the rat.
In this animal, we have employed intratympanic administration of
histamine to impair ciliary activity in the tubotympanum and to induce
mucosal swelling; this protocol prolongs the mucociliary clearance time
from the tympanic cavity (7). The rationale for this
protocol was that with malfunctioning of the mucociliary physiology of
the eustachian tube there would be a decreased barrier for pneumococci
to enter the tympanic cavity.
Practical employment of the protocol in evaluating protection against
pneumococcal disease was tested in two different immunization schemes:
intranasal priming with vital pneumococci and subcutaneous vaccination
with Pn 14 tetanus toxoid-conjugated polysaccharide (PS14TT).
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MATERIALS AND METHODS |
Animals and experimental conditions.
Female specific
pathogen-free rats of the Rivm:WU(CPB) strain, 6 weeks of age, were
obtained from breeding facilities of the National Institute of Public
Health and the Environment, Bilthoven, The Netherlands. During the
experiments, animals were housed individually in Macolon III filter top
cages, were given SSP-Tox standard diet (Hope Farms, Woerden, The
Netherlands) and drinking water ad libitum, and were kept at a 12-h
light-12-h dark regime. Disturbances were minimized. The appropriate
number of experimental units was determined by using an analysis of
statistical power (8). All procedures with the animals were
performed under the supervision of the Institute's Council for
Experiments on Animals, according to Dutch legislation.
Bacterial strains and culture.
Pn 19F and Pn 23F were
obtained from the National Institute of Public Health and the
Environment. Pn 6B (BG 7322) was obtained from A. Virolainen
(University of Alabama). Pn 14 (IHU 30544) was a blood isolate from a
Finnish patient. All serotypes were passed intraperitoneally through
rats to increase virulence. Bacteria were isolated from peripheral
blood after 18 h and stored in 20% glycerol at 
70°C.
Pneumococci were serotyped by the Quellung reaction with antisera from
the Statens Seruminstitut, Copenhagen, Denmark. Pneumococci were
routinely grown overnight on blood agar plates (Oxoid, Haarlem, The
Netherlands). Before inoculation, the bacteria were cultured at 37°C
in 20 ml of Todd-Hewitt broth containing 2% inactivated horse serum
and gently shaken (120 cycles/min). After 3 to 4 h, the bacteria
reached log phase and were collected. After centrifugation at 3,500 rpm
for 10 min, the supernatant was discarded. The pellet was resuspended
in 10 ml of PBS, and the concentration of vital bacteria in the
suspension was determined by culturing serial dilutions.
Experimental manipulations.
For invasive procedures, rats
were anesthesized by intraperitoneal injection (0.18 ml/100 g of rat
weight) with a mixture of ketamine (35 mg/ml; Kombivet, Etten-Leur, The
Netherlands), xylazine (6 mg/ml; Bayer AG, Leverkusen, Germany), and
atropine (0.1 mg/ml; Eurovet, Bladel, The Netherlands). Halothane
anesthesia was used for short noninvasive procedures (e.g., intranasal
instillation and diagnostic procedures).
Histamine (dihydrochloride; Sigma Chemical Co., St. Louis, Mo.) was
administered through the tympanic membrane with a 30G1/2
needle under
otoscopic surveillance. In the standard challenge
procedure, 0.035 ml
of a 10
4 or 10
3 M solution was given on 2 subsequent days before inoculation
of bacteria. Suspensions of bacteria
were prepared in a concentration
of 0.7 × 10
9 to
3.8 × 10
9 CFU/ml, and 50 µl of such a suspension
was inoculated intranasally
with a Teflon infusion catheter. In pilot
experiments, Pn 14 produced
relatively high infection rates and was
therefore selected for
further optimization of the model. Standard
evaluation was on
postinfection day
6.
Protection assays.
Immunization was achieved in two ways. In
the first protocol, animals were primed with a standard intranasal
inoculation of homotypic bacteria 14 and 28 days before the standard
challenge procedure, but without histamine. In the second protocol,
animals were vaccinated subcutaneously with a vaccine preparation
containing 0.1 µg of PS14TT per 0.5 ml of 0.1% (wt/vol)
ALPO4 suspension (24). This vaccine was given 7 and 35 days before the standard challenge procedure.
Monitoring and evaluation.
Otomicroscopy and tympanometry
were performed before experimental manipulation and at regular
intervals (2 to 3 days) in the course of an experiment, to monitor the
condition of the middle ear. Tympanometry was done with a GSI38
tympanometer (Lucas Grason-Stadler Inc., Milford, N.H.).
Experiments were typically terminated 6 days after inoculation of
bacteria, by bleeding the animals under anesthesia. Tympanic
bullae
were rinsed, and bacterial culture of the lavages was performed
on
standard selective media for identification of intratympanic
flora.
These included sheep blood and chocolate agar, both also
combined with
clindamycin and Endo and negram agars. Suspected
pneumococci isolates
were serotyped, and only the confirmed presence
of pneumococci in the
middle ear was valued as final evidence
for Pn-induced otitis media.
Although otoscopy, tympanometry,
and histopathology are indicative of
otitis media, these parameters
do not discriminate for the causative
pathogenic agent. This is
of importance since a potential source of
bias is formed by symptoms
of otitis media caused by contaminating
flora. The infection rate
determining the outcome of an experiment is
defined as the percentage
of animals in an experimental unit with a
Pn-positive middle ear
lavage.
Tympanic bullae were collected and processed according to standard
histologic and electron microscopy (EM) procedures. Standard
histologic
procedures included 4% formalin fixation, decalcification
in 10% EDTA
solution, and paraffin embedding. Standard EM procedures
included
perfusion fixation with a phosphate-buffered fixative
of 2% (wt/vol)
paraformaldehyde and 2.5% (wt/vol) glutaraldehyde
and decalcification
in 5% EDTA in fixative and postfixation with
1% osmium tetroxide of
selected tissue blocks, followed by dehydration
and embedding in epoxy
resin glycidether 100 (Merck). Ultrathin
sections were prepared from
selected areas, contrasted with aqueous
uranyl acetate (2% [wt/vol])
and lead citrate (1% [wt/vol]), and
examined in a Philips EM 201 operating at 60
kV.
Detection of immunoglobulin A (IgA)-positive cells was done on paraffin
sections with an indirect immunoperoxidase procedure:
a mouse anti-rat
IgA monoclonal antibody (
26) was detected with
a
peroxidase-labeled rat anti-mouse IgG (Jackson Laboratory).
Peroxidase
activity was visualized with 3,3'-diaminobenzidine
as substrate. Cell
nuclei were counterstained with hematoxilin.
IgA-positive plasma cells
in the mucosa of the tympanic bulla
were counted with a light
microscope and expressed as the number
per area. Image processing and
analysis were performed with IBAS
(Kontron Elektronik, Munich,
Germany). From each specimen, four
serial sections (each 20 µm) were
examined.
Anti-Pn 14-specific polysaccharide (PS14) and anti-cell wall
polysaccharide (CPS) antibodies in serum were detected by enzyme-linked
immunosorbent assay as described previously (
24). Before
analysis
of anti-PS14 antibodies, sera were preincubated with 120 µg
of
CPSs (Statens Seruminstitut) per ml at 37°C for 30 min. All
samples
were serial diluted and tested in duplicate for
antigen-specific
IgG; the starting dilution was 1/10, and there were
seven subsequent
threefold serial dilutions. The spectrophotometry
range was 0.050
(background) to 2.100. From the measured serial
absorbances, a
linear curve fit and 50% of the maximum and minimum
optical density
absorbances were calculated (KinetiCalc; Bio-Tek).
These 50% absorbances
were log
10 converted to normalize
distributions. From these log
10 conversions, differences
between values before and after immunization
were
calculated.
The presented results are the averages of
n independent
observations in immunized and sham-immunized groups. The statistical
difference of ratios of otitis media between experimental groups
was
calculated with a Fisher's exact test. The statistical significance
for differences of IgG titers between experimental groups was
calculated with a one-tailed Student's
t test.
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RESULTS |
Morphologic effects of intratympanic histamine.
The effect of
histamine on the histology of the tympanic cavity is characterized by
hyperemia (vascular dilatation), intramucosal sanguineous exudation,
and submucosal hemorrhage. Histamine-facilitated middle ear challenge
with Pn 14 results in an inflammatory reaction, marked by an
infiltration of polymorphonuclear leukocytes in the bullar mucosa (Fig.
1).
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FIG. 1.
Representative illustrations of the mucosa of the
tympanic cavity. Left, normal mucosa; middle, sub- and intramucosal
sanguinous exudate (h) and dilated vessels (v) as a result of histamine
treatment (24 and 48 h before intranasal mock infection); right,
infiltrated mucosa, mainly with polymorphonucleated cells, as a result
of middle ear infection (histamine 24 and 48 h before intranasal
inoculation with Pn 14). Dilated vessels and hemorrhage are still
visible. Tympanic bullae were prepared on day 6 after infection.
Arrows, mucosa extension; b, bone of bulla wall. Bar = 0.2 mm.
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|
EM analysis of the epithelium of the eustachian tube shows that
histamine causes epithelial cells to lose their columnar appearance
(Fig.
2b), which is present in control
animals (Fig.
2a). Some
cells show loss of cilia, and in these cells,
cilia-associated
basal bodies lose their submembranous organization and
are found
scattered throughout the cytoplasm (Fig.
2c to d). There are
mucous
depositions on the apical membranes, even between cilia
remnants,
indicating loss of clearance function (Fig.
2b). These
effects
are more pronounced in the tympanic segment than in the
nasopharyngeal
segment of the eustachian tube.
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FIG. 2.
Transmission EM of ciliated epithelium of the eustachian
tube. (a) Sham-treated specimen. (b to d) Effects of histamine
treatment. Histamine was administered through the tympanic membrane 48 and 24 h before sacrifice. Histamine induces loss of cilia
(indicated by the asterisk in panel b; this area is magnified in panel
c). Mucous deposition is present after histamine treatment over and
between cilia (b) and microvilli (c); in panel b, this mucous
deposition results in clumping of cilia (arrow). These depositions are
not observed after sham treatment (arrow in panel a). Microvilli (open
arrows) are present with (c) and without (d) mucus on epithelial cells
which have lost their cilia. These cells show numerous basal bodies in
a disordered manner (arrowheads). Normal apical organization of
cilia-associated basal bodies is shown in a cell in panel d, which also
shows normal cilia (arrow); this apparent normal cell resides adjacent
to a pathologic epithelial cell. Below the apical plasma membrane of
pathologic cells there are vacuoles filled with a mucous substance
(indicated by the stars in panels c and d). Bars, 3 µm (a and b), 1 µm (c), and 0.5 µm (d).
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|
Parameters determining the infection rate.
In a single dose,
histamine is effective from 103 M (Table
1). Repeated dosage is effective from
104 M; lower concentrations were not tested in the
repeated dosage scheme. A repeated dosage of 104 or
103 M on 2 subsequent days before intranasal
administration of pneumococci proved to be adequate for a standard
challenge procedure.
With Pn 14, infection rates are not dependent on the concentration of
the intranasally instilled suspension in a range of
10
5 to
10
8 CFU (Table
1); a lower concentration is ineffective. In
the
standard procedure, presence of bacteria in the tympanic cavity
was
evaluated 6 days after challenge. Thereafter, the infection
rate
declines. This clearance of the infection is time dependent
(Table
1).
With the standard challenge procedure, middle ear infections are
obtained with all four tested Pn serotypes, Pn 6B, Pn 14,
Pn 19F, and
Pn 23F (Table
1), although there may be differences
in virulence (not
tested).
In the absence of a competitive quantity of virulent bacteria,
histamine treatment produces an opportunity to other (resident)
flora
to enter the tympanic cavity. With the standard challenge
procedure,
there is a high and significant inverse correlation
(
r2 = 0.63) between the infection rates for
Pn 14 and for other flora
(Table
2). This
inverse correlation is also obvious between infection
rates with other
Pn serotypes and opportunistic flora (data not
shown). Occasionally,
there were concomitant infections of Pn
and contaminating flora, but in
these cases, middle ear lavages
contained only low concentrations of
vital pneumococci, as indicated
by low-density colony growth in
culture.
Employing the otitis media model in protection assays.
The
value of this model as a read-out tool for protection after vaccination
was tested in two immunization protocols. In the first protocol, the
animals were intranasally primed with homotypic bacteria before the
standard challenge procedure with histamine and Pn 14. This priming
protocol resulted in a reproducible and significant decrease of
infection rate (Table 3). Sera of these animals, drawn at the time of challenge (day 28), showed a moderate but
significant increase in anti-CPS titers compared to those of
sham-primed animals (Table 3), which continued to increase until the
time of section (day 33). However, no anti-PS14 IgG responses
were found in sera of these rats (Table 3). Immunochemical analysis of bullae of three randomly selected animals from both groups
showed significantly higher numbers of IgA-positive cells in the mucosa
of primed animals than in that of sham-primed animals (Table 3).
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TABLE 3.
Effect of priming with homotypic pneumococci on standard
middle ear challenge with histamine and Pn 14 (infection rate and
immunologic parameters)
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|
The second protocol included subcutaneous vaccination with PS14TT
before standard challenge procedure with histamine and Pn
14. This
protocol also results in a significant reduction of the
middle ear
infection rate compared to that in sham-vaccinated
animals (Table
4), associated with a high and
significant increase
in anti-PS 14 specific IgG in the serum, measured
at the moment
of the booster vaccination (Table
4). Anti-CPS titers in
the
serum showed a small and not significant increase in the vaccinated
animals (Table
4).
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TABLE 4.
Effect of vaccination with PS14TT vaccine on standard
middle ear challenge with histamine and Pn 14 (infection rate and
Pn-specific serum IgG titers)
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| |
DISCUSSION |
Histamine pretreatment protocol as model for human disease.
Histamine is one of the many inflammatory mediators found in middle ear
effusions associated with otitis media (19). This histamine
may be generated by degranulation of mast cells in local mucoperiosteum
(1), e.g., as an inflammatory response triggered by viral
infections (9); in patients with allergic conditions, there
is an association between otitis media and histamine release from
peripheral blood basophils (9). In vitro, histamine can regulate ciliary activity of eustachian tube and middle ear mucosa (7). In guinea pigs, intratympanic injection of histamine, with a similar dose as used in this study, induces the accumulation of
middle ear effusion (7). Histamine also contributes to
dysfunction of the eustachian tube by affecting the microcirculation
and vascular permeability in its mucosa (5, 20). Dysfunction
of the eustachian tube after administration of histamine is indicated
by an increased mucociliary clearance time (7) and increased
perfusion pressure and opening pressure (19). These findings
are also relevant for our model, as indicated by the EM observation of
structural (Fig. 2b to d) and functional loss of epithelial cilia in
the eustachian tube and the light microscopical observation of mucosa pathology in the tympanic cavity (Fig. 1). Combined, these observations indicate that histamine can optimize conditions for bacterial passage
through the eustachian tube, and this factor may therefore be a major
mediator in the pathogenesis of bacterial otitis media. We used a
concentration of histamine that was only 10 to 100 times higher than
that detected in otitis media effusions (1), and therefore,
this protocol may well mimic a major step in the natural pathogenesis
of otitis media.
The histamine pretreatment protocol facilitates the ascending infection
of pneumococci from the nasopharyngeal cavity to the
middle ear, thus
offering an appropriate model for testing acquired
immunity of the
eustachian tube and middle ear mucosa to these
bacteria after
vaccination. For this reason, this model is preferred
to common
clearance models employing direct inoculation of bacteria
in the middle
ear cavity, thus circumventing mucosal immunity
in the eustachian tube
(
12,
14,
18,
25). Other models
employ obstruction of the
eustachian tube (
17) or coinfection
of bacteria with
influenza virus A or adenovirus, the latter in
chinchillas, to obtain
colonization of the tympanic cavity through
the eustachian tube
(
10,
21). Compared to most of these approaches,
histamine
pretreatment is a very controllable protocol, facilitating
Pn-induced
otitis media to a relative high
rate.
Employing the model in immunization assays.
We hypothesized
that a model employing entry of bacteria through the eustachian tube
would enable evaluation of immunity. Indeed, both after priming of the
intranasal mucosa with homotypic pneumococci and after subcutaneous
immunization with a PS14TT vaccine, there were significant reductions
in infection rates with pneumococci (Tables 3 and 4). These reductions
were associated with mucosal and systemic immunologic responses. The
low to moderate antibody responses after intranasal exposure to
pneumococci point to local mucosal immunity as the effector of the
reduced infection rate. This is supported by the increased number of
IgA-positive plasma cells in the mucosa of the tympanic cavity. On the
contrary, the high anti-PS14 titers obtained after subcutaneous
vaccination indicate systemic immunity as an effector in this case.
Histamine-facilitated induction of bacterial otitis media is thus
validated as a tool for the evaluation of protecting
vaccines.
| |
ACKNOWLEDGMENTS |
We thank P. W. Wester for his critical support, B. J. van Middelaar, D. J. Elberts, and C. Moolenbeek for their skilled
assistance in the animal facilities, L. A. Oomen for the
enzyme-linked immunosorbent assays, J. Venema, H. C. W. Thuis, and R. Boot for their contributions with the bacteriological
analysis, and G. Riool and S. de Waal for technical assistance with the EM.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Laboratory for
Pathology and Immunobiology, National Institute of Public Health and the Environment (RIVM-LPI), P.O. Box 1, NL-3720 BA Bilthoven, The
Netherlands. Phone: 31 30 2743485. Fax: 31 30 2744437. E-mail: L.van.der.ven{at}rivm.nl.
Editor:
E. I. Tuomanen
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Infection and Immunity, November 1999, p. 6098-6103, Vol. 67, No. 11
0019-9567/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
