CLINICAL MICROBIOLOGY REVIEWS, Oct. 2008, p. 666–685 Vol. 21, No.

4
0893-8512/08/$08.00⫹0 doi:10.1128/CMR.00012-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Animal Models of Streptococcus pneumoniae Disease

Damiana Chiavolini,§ Gianni Pozzi, and Susanna Ricci*
Laboratory of Molecular Biology and Biotechnology (LA.M.M.B.), Department of Molecular Biology, University of Siena, Siena, Italy

INTRODUCTION .......................................................................................................................................................666
ANIMAL MODELS OF PNEUMOCOCCAL DISEASE .......................................................................................667

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Considerations of Animal and Pneumococcal Strains ......................................................................................667
ANIMAL MODELS OF PNEUMONIA ...................................................................................................................668
Mouse Models .........................................................................................................................................................668
i.t. models.............................................................................................................................................................669
i.n. models............................................................................................................................................................669
Remarks on murine pneumococcal pneumonia..............................................................................................670
Rat and Rabbit Models..........................................................................................................................................670
ANIMAL MODELS OF SEPSIS ..............................................................................................................................671
Mouse Models .........................................................................................................................................................672
Rat and Rabbit Models..........................................................................................................................................673
ANIMAL MODELS OF MENINGITIS ...................................................................................................................673
Mouse Models .........................................................................................................................................................674
Direct induction of meningitis ..........................................................................................................................674
Hematogenous meningitis..................................................................................................................................675
Nonhematogenous meningitis ...........................................................................................................................675
Rat and Rabbit Models..........................................................................................................................................675
i.cist. models for rats..........................................................................................................................................675
Hematogenous and otologic models for rats...................................................................................................676
i.cist. infection in rabbits...................................................................................................................................677
ANIMAL MODELS OF OTITIS MEDIA................................................................................................................677
Chinchilla, Gerbil, and Guinea Pig Models........................................................................................................678
Rat and Mouse Models ..........................................................................................................................................678
CONCLUSIONS .........................................................................................................................................................679
ACKNOWLEDGMENTS ...........................................................................................................................................679
REFERENCES ............................................................................................................................................................680

INTRODUCTION age die of pneumonia and invasive diseases in developing

Streptococcus pneumoniae (the pneumococcus) is a major
human pathogen that colonizes the upper respiratory tract
and causes both life-threatening diseases such as pneumo-
nia, sepsis, and meningitis and milder but common diseases,
like sinusitis and otitis media (91). Colonization of the na-
sopharynx by S. pneumoniae begins early on in life and peaks
(up to 55%) at the age of 3 years (31). Colonization not only
provides the basis for pneumococcal horizontal spread but is
also necessary for invasive disease (31). The pneumococcus
produces a plethora of virulence factors that allow bacteria
to spread from the upper to the lower respiratory tract,
leading to pneumonia and invasive disease (126). The latest
report from the Centers for Disease Control and Prevention
in the United States estimated that in 2006 pneumococci
caused 41,400 invasive infections and 5,000 deaths, most of
which were due to bacteremic pneumonia (55). However,
each year at least 1 million children younger than 5 years of
* Corresponding author. Mailing address: LA.M.M.B., Università di
Siena, Policlinico Le Scotte, V lotto 1° piano, Viale Bracci, 53100
Siena, Italy. Phone: 39 0577 233100. Fax: 39 0577 233334. E-mail:
riccisus@unisi.it.
§ Present address: Evans Biomedical Research Center, 650 Albany
Street, Boston University School of Medicine, Boston, MA 02118.
countries (177). Mortality rates for pneumonia and menin-
gitis are especially high for young children, the elderly, and
immunocompromised individuals (55), including patients
with human immunodeficiency virus (135).
Pneumococcal diseases are treated with ␤-lactams (penicil-
lin G, amoxicillin, cephalosporins), respiratory fluoroquino-
lones, macrolides, and vancomycin (159). However, efforts to
treat pneumococcal diseases have been complicated by in-
creasing resistance to antibiotics. A recent work reported high
rates of resistance for some antimicrobials such as penicillin
(34.2%), trimethoprim-sulfamethoxazole (31.9%), and eryth-
romycin (29.5%) (74). For the prevention of pneumococcal
disease, there is a licensed 23-valent polysaccharide vaccine
which is effective against 23 out of 90 pneumococcal serotypes
but does not confer protection in children younger than 2 years
of age or in the elderly. A 7-valent conjugate vaccine has also
been available since 2000 and is effective in children, although
its cost is high and protection is induced only against serotypes
included in the vaccine formulation (32). Both 9- and 11-valent
conjugate vaccines will be licensed in the near future (16).
Protein-based or protein subunit vaccines common to different
capsular serotypes represent attractive alternatives to improve
protection and to address the current limitations of polysac-
charide vaccines (16).

666
VOL. 21, 2008
ANIMAL MODELS OF PNEUMOCOCCAL DISEASE
Animal experimentation is an essential tool for the study of
infectious diseases. Numerous animal models of diseases
caused by S. pneumoniae are currently available for clarifying
mechanisms of disease pathogenesis, testing novel drugs and
vaccine candidates, and characterizing the role of bacterial and
host factors. Recent review articles have specifically addressed
the value of animal models to test pneumococcal protein vac-
cines (225) and the utility of murine models of pneumonia to
assess the efficacy of antimicrobials (175). Some authors have
examined mouse genetic susceptibility to pneumococcal dis-
ease (121), while others have discussed the value of knockout
mice to investigate the pathophysiology of bacterial meningitis
(191). In 1999, Giebink effectively reviewed the utility of the
chinchilla model for the study of middle ear (ME) pneumo-
coccal infection (90), while Sabirov and Metzger have lately
evaluated the importance of the mouse to test mucosal vac-
cines against otitis media (211). A recent review on animal
models of invasive pneumococcal disease focused on the im-
portance of sepsis and pneumonia in experimental animal
models for studying vaccine efficacy and also provided very
useful considerations on technical aspects (39). Meningitis and
otitis media models were mentioned by the authors, although
to a lesser extent (39). The present report is a more compre-
hensive review of animal models that have significantly con-
tributed to our knowledge of all the clinically relevant pneu-
mococcal diseases, highlighting the importance of readouts,
defining the advantages and the drawbacks, and suggesting
potential applications of each one of these systems. Models of
colonization are briefly mentioned throughout the text but not
discussed in detail. The reader interested in animal models of
pneumococcal colonization should refer to a recently pub-
lished review (157). We fully appreciate the fundamental im-
portance of nasopharyngeal carriage as a crucial step for inva-
sive disease, but we have chosen to focus on animal models of
acute disease, such as pneumonia, sepsis, meningitis, and otitis
media. For easier reading, major experimental animal models
are listed in tables according to the pneumococcal disease and
the animal species. Representative models were chosen based
on historical importance, innovative improvement of preexist-
ing models, widespread use within the pneumococcus research
community, and usefulness for future studies.
Considerations of Animal and Pneumococcal Strains
The choice of both animal and bacterial strains should be
carefully considered before approaching the study of pneumo-
coccal disease in vivo. It is beyond the scope of this review to
include a detailed discussion on animal or S. pneumoniae
strains to be used in experimental models of disease; however,
we provide general considerations on the subject.
Mouse and rat inbred strains afford more-uniform responses
to experimental treatments due to their tightly controlled im-
mune system. For this reason, they have extensively been em-
ployed as models for infection, vaccination, and drug efficacy
studies. Outbred mouse strains are instead generated to main-
tain maximum heterozygosity. Their phenotypic diversity, de-
spite being lower than that of humans, may be important to
mimic the natural variation in response to infection. In addi-
MODELS OF PNEUMOCOCCAL DISEASE 667
tion, their lower cost makes them attractive alternatives to
inbred strains.
Experimental pneumococcal disease has been investigated
by use of different rodents and the rabbit, but undoubtedly the
mouse represents the most commonly used animal model. In-
bred mouse strains, including BALB/c, C57BL/6, DBA, and
CBA mice, have largely been exploited for the analysis of
pneumococcal pneumonia (12, 113, 131, 176, 186, 201, 234),
sepsis (15, 42, 43, 163, 186), meningitis (86, 95, 136, 173, 230),
and otitis media (162, 164). Experimental sepsis has also been
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induced in immunodeficient mice, such as the CBA/N strain,
which carries the Xid-linked immune deficiency (38, 43).
CBA/N mice are unable to produce antibodies against pneu-
mococcal polysaccharides and phosphocholine and are thereby
highly susceptible to infection by S. pneumoniae (41). How-
ever, outbred mice, such as MF1, CD-1 (Swiss), Swiss-Webster,
and NMRI, have increasingly become more popular. They
have widely been used for analyzing pathogenicity mechanisms
of pneumonia (50, 58, 114, 120, 125, 131), sepsis (109, 131,
251), and meningitis (59, 95, 160, 268). In addition, outbred
mice have also been employed to evaluate active and passive
protection against pneumonia and sepsis (4, 115, 163) and the
efficacy of antibiotic therapy against invasive pneumococcal
disease (13, 141, 169, 220, 223). The host genetic factors con-
trolling the response to infection by S. pneumoniae are still
unknown. Mouse susceptibility and resistance to pneumococ-
cal disease have been investigated in more detail in several
studies (72, 92, 130, 215) and discussed in a few reviews (41,
121). The rat has also been exploited for studying invasive
pneumococcal disease and otitis media. Outbred strains, in-
cluding Wistar, Sprague-Dawley, and CD (derived from Spra-
gue-Dawley) rats, have been extensively utilized as experimen-
tal models (2, 49, 71, 79, 101, 103, 145, 147, 166, 193, 214, 221,
242, 253). The New Zealand White rabbit is the most com-
monly used strain to induce pneumococcal pneumonia, sepsis,
and meningitis (26, 57, 69, 98, 106, 119, 196, 246). Finally, the
chinchilla (only outbred strains are available), gerbil, and
guinea pig are commonly used animal models of experimental
otitis media (81, 89, 90, 260).
There is large variation in the outcomes of infection by S.
pneumoniae in experimental animal models, depending on the
pneumococcal strain used. It is well accepted that the virulence
of S. pneumoniae strains is strongly influenced by the capsular
serotype in both humans and mice (38, 129, 215); however, the
genetic background of the strain also seems to be relevant for
disease development (29, 129, 215). Certain human isolates are
poorly virulent in vivo, whereas other pneumococcal strains
that are highly virulent in mice have less relevance for human
disease (38, 39). Typically, strains of capsular serotypes 2, 3, 4,
5, and 6 are virulent in mice, whereas strains of types 14, 19,
and 23 are relatively avirulent (21, 39). The low virulence of
many pneumococcal strains in rodents can be balanced by
either using larger challenge doses (40) or neutropenic hosts
(19, 49, 169, 221, 223).
Experimental pneumococcal disease has been induced with
both historical laboratory strains and clinical isolates. The use
of classic strains, able to generate consistent features of dis-
ease, allows comparison of results among research groups. Of
particular relevance is the type 2 D39 reference strain (142),
which several research groups have employed to study the
668 CHIAVOLINI ET AL. CLIN. MICROBIOL. REV.

TABLE 1. Mouse models of pneumonia

Route of Pneumococcal
Mouse strain(s) Aim(s) of study Readouts Utility Reference(s)
infection serotype(s)

i.t. C57BL/6 3 Antibiotic efficacy, CFU, histology, survival, Drug efficacy and 12
model description pharmacokinetics pharmacokinetics
i.t. ddY NSa Gastric juice in Survival, CFU, PMNb, Pathogenesis of 114
pneumonia lung histology pneumonia in the
elderly
i.n. MF1 2 Role of pneumolysin CFU, lung histology Host-parasite interaction, 50
and autolysin, model disease pathogenesis,
description vaccine and drug

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studies
i.n. Neonatal and 1 Immunization in early Antibody titers, CFU Neonatal and early-life 115
infant NMRI life vaccinology
i.n. BALB/c and OF1 14 (for BALB/c) Therapy in neutropenic CFU, survival, Antibiotic efficacy, 201, 223
and 3, 9 (for mice pharmacokinetics immunotherapy studies
OF1)
i.n. CBA/J 3, 11, 19 Model of penicillin- Survival, CFU, Antibiotic efficacy in 234
resistant pneumonia histology, immunocompetent
pharmacokinetics mice
Aerosol nebulizer CBA/J 28 Model of chronic CFU, serum Disease pathogenesis 113
obstructive immunoglobulin G
pneumonia
Aerosol nebulizer CBA/J 3 Low-dose model, drug Survival, CFU, histology Efficacy of tissue-directed 176
efficacy antimicrobials
a
NS, not specified.
b
PMN, polymorphonuclear cells.

pathogenesis of pneumococcal disease and test vaccine anti-
gens over the years (25, 40, 50, 92, 103, 104, 109, 123, 124, 131,
180–182, 186). Serotype 3 strains, in particular A66 (9) and
WU2 (43), are also widely used for inducing sepsis in the
mouse and the rat (2, 40, 43, 109, 129, 163) and to study
invasive pneumococcal disease in the rabbit (246, 247). More
recently, laboratory strains with sequenced genomes, such as
the type 4 TIGR4 strain (238) and the type 19F G54 strain
(75), have also been employed by different research groups (58,
59, 80, 131, 187, 197, 215). The availability of clinical isolates is
also important in attempting the reproduction of human dis-
ease in laboratory animals. Several groups have preferred se-
rotype 3 and 6 strains isolated from patients with pneumococ-
cal meningitis and have successfully induced this disease in the
mouse, the rat, and the rabbit (26, 35, 69, 76, 85, 95, 145, 192,
268, 269). Clinical isolates of serotypes 3, 6, 9, and 19 have also
been employed in laboratory animal models to elucidate fea-
tures of pneumococcal pneumonia and sepsis (12, 14, 40, 49,
196, 223, 234, 251). For experimental otitis media, strains of
serotypes 3, 4, 6, 7, 9, 11, 14, 19, and 23, which are the ones
most commonly isolated from children with acute otitis, have
been used (18, 80, 81, 89, 101, 154).
ANIMAL MODELS OF PNEUMONIA
S. pneumoniae is the leading causative agent of community-
acquired pneumonia (159). In 2006, bacteremic pneumonia
constituted more than 66% of invasive pneumococcal disease
in the United States (55). Typically, pneumococcal pneumonia
is lobar. However, other radiographic patterns are also com-
monly found, including lobular bronchopneumonia and occa-
sionally interstitial or mixed types of pneumonia (127, 188).
The proportion of pneumococcal pneumonia associated with
bacteremia is estimated to be approximately 20 to 25% (7).
The pathogenesis of pneumococcal pneumonia is a complex
interplay between pneumococcal virulence and the host im-
mune response (91). Since the late 1970s, when an important
model of pneumococcal pneumonia was set up for the rat (14),
many different animal models of pneumonia have been devel-
oped. Pneumonia due to S. pneumoniae has been studied
largely by use of the mouse, probably the most commonly
employed system today for the characterization of this disease,
due to the ease of manipulation and data reproducibility in
large numbers of animals.
Mouse Models
Different research groups have reproduced the features of
pneumococcal pneumonia in the mouse (Table 1). Mouse
models of pneumonia allow the analysis of different parame-
ters, including animal survival after infection, the presence of
bacteria in lungs and blood, levels of inflammation, and histol-
ogy of lung tissue. Additionally, quantification of antibody ti-
ters and antimicrobials performed in vaccine and drug phar-
macokinetic studies, respectively, are also feasible. Two main
routes of infection are currently used to induce pneumonia: the
intratracheal (i.t.) and the intranasal (i.n.). The i.t. model re-
quires a complex and invasive technique for disease induction
but offers the advantage of allowing 99% delivery of the bac-
terial inoculum to the lungs (208). The i.n. route includes both
the standard aspiration method (50) and the aerosol nebulizer
system (176). The model of infection through i.n. aspiration is
the most commonly used, as it is fast and easy to perform
without invasive surgical procedures and also because it mim-
ics the natural route of infection in humans. The i.n. aerosol
model instead requires an exposure chamber with a nebulizer,
but it is suitable for the simultaneous infection of many mice.
A key aspect of i.n. aspiration models is the need for anesthesia
VOL. 21, 2008
to allow infection of the lower respiratory tract (58), in com-
parison to absence of anesthesia in both colonization (157,
258) and i.n. aerosol (113, 176) pneumonia models. When
animal sedation is required, the choice of anesthetic should
also be evaluated for experimental design and result interpre-
tation. It has been reported that pentobarbital and halothane
have distinct effects on the pathogenesis of pneumococcal
pneumonia in mice (207).
i.t. models. The i.t. pneumonia model was first described by
Azoulay-Dupuis et al. to test the efficacies of different antibi-
otics (12). It is based on an inoculation technique previously
described for the study of pneumonia caused by Staphylococcus
aureus and Klebsiella pneumoniae (78). Briefly, mice were sus-
pended vertically on a board by supporting the lower incisors
on a wire loop and retaining the lower incisors with a rubber
band. The oropharynges were transilluminated, and the tra-
cheas were cannulated with needles attached to microliter sy-
ringes containing the pneumococcal suspension (12). This
model is still used in several studies on drug efficacy, host
response to infection, and the role of pneumococcal virulence
factors in the disease (1, 11, 19, 168, 169, 208, 237). Another i.t.
mouse model, based on pneumococcal inoculation via the oro-
pharynx, was developed by Iwasaki et al. to investigate the
combined effects of bacteria and gastric juice commonly ob-
served for elderly patients with aspiration pneumonia (114).
Finally, studies on neutrophil migration after induction of
pneumonia were carried out by injection of bacteria directly
into the exposed trachea (167, 232).
All i.t. infection models have the advantage of allowing the
delivery of the entire bacterial inoculum into the lower respi-
ratory tract and directly causing pneumonia without interme-
diate pathogenic steps. By using different mouse strains, it is
possible to induce either acute or subacute forms of disease
(13). Swiss mice develop acute pneumonia and die rapidly (2 to
4 days), while C57BL/6 animals develop a subacute progressive
disease leading to death in 8 to 10 days (13).
i.n. models. In the late 1980s, two studies demonstrated the
importance of pneumolysin (25) and autolysin (24) in pneu-
mococcal virulence by using in vivo models, including an i.n.
pneumonia model based on bacterial instillation into the
mouse nostrils. However, the first detailed i.n. aspiration
model was described by Canvin et al. in 1995, who introduced
the practice of infecting animals with mouse-passaged pneu-
mococci (Table 1) (50). Pneumococci recovered from the
blood of intraperitoneally (i.p.) infected animals were inocu-
lated into the nostrils of anesthetized mice, which rapidly de-
veloped bronchopneumonia with bacteremia (50). This model
has been employed largely in studies on host immune response
to pneumococcal lung infection (23, 70, 122, 123, 130), mouse
susceptibility and resistance to the disease (92), invasion by S.
pneumoniae of bronchoepithelial cells in vivo (124), the role of
virulence determinants in pneumonia (30, 58, 120, 125, 158),
and the efficacy of antibiotics and anti-inflammatory drugs
(250, 252). Furthermore, many investigations on protection
from i.n. challenge after immunization with vaccine candidates
have been performed. Pneumolysin (4), PspA (40, 257), PspC
(15), and phosphorylcholine (241) have successfully been em-
ployed in vaccine studies followed by i.n. challenge. Whereas
PsaA has been able to confer protection against colonization
(37, 183), it did not elicit protection against focal pneumonia
MODELS OF PNEUMOCOCCAL DISEASE 669
(40). PsaA has also been reported to be unable to provide
significant protection against i.p. challenge (180). In passive-
immunization studies, capsular polysaccharide antibodies ob-
tained from vaccinated infants were also able to protect mice
against lung infection (213). The protective efficacies of novel
protein targets and live attenuated pneumococci (with dele-
tions in genes encoding the capsule, pneumolysin, and PspA)
have also been tested as vaccine candidates in this type of
pneumonia challenge model (100, 205). Of importance for
vaccine studies was also an early-life infection model (115). As
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children younger than 2 years of age are major targets of
pneumococcal vaccination, the mouse model of i.n. infection
with aspiration set up for adult animals (50) was adjusted to
neonatal (1-week-old) and infant (3-week-old) mice, which
mimic the immune systems of human neonates and infants,
respectively. By using this approach, several aspects of vacci-
nology in early life were investigated, including the efficacies of
polysaccharide conjugate vaccines delivered by the mucosal
route (115), the role of T-cell responses to pneumococcal con-
jugates (116), and the importance of maternal antibodies for
protecting offspring against S. pneumoniae (204).
Other i.n. aspiration models of pneumonia have been devel-
oped for mice rendered neutropenic after treatment with cy-
clophosphamide: Ramisse et al. described the role of human
immunoglobulins in passive and active immunotherapy against
pneumonia (201), while Soriano et al. focused on the pharma-
cokinetics of penicillins and cefotaxime in the disease (223).
While both systems described above employ immunosup-
pressed animals, Tateda et al. established an i.n. model of
penicillin-resistant pneumococcal pneumonia in immunocom-
petent CBA/J mice (234). This experimental system represents
the first example of penicillin-resistant pneumonia in uncom-
promised mice, which are generally quite insensitive to infec-
tion by penicillin-resistant strains (169). The last model was
employed both for therapeutic studies (229, 233) and for an-
alyzing the host inflammatory response during pneumonia
(228).
Finally, experimental pneumococcal pneumonia in mice can
also be induced by the i.n. aerosol route using an exposure
chamber and a nebulizer. The technique less closely resembles
the development of pneumococcal pneumonia in humans,
where the disease generally follows aspiration of bacteria from
the upper respiratory tract. The first example of aerosol-in-
duced pneumococcal pneumonia originates from an early work
by Coil et al., who described that splenectomized mice were
more susceptible to type 3 pneumococci delivered by means of
an aerosolized suspension (61). Another attempt at an i.n.
aerosol pneumonia model was made to analyze the develop-
ment of pneumonia after airway obstruction in mice persis-
tently colonized by S. pneumoniae (113). This model intends to
mimic the occurrence of pneumonia in humans with altered
mucociliary clearance mechanisms, chronic obstructive pulmo-
nary disease, or concomitant viral infections (113). However, a
more exhaustive model was established by Nuermberger et al.
(176). They used a low bacterial inoculum to generate a murine
disease with a resemblance to human pneumonia that was
closer than those seen for traditional mouse i.n. and i.t. mod-
els, which rely on larger doses to produce fulminant infections
due to concomitant sepsis (12, 50). This model is especially
suitable for evaluating the efficacy of antimicrobials at the site
670 CHIAVOLINI ET AL. CLIN. MICROBIOL. REV.

TABLE 2. Rat models of pneumonia

Route of Pneumococcal
Rat strain Aim(s) of study Readouts Utility Reference(s)
infection serotype(s)

i.t. R strain albino 3 Antimicrobial therapy, CFU, histology Pathogenesis of lobar 14

model description pneumonia, drug
efficacy studies
i.t. Sprague-Dawley 3 Model in cirrhotic and Survival, CFU, Pneumonia in 71, 166
chronically hematology and alcoholic and
intoxicated rats chemistry tests cirrhotic patients
Intrabronchial Wistar 3 Effect of pneumolysin Lung histology Pathogenesis of lobar 79

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on lungs pneumonia,
detailed surgery
information
Intrabronchial Infant CD 6 Model of penicillin- Gross pathology and Drug efficacy studies 221
resistant pneumonia CFU
in neutropenic rats
Intrapulmonary CD 2, 6B Model of penicillin- CFU, lung histology, Antibiotic efficacy 49
resistant pneumonia pharmacokinetics studies
in neutropenic rats
Intrapulmonary Infant Sprague- 1, 3, 4, 5, 6B, 7F, Low-dose pneumonia Survival, CFU, lung Pathogenesis of 214
Dawley 9V, 14, 19F, model histology pneumonia in
23F children, vaccine
studies

of infection rather than in the bloodstream, as the experimen-
tal approach allows the development of an indolent subacute
pneumonia (175). The main disadvantage of this model is the
use of neutropenic mice, which might not optimally represent
the human host (175, 176).
Remarks on murine pneumococcal pneumonia. i.t. models
generally cause lobar pneumonia (12), while systems based on
i.n. aspiration of bacteria induce bronchopneumonia (50). In
both cases, there are concomitant pneumonia and sepsis: in-
fection readily leads to death of animals, which most likely
succumb to septic shock rather than to pulmonary disease.
Clearly, models of focal pneumonia in the absence of gener-
alized sepsis are more helpful to study the pathogenesis of
pulmonary disease, vaccine protection, and antibiotic efficacy
exclusively in the lungs. In addition, they better reproduce the
clinical manifestations in humans, where pneumonia is usually
not associated with bacteremia (7). The use of pneumococcal
strains of serotypes 14, 19, and 23, which are poorly virulent in
mice, is a necessary requisite for causing this type of disease. So
far, type 19 strains have been employed to induce progressive
focal pneumonia without sepsis (40, 234). Alternatively, neu-
tropenic mice infected with low doses of virulent pneumococ-
cal strains (e.g., type 3) also develop primary pneumonia with
delayed dissemination (approximately 4 days after infection)
(176).
Rat and Rabbit Models
Rat and rabbit models are uncommonly employed compared
to the mouse. Their larger size allows the collection of more-
substantial samples, but one drawback is that experimental
groups are normally smaller, thus potentially compromising
statistical significance. Sophisticated surgical procedures are
required to induce pneumonia via i.t., intrabronchial, or in-
trapulmonary routes of infection, and to our knowledge, no i.n.
models of pneumococcal pneumonia have been set up with
these animals.
Table 2 summarizes the principal characteristics of rat mod-
els of pneumococcal pneumonia. Main readouts include ani-
mal survival, observation of gross pathology, lung histology,
and bacterial counts in lungs and blood. The rat has been
employed mainly for clarifying features of pneumonia occur-
ring in asplenic, neutropenic, cirrhotic, or alcoholic subjects,
thus representing a valuable experimental system for studying
pneumonia in the immunocompromised host. Alcoholism is a
predisposing factor for the development of pneumococcal
pneumonia (140), and mortality rates from bacteremic pneu-
monia double (up to 40%) in alcoholic patients with hepatic
insufficiency (8). The models in ethanol-intoxicated (71) and
cirrhotic (166) rats were developed over 15 years ago by using
the i.t. route of infection to deliver pneumococci into the left
lobe of the lung following intubation of the main stem bron-
chus (14). Infection caused lobar pneumonia and sepsis (14).
The model in ethanol-intoxicated animals was based on con-
tinuous ethanol feeding leading to chronic intoxication (71),
while experimental cirrhosis was induced in rats by adminis-
tration of a hepatotoxin that caused cirrhosis and ascites (166).
In both cases, rats showed increased susceptibility to pneumo-
coccal pneumonia. These models have been used by the same
research group in studies on phagocytosis and antibiotic effi-
cacy (84, 184, 199). A sophisticated surgical method based on
the delivery of bacteria into the apical lobe bronchi of adult
rats was described in the early 1990s (79). Briefly, rats were
anesthetized and intubated before the thoracic cage was
opened to reveal the apical lobe of the lung. The apical lobe
bronchus was exposed and partially sutured to impair muco-
ciliary clearance, and the inoculum was injected through the
bronchial wall into the lumen toward the lung periphery (79).
Histological examination of rat lungs showed clear consolida-
tion of the apical lobes (lobar pneumonia) (79). Two other
VOL. 21, 2008
TABLE 3. Rabbit models of pneumonia
Route of Pneumococcal
Rabbit strain Aim of study
infection serotype(s)
i.t. New Zealand 2, 3 Inflammation induced by
White cell wall
Intrabronchial New Zealand 9, 19 Model of penicillin-
White resistant pneumonia
Intrabronchial New Zealand NSa Ventilator-associated
White pneumonia
Intrapulmonary New Zealand NS Neutrophil activity in
White lungs
a
NS, not specified.
b
CT scan, computerized tomography scan.
c
ICU, intensive care units.
d
FDG, 18F-labeled fluoro-2-deoxy-D-glucose.
models of lobar pneumonia induced either intrabronchially in
infant rats (221) or intrapulmonarily in adult rats (49) ren-
dered neutropenic were employed to study the efficacy of an-
tibiotics against penicillin-resistant lobar pneumonia (49, 221,
222). As already described for the mouse, the use of immuno-
compromised animals is necessary to produce pneumonia by
penicillin-resistant strains of S. pneumoniae in rats. The above-
described infant rat model is based on a nonsurgical intrabron-
chial instillation of bacteria resuspended in cooled melted agar
(as an adjuvant and growth medium) (221). Another group
developed a refined intrapulmonary pneumonia model for in-
fant rats based on the nonsurgical inoculation of a low bacte-
rial inoculum (1 to 400 CFU per rat) (214). The right chests of
pups were prepared with ethanol, and rat-passaged bacteria
were delivered transthoracically into the mid-right lungs. Prior
to inoculation, pneumococci were entrapped in cooled agar
particles to provide both protection from clearance and adju-
vant activity. The virulences of 10 different serotypes prevalent
in children were compared (214). Despite being a model of
focal pneumonia, it differs from the one described for the
mouse by Briles et al. (40) in that experimental pneumonia
may be accompanied by bacteremia and occasionally by men-
ingitis, mimicking the disease seen for children of developing
countries. The model was utilized in both neonatal (4-day-old)
and infant (3-week-old) rats to assess passive protection
against intrapulmonary challenge with S. pneumoniae (155,
156, 224).
The rabbit has also been employed to study pneumococcal
pneumonia, although fewer models are available. Readout pa-
rameters include animal survival, determination of leukocyte
numbers in lungs and blood, histology of the lungs, and assess-
ment of drug concentration in serum (Table 3). All models
described below are based on i.t., intrabronchial, or intrapul-
monary routes and are useful systems to study disease patho-
genesis and perform drug efficacy studies. However, all of them
necessitate surgical expertise for disease induction. An early i.t.
model was established in 1987 and it is based on the instillation
of pneumococci in rabbits by means of a catheter inserted into
the animal trachea (246), as previously described for experi-
mental pneumonia caused by S. aureus (185). By using this
MODELS OF PNEUMOCOCCAL DISEASE 671
Readouts Utility Reference
CFU, protein content, Disease pathogenesis, efficacy 246
leukocytes in lungs of antimicrobial and
adjunctive therapy
Survival, CT scanb, lung Efficacy and 196
histology, pharmacokinetics of drugs
pharmacokinetics (model of human therapy)
CFU, lung histology Pneumonia in ICUc 57
Downloaded from http://cmr.asm.org/ on October 22, 2020 at NUS CENTRAL LIB
FDGd uptake in lungs Acute and chronic lung 119
inflammation in
pneumonia
approach, analyses of the roles of pneumococcal cell surface
components in the induction of pulmonary inflammation
(246), of host oligosaccharides in preventing pneumococcal
colonization of the nasopharynx and subsequent lung infection
(110), and of the platelet-activating factor in the pathogenesis
of pneumonia (47) were performed. Two further models were
developed for immunocompetent rabbits based on intrabron-
chial instillation of bacteria (57, 196). The former reproduces
ventilator-associated pneumonia (57), while the latter mimics
penicillin-resistant pneumonia (196). In the model of antibiot-
ic-resistant pneumonia, pathology—if untreated—was charac-
terized by initial unilobar lesions and subsequent consolidation
of other lobes and confluent bronchopneumonia with bactere-
mia (196). The system mimics lethal pneumococcal pneumonia
in patients and allows simulation in the rabbit of human anti-
biotic pharmacokinetics. As this model simulates “human-like
treated pneumonia,” it constitutes an extremely valuable tool
for assessing drug efficacy and pharmacokinetics in humans
(67, 195, 196), and it may also be useful for evaluating the
selection of resistant pneumococcal strains in vivo (68). As
aforementioned, the same research group also developed a
novel infection model based on the induction of ventilator-
associated pneumococcal pneumonia in adult rabbits (57). This
approach proved to be instrumental to assess antibiotic efficacy
against pneumonia associated with mechanical ventilation,
which is a serious health concern in intensive care units (56).
Finally, positron emission tomography was also exploited to
study intrapulmonary experimental pneumonia localized in the
upper lung lobes of rabbits (119).
ANIMAL MODELS OF SEPSIS
The definition of sepsis and related terms (e.g., bacteremia,
septicemia, severe sepsis, septic shock) has been controversial.
In 1989, sepsis was defined as the host systemic response to
infection according to specific measurements of clinical param-
eters (e.g., blood leukocyte counts, body temperature, and
heart and respiratory rates), in contrast to bacteremia, which is
defined only according to the presence of bacteria in the blood
(33). S. pneumoniae is an important cause of sepsis in humans,
672 CHIAVOLINI ET AL.
TABLE 4. Mouse models of sepsis
Route of Pneumococcal
Mouse strain(s) Aim(s) of study
infection serotype
i.v. CBA/N, DBA/2, 3 Protection by anti-PCa
BALB/c antibodies, model
description
i.v. CD-1 3 Host response to sepsis
i.p. CBA/N, BALB/c 3 Protection by anti-PC
antibodies, model
description
a
Anti-PC, anti-phosphocholine.
generally occurring as a form of an advanced stage of pneu-
monia (60). Predisposing conditions are splenectomy (219),
hepatic cirrhosis, respiratory insufficiency, and cardiovascular
disease (140). In children of less than 3 years of age, a common
clinical manifestation is occult bacteremia (without a focus)
(153). In discussions of experimental animal models developed
to study pneumococcal disease, the terms sepsis and bactere-
mia are commonly found and used interchangeably. Experi-
mental sepsis by S. pneumoniae can be generated either by
inoculating bacteria directly into the bloodstream or by injec-
tion into the peritoneal cavity. While sepsis induced by the i.p.
route is secondary to peritoneal infection, with the concomi-
tant strong peritoneal inflammation, intravenous (i.v.) inocu-
lation produces a “cleaner” model, where the clearance of
bacteria by the reticuloendothelial system can be easily stud-
ied. In addition, i.p. infection represents neither an important
clinical manifestation of invasive pneumococcal disease nor
the natural route of infection by S. pneumoniae in humans.
Mouse Models
Besides a few early studies carried out in guinea pigs to
analyze the role of spleen and complement in experimental
pneumococcal sepsis (44, 45), the mouse is the most exten-
sively used animal model for inducing sepsis by S. pneumoniae.
Most research groups have analyzed the occurrence of murine
sepsis principally by determining the presence of bacteria in
the blood and by observing animal survival after infection
(Table 4). Sepsis in the mouse can be induced by either the i.p.
or the i.v. route of infection. Sepsis can also develop secondary
to pneumonia induced by i.n./i.t. routes or to meningitis fol-
lowing intracranial injection, but the kinetics of bacterial tran-
sit from lungs or brain into the bloodstream cannot be con-
trolled experimentally. Consequently, performing viable
counts in the blood after i.n./i.t. or intracranial inoculations
does not constitute a direct measure of bacterial clearance
operating in those districts and it is not the finest approach to
evaluate immune mechanisms and efficacy of drugs or vaccines
in experimental pneumonia or meningitis. As above men-
tioned, the i.v. model is a direct and valuable system for in-
ducing infection in the bloodstream and thus for studying the
mechanisms of bacterial clearance, although the technique can
be time-consuming and difficult to perform compared to i.p.
infection. To facilitate i.v. injection of bacteria into the mouse
CLIN. MICROBIOL. REV.
Readouts Utility Reference
Survival, CFU Bacterial clearance 43
mechanisms, passive and
active protection
CFU, hematological Pathogenesis of sepsis 251
parameters,
cytokines,
histology
Downloaded from http://cmr.asm.org/ on October 22, 2020 at NUS CENTRAL LIB
Survival, CFU Passive and active protection, 43
drug efficacy and virulence
studies
tail vein, researchers have used an infrared lamp to allow
vasodilatation (109). i.p. inoculation is technically easier but
presents disadvantages including the risk of causing tissue
damage or pain to the animal and may not allow the study of
bacterial clearance by the reticuloendothelial system (38).
Briles et al. observed that certain pneumococcal strains that
were lethal following injection of mice by the i.p. route were
avirulent by the i.v. route, indicating that i.p.-induced sepsis is
much more severe (38, 43). For this reason, the i.p. route is
widely used to mouse passage pneumococci in order to render
them more virulent (50).
Studies performed by the group of Briles et al. in the early
1980s employed the i.v. mouse sepsis model mainly for vaccine
studies. These works largely contributed to demonstrate S.
pneumoniae clearance from the bloodstream promoted by nat-
urally occurring anti-phosphocholine antibodies (43, 163, 261).
The model was employed and is still used by this group and
others, resulting in several investigations on the role of pneu-
mococcal virulence factors during sepsis and in studies aimed
at analyzing the efficacy of vaccines (15, 20, 30, 38, 42, 109, 131,
186, 200, 202, 227, 245). For instance, thanks to the use of the
i.v. infection route, it was possible to unambiguously establish
the role of PspC as a key virulence factor in pneumococcal
sepsis (109). In a work by Loeffler et al., the therapeutic value
of a novel antimicrobial, the Cpl-1 phage enzyme, was proven
by using an i.v. mouse sepsis model (149). One of the most
detailed studies of the pathogenesis of sepsis in the mouse was
made by Wang et al., who investigated the systemic host re-
sponses to sepsis in normal and immunosuppressed mice in-
fected by the i.v. route (251). A complete analysis of different
parameters, such as bacterial loads in blood and lungs, lung
injury, biochemical markers, and hematological and inflamma-
tory mediators, was carried out (251).
The i.p. route of infection is one of the earliest techniques
employed to induce experimental sepsis. This method was used
by Briles et al. in early studies of blood clearance mechanisms
(43). i.p. challenge models are still widely employed in studies
on the efficacies of vaccine candidates, including the surface
proteins PspA, PspC, and PsaA; a toxoid derivative of pneu-
molysin (PdB); the caseinolytic protease (ClpP); the compo-
nents of two iron uptake transporters, PiaA and PiuA; and the
more recently discovered pilus protein subunits (46, 51, 87,
118, 180, 181). The i.p. model also proved to be useful to clarify
VOL. 21, 2008
TABLE 5. Rat and rabbit models of sepsis
Route of Pneumococcal
Animals Aim(s) of study
infection serotype
i.v. Sprague-Dawley 25 Model description for
rats splenectomized rats
i.v. Sprague-Dawley 3 Role of complement in Survival, CFU
rats cirrhotic rats
i.p. New Zealand White 1 Role of lysosomes in
rabbits sepsis
Intracardiac New Zealand White 3 Role of spleen in
rabbits clearance, model
description
i.v. Chinchilla rabbits NSa Pharmacokinetics of
cephalosporins
a
NS, not specified.
the role of pneumococcal virulence factors in systemic infec-
tions (182, 262) and to evaluate efficacy of antibiotics (53, 141).
The i.p. sepsis model was also employed to assess passive
protection conferred by a mouse hyperimmune serum (263)
and by human anticapsular (117) and anti-pneumolysin anti-
bodies (171). Passive immunization with anticapsular antibod-
ies in infant (13- to 15-day-old) mice demonstrated that anti-
capsular opsonophagocytic titers are better predictors of
protection than are enzyme-linked immunosorbent assay im-
munoglobulin G titers (117). Very few studies focused on the
host response to pneumococcal sepsis by describing the func-
tion of tumor necrosis factor alpha in controlling systemic
infection (22, 178) and increasing the permeability of the
blood-brain barrier (BBB) (244).
Rat and Rabbit Models
Experimental models of sepsis in the rat and the rabbit have
been used mainly from the beginning of the 1970s throughout
the 1980s, but presently they have mostly been replaced by the
mouse due to the latter’s greater ease in manipulation and
smaller size. Similarly to that induced in the mouse model,
sepsis induced in the rat and rabbit models is studied by ob-
serving animal survival over time and by determining the pres-
ence and number of bacteria in the blood (Table 5). As in the
case of rat models of pneumococcal pneumonia (Table 2), rat
models of sepsis are particularly valuable for studying the dis-
ease in immunocompromised animals. The first model of sep-
sis in the rat was set up in 1972 by Leung et al., who infected
splenectomized rats by injecting pneumococci into the tail
veins (147). Despite being an early work, the authors made
significant observations, including the importance of (i) the use
of i.v. rather than i.p. challenge to show a difference between
normal and immunocompromised animals in susceptibility to
infection and (ii) vaccination to protect splenectomized rats
from fatal sepsis (147). The model was also employed to eval-
uate the efficacies of penicillin and steroid drugs (99), of hu-
man gamma globulins (179), and of a type 6 capsular polysac-
charide vaccine (111) to treat and/or prevent pneumococcal
postsplenectomy sepsis. Starting from a previously described
model of pneumococcal pneumonia in cirrhotic rats (166), a
MODELS OF PNEUMOCOCCAL DISEASE 673
Readouts Utility Reference
Survival, CFU Sepsis in 147
immunocompromised
hosts
Sepsis in cirrhotic 2
patients
Lysosomal enzymes in plasma Clinical and metabolic 98
and organs, CFU, vital parameters during
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parameters sepsis
CFU, splenic mass, and blood Sepsis in 106
flow splenectomized
patients
CFU, drug concentrations in Drug efficacy studies 82
plasma
similar experimental strategy was applied to investigate pneu-
mococcal sepsis in cirrhotic rats (2). Patients with alcohol-
induced hepatic cirrhosis are predisposed to pneumococcal
infection, partially due to decreased liver production of com-
plement factors. In this model, cirrhotic rats are also hypo-
complementemic compared to healthy animals (2, 166). By
using this approach, a detrimental role of pneumolysin was
observed in both depleting complement components and re-
ducing serum opsonic activity (3), resulting in enhanced pneu-
mococcal virulence in the cirrhotic host (2). To our knowledge,
there are no reports of i.p. models of sepsis in the rat.
One of the first models of pneumococcal sepsis in the rabbit
was described in 1970 by Guckian et al., and it represents a fine
example of analysis of clinical parameters (e.g., body temper-
ature, cardiac output, arterial blood pressure) detected during
the course of this disease (98). Infection was established by the
i.p. route, and the model was employed to analyze the roles of
lysosomes and cathepsin inhibitor in plasma (98), metabolic
alterations in the course of disease (96), and the effects of
splenectomy and drug therapy in the disease (97). An intra-
cardiac model of experimental sepsis in New Zealand White
rabbits was utilized to investigate the key role played by the
spleen during the disease (106). This represents another ex-
ample of refined surgery and measurements of clinical param-
eters in the rabbit in the course of infection. Effects of
hemisplenectomy, total splenectomy, spleen transplant, and
spleen repair on the clearance of S. pneumoniae during exper-
imental sepsis were evaluated (62, 106). Finally, an i.v. model
of sepsis was established in chinchilla rabbits to study the
pharmacokinetics of cephalosporins (82).
ANIMAL MODELS OF MENINGITIS
S. pneumoniae is one of the major infectious agents respon-
sible for acute bacterial meningitis, which can be fatal in 5% to
40% of patients (137, 146). Up to 30% of survivors suffer from
neurological sequelae (e.g., learning, hearing, and memory im-
pairment, seizures, and motor deficits) due to brain damage
(146). In patients with meningitis, brain injury can manifest
with several patterns, including vasculitis, cortical necrosis, and
neuronal apoptosis in the hippocampus (137, 146). Develop-
674 CHIAVOLINI ET AL. CLIN. MICROBIOL. REV.

TABLE 6. Mouse models of meningitis

Pneumococcal
Route of infection Mouse strain(s) Aim of study Readouts Utility Reference
serotype(s)

Intracerebral (ventricular) CD-1 NSa Drug efficacy CFU in brain and Therapeutic studies 220
organs
Intracerebral (right C57BL/6 3 Model description Immunohistochemistry, Meningitis pathophysiology, 86
forebrain) CFU, clinical scores, drug and virulence
behavioral tests studies
i.cist. (cisterna magna) C57BL/6 3 Role of eNOSb Survival, clinical scores, Meningitis pathophysiology, 136
cytokines, and hearing loss, therapeutic

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histology studies
Intracerebral MF1 2, 3, 4 Model description Histology, survival, Virulence studies, host- 59
(subarachnoidal) CFU parasite interaction
i.cist. (cisterna magna) Infant BALB/c 3 Model of brain Survival, CFU in CSF, Meningitis in children, 95
C57BL/6, damage histology, and brain damage
CD-1 apoptosis
i.p. Infant C57/129 6 Role of ICAM-1 Survival, CFU in Hematogenous meningitis, 230
organs, cytokines drug studies
i.n. Swiss 6 Model description Brain histology, CFU Pathogenesis via natural 268
in CSF, cytokines infection, drug studies
i.n. Swiss 2 Model description CFU in brain and Disease pathogenesis via 160
other organs natural infection
a
NS, not specified.
b
eNOS, endothelial nitric oxide synthase.

ment of meningitis generally initiates from the colonization of
the nasopharynx by S. pneumoniae, which reaches the lungs
and then invades the bloodstream with subsequent crossing of
the BBB (146). Meningitis can also be caused either by con-
tiguous spread of pneumococci infecting the sinuses or the ME
or by accidental traumatic inoculation of bacteria into the
central nervous system (CNS) (137). Experimental animal
models are essential tools to study the pathogenesis of menin-
gitis and the efficacies of different drugs against the disease. If
the mouse is a key laboratory animal for the induction of
experimental pneumonia and sepsis, rabbits and rats represent
the counterparts for the study of experimental meningitis.
Mouse Models
As previously mentioned, the mouse has become an exper-
imental system for studying pneumococcal meningitis only re-
cently, while most studies between the mid-1970s and the 1990s
were carried out in the rat and the rabbit. Two major types of
S. pneumoniae murine meningitis models exist: (i) direct infec-
tion by the intracerebral or the intracisternal (i.cist.) route and
(ii) infection induced by the i.p. or i.n. route. Direct bacterial
inoculation into the CNS mimics the contiguous spread of
bacteria from the nasopharynx or the ME or the inoculation of
pneumococci into the brain due to trauma. This system allows
the study of host-pathogen interactions once infection is estab-
lished in the meninges but does not enable the analysis of the
different pathogenic steps occurring from colonization to dis-
ease in the CNS (137). In contrast, meningitis induced via i.n.
or i.p. routes is useful for the analysis of pathogenesis accord-
ing to the natural way of infection but has the drawback that
approximately 50% of infected animals will die due to sepsis
without ever developing meningitis (243, 268). Characteriza-
tion of meningitis in the mouse model has been performed by
analyzing different parameters, including animal survival; as-
sessment of clinical scores; viable bacterial counts in the brain,
cerebrospinal fluid (CSF), and other organs; histological anal-
ysis of the brain tissue; and determination of leukocyte and
cytokine levels in CSF and serum (Table 6). One disadvantage
presented by the mouse model is the difficulty of collecting
CSF samples (only up to 10 ␮l can be obtained) due to the
small size of the animal (52).
Direct induction of meningitis. The meningitis model of
Gerber et al., described in 2001 and based on a previously
established Listeria monocytogenes meningoencephalitis model
(218), is an attractive system for analyzing pneumococcal men-
ingitis following the infection of inbred mice into the right
frontal lobe of the brain (86). Meningitis was studied by per-
forming bacterial counts for different organs, histology of the
brain, clinical score calculations, and behavioral tests. This
model has been employed to study the efficacy of rifampin
against meningitis (173) and has also been used for analyzing
host and pneumococcal factors involved in disease pathogen-
esis (35, 76, 198, 255). In another work by the same research
group, mice surviving pneumococcal meningitis presented with
clear deficits in motor skills, spatial memory, and learning
when subjected to behavioral tests (e.g., Morris water maze
test, tightrope test) (254). Therefore, this mouse model also
enables the assessment of postinfectious sequelae (254). The
induction of experimental meningitis in the mouse was also
accomplished by inoculating pneumococci directly into the cis-
ternae magnae (i.cist.) of anesthetized C57BL/6 mice (136). By
use of this model, Koedel et al. described the role of nitric
oxide synthase in the disease, and the same system has been
used to study innate immune responses to infection, including
cytokine expression patterns and the role of caspase-1, MyD88,
and complement factors (133, 134, 136, 138, 209). As all mice
VOL. 21, 2008
infected by the i.cist. route presented with hearing loss, this
system allowed the establishment of the first experimental ex-
ample of pneumococcal meningitis-associated hearing loss in
the mouse (133). The two models described above finely re-
semble pneumococcal meningitis in humans and offer the op-
portunity to study the pathophysiologic changes during men-
ingitis, the effects of drug administration, and postinfection
neurological sequelae.
Most mouse models of meningitis employ inbred mice, but
studies employing outbred strains also exist. Shapiro et al.
induced meningitis by the intracerebral-ventricular route in
outbred CD-1 mice and studied the therapeutic efficacy of
clinafloxacin (220). Another model of meningitis in outbred
MF1 mice was developed by our research group by inoculating
S. pneumoniae strains of different capsular serotypes via the
intracranial-subarachnoidal route (59). The establishment of
meningitis was confirmed by histological analysis of the brain,
and animal survival and bacterial counts in different organs
were also evaluated. Because levels of disease development
and outcomes were comparable among strains of serotypes 2,
3, and 4 (59), this model may be instrumental for screening the
virulences of different serotypes and possibly for assessing at-
tenuation of isogenic mutants. The model is based on a mod-
ified version of a method describing the induction of crypto-
coccal meningoencephalitis in the mouse (28). Recently, a
novel model of brain damage in pneumococcal meningitis has
been described for infant (11-day-old) mice of both inbred
(C57BL/6, BALB/c) and outbred (CD-1) strains: all mice in-
fected via the i.cist. route rapidly developed the disease and
some animals also exhibited brain damage (95). Among the
mouse strains tested, BALB/c mice showed the largest suscep-
tibility to infection, while C57BL/6 mice were the most resis-
tant and the ones with major neuronal injury both in the cortex
and in the hippocampus. The model was established from
previous studies of brain injury to infant rats (143) and is
promising for the study of neuronal damage in children (95).
Hematogenous meningitis. Tan et al. developed one of the
first i.p. meningitis model for inbred infant mice in order to
study the role of intercellular adhesion molecule 1 (ICAM-1)
in this disease (230). Similarly, two additional murine models
of meningitis via the i.p. route were described later on by
Iizawa et al., who analyzed the efficacy of cefozopran against
the disease (112), and by Tsao et al., who focused on elucidat-
ing the role of tumor necrosis factor alpha in regulating the
opening of the BBB during experimental meningitis (243). In
the latter model, antibiotics were given soon after infection to
avoid mouse death due to septic shock before the development
of meningitis.
The model of meningitis induced by the i.n. route mimics the
natural way of infection. The work of Kostyukova et al. repre-
sents one of the first attempts to analyze the roles of different
pneumococcal virulence factors (e.g., hyaluronidase, capsule,
and pneumolysin) in the pathogenic steps leading to meningitis
following i.n. infection (139). However, the first detailed model
of hematogenous meningitis should be accredited to a work
conducted by Zwijnenburg et al., who infected Swiss mice via
the i.n. route using a mixture of bacteria and purified pneu-
mococcal hyaluronidase to favor systemic invasion from the
nasopharyngeal mucosa (268). As aforementioned, the disad-
vantage of this system relies in the fact that only 50% of
MODELS OF PNEUMOCOCCAL DISEASE 675
animals developed the disease (268). The same research group
largely used the above-described model to study the role of
interleukin-1 (IL-1), IL-10, and IL-18 in the disease (264, 266,
267). Recently, the model was also employed to assess the
therapeutic effects of the inhibitor of complement factor C1,
which was effective at promoting bacterial clearance and re-
ducing both mouse illness and inflammation in the CNS (265).
Finally, the model described by Marra and Brigham is also
based on i.n. infection of Swiss mice with pneumococci (with-
out hyaluronidase) (160). The course of meningitis and bacte-
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rial counts in brains, lungs, and blood following infection with
different challenge doses were evaluated in that study (160).
Nonhematogenous meningitis. A study conducted by van
Ginkel et al. reported a novel in vivo route for pneumococci to
enter the CNS along olfactory nerves: nasal colonization by S.
pneumoniae resulted in bacterial entry into the brain via ret-
rograde axonal transport without causing bacteremia (249). To
our knowledge, this is the first report describing a nonhemat-
ogenous meningitis caused via i.n. infection of mice.
Rat and Rabbit Models
Rat and rabbit models are still currently employed by several
research groups to study pneumococcal meningitis. They rely
on the induction of disease via the i.cist. route, with the excep-
tion of two models of either hematogenous or otologic men-
ingitis developed in the rat (206, 253). As already mentioned
for the pneumonia models in rats and rabbits, more-sophisti-
cated surgical procedures are here employed to cause the dis-
ease; however, animal size has the advantage of allowing large
samples of and multiple sampling for CSF and blood compared
to what is possible with the mouse model. Except for an oto-
logic model (253), none of the methods used to inoculate these
animals mimic the natural route of infection occurring in hu-
mans, but they enable the study of disease once infection is
established in the meninges (Table 7). The diseases induced in
the two animal species differ in some aspects, including brain
damage. Apoptosis in the dentate gyrus and cortical necrosis
are the predominant features of damage in the rabbit (269)
and in the adult rat (103), respectively. Conversely, the infant
rat is unique, as it reproducibly manifests both hippocampal
apoptosis and cortical necrosis (143), similar to what is ob-
served for patient brain damage (137, 146).
i.cist. models for rats. Both infant and adult rats have been
employed extensively in different studies on pneumococcal
meningitis and represent valuable tools for studying disease
characteristics, host immune response to infection, and efficacy
of antimicrobials and anti-inflammatory drugs (Table 7). In-
fant (6- to 12-day-old) rats are generally more susceptible than
adults to pneumococcal infection, and despite their smaller
size, repeated collection of CSF and blood is still feasible. As
aforementioned, another advantage of infant compared to
adult rats is the possibility of inducing brain damage both in
the hippocampus (apoptosis) and in the cortex (necrosis)
(143). Thus, the infant rat represents a robust and reliable
laboratory animal to study brain damage (27, 143, 145).
In the early 1990s, the i.cist. infant rat meningitis model was
used by different groups, mainly for therapeutic studies (242,
259). In 1990, Tsai et al. established an important i.cist. model
in infant Sprague-Dawley rats for the study of cefepime phar-
676 CHIAVOLINI ET AL. CLIN. MICROBIOL. REV.

TABLE 7. Rat and rabbit models of meningitis

Route of Pneumococcal
Animal Aim(s) of study Readouts Utility Reference(s)
infection serotype(s)

i.cist. Infant Sprague- 3 Drug efficacy, MMPa in Drug pharmacokinetics, Therapeutic studies, host 145, 242
Dawley rat brain damage histology, CFU, immune response,
MMP in CSF brain damage
i.cist. Wistar rat 3, 6 Microvascular changes ICPb, rCBFc, brain Meningitis 193
edema pathophysiology
i.cist. Wistar rat 2 Damage to brain Ciliary beat frequency, Virulence studies, host 103
ependyma CFU, CSF response to infection,

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parameters, SEMd of brain damage
brain
i.p. Infant Sprague- 3 Pathogenesis of inner CFU in blood and CSF, Meningitis secondary to 206
Dawley rat ear invasion histology of ME and ear infection, drug
brain efficacy studies
Otologic, i.p. Wistar rat 2 Cochlear implantation in Gram staining, Post-cochlear 253
meningitis histology, CFU implantation
counts meningitis, therapeutic
studies
i.cist. New Zealand White 3 Model description, drug ␤-Lactam Disease pathophysiology, 69
rabbit study concentrations in host response, drug
CSF and virulence studies
i.cist. New Zealand White 3 Hearing assessment Hearing loss, histology Hearing loss due to 26
rabbit of inner ear and meningitis, drug
brain efficacy
a
MMP, metal metalloproteinases.
b
ICP, intracranial pressure.
c
rCBF, regional cerebral blood flow.
d
SEM, scanning electron microscopy.

macokinetics (242). However, the inoculation technique was
fully described later in studies conducted by Leib et al. (145)
and is based on a previously described model for meningitis
caused by group B streptococci (132). Briefly, pups were in-
fected i.cist. with 10 ␮l of bacterial suspension by use of a
32-gauge needle and then monitored for seizures due to injec-
tion (132, 145). Development of meningitis was proven by
culturing bacteria present in the CSF (132, 145). The menin-
gitis model in the infant rat has largely been used by Leib and
coworkers in studies on the involvement of matrix metallopro-
teinases (143, 145, 165) and endothelin (194) in neuronal
damage. By means of this model, the efficacies of several ther-
apeutic approaches against meningitis and/or postinfectious
sequelae, including antioxidants (6), adjunctive therapy drugs
(27, 143, 144, 194), and antimicrobials such as daptomycin (94)
and phage lysins (93), have been evaluated. It is interesting
that in this experimental system, the use of dexamethasone as
an adjuvant therapy increased hippocampal cell injury and
reduced the learning capacities of rats (144). More recently,
the same model was also employed to analyze the key role of
choline in the pathogenesis of meningitis and brain damage
(83).
The adult rat model of pneumococcal meningitis was origi-
nally described in detail in a work carried out to study the
microvascular alterations during the establishment of menin-
gitis (193). Wistar rats were anesthetized, tracheotomized, and
artificially ventilated before the insertion of catheters into the
cisternae magnae. A craniotomy was then performed in the
right parietal bone to place a probe for measuring regional
cerebral blood flow and intracranial pressure, and once these
parameters reached stable baseline levels, meningitis was in-
duced by injecting live pneumococci in a volume of 75 ␮l (192,
193). The method was instrumental to assess the detrimental
effect of the oxidant peroxynitrite in the disease (128) and the
function of transforming growth factor ␤2 in suppressing
cerebrovascular alterations and brain edema in early experi-
mental meningitis (192). Another adult rat i.cist. model was
developed by Hirst et al., who investigated the effects of pneu-
mococcal infection on cerebral ciliated ependyma during ex-
perimental meningitis. An accurate description of the animal
surgical preparation is given, based on the implantation of a
19-gauge cannula into the cisterna magna, which allowed both
pneumococcal infection and CSF sampling during animal fol-
low-up. A detailed report on treatment and analysis of CSF,
blood, and brain tissue is also provided (103). This technique
was successfully employed to show that S. pneumoniae caused
loss of and damage to ependymal cells and ciliary functions,
which may contribute to several neuropathological aspects of
meningitis (103). Recently, the same research group also
showed that both autolysin and pneumolysin play key roles in
the pathogenesis of pneumococcal meningitis (104).
Hematogenous and otologic models for rats. Hematogenous
and otologic models for rats were established to study menin-
gitis secondary to sepsis or otitis media. In the model of Ro-
driguez et al., infant Sprague-Dawley rats were inoculated by
the i.p. route, and some of the animals infected developed
meningitis (about 50%) and cochlear inflammation (20 to
80%). This model may be useful to study the pathogenesis of
inner ear invasion by S. pneumoniae during hematogenously
acquired experimental meningitis (206). Recently, another
VOL. 21, 2008 MODELS OF PNEUMOCOCCAL DISEASE 677

TABLE 8. Animal models of otitis media

Route of Pneumococcal
Animal(s) Aim(s) of study Readouts Utility Reference
infection serotype(s)

Intratympanic Chinchilla 23 Model description, Exudation in ME, Disease pathogenesis, 89

antibody antibody titers, host response, vaccine
response CFU and drug studies
Intrabullar Mongolian gerbil, 3 Model description Otoscopy, IEa Antibiotic efficacy and 81
chinchilla histology, CFU kinetics, vaccine and
virulence studies
Intratympanic Guinea pig 23F Model description CFU in ME fluid, Drug efficacy studies, 239

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ME histology disease pathogenesis
Intrabullar Sprague-Dawley 3, 6A Model description Survival, CFU in ME Evaluation of drugs and 101
rat and blood vaccines
i.n. Rivm:WU (CBP) 14 Model description, ME histology, TEMb, Immunization and 248
rat vaccine study antibody titers correlates of
protection in a
natural-route model
Intrabullar BALB/c, C57BL/6 3, 6, 9, 19 Model description CFU in blood and Host inflammatory 164
mice ear, survival, response, drug and
otomicroscopy vaccine studies
i.n. BALB/c mice 19F Prevention of Bacterial Otitis media in children, 162
otitis media by multiplication in vaccine studies
phage lysin vivo
a
IE, inner ear.
b
TEM, transmission electron microscopy.

model was set up in adult rats inoculated via both otologic
(inner ear and ME) and i.p. routes of infection to establish
whether cochlear implantation increases the risk of pneumo-
coccal meningitis (253). The use of three infection routes cov-
ers the potential routes used by S. pneumoniae to reach the
CNS: direct spread from the inner ear, contiguous or hema-
togenous spread from the ME, and invasion of the CNS from
the bloodstream. Meningitis was induced in all animals with
cochlear implants, regardless of the infection route (253).
i.cist. infection in rabbits. Rabbit models of pneumococcal
meningitis have contributed substantially to our present knowl-
edge of the disease and have significantly helped the evaluation
of antimicrobial drug efficacy. The i.cist. rabbit model is an
advantageous system that allows accurate analysis of disease
features, as well as multiple sampling and large samples of CSF
and blood (235). Due to the large quantity of CSF available
from this animal, many types of analysis can be performed,
providing information on bacterial and leukocyte counts and
concentrations of glucose, proteins, and other metabolites (Ta-
ble 7). A limitation of the model is that experimental groups
are generally small due to animal size, representing an issue for
the performance of dependable statistical analysis. Another
obvious drawback relates to the unnatural method of infection,
based on the instillation of relatively large numbers of bacteria
into the cisterna magna.
The adult rabbit model, originally developed by Dacey and
Sande in 1974, is one of the first important models of pneu-
mococcal meningitis based on an extremely accurate technique
for inducing the disease. Briefly, animals were secured in a
frame through a dental helmet attached to the skull: their
cisternae magnae were then punctured with needles mounted
in a geared electrode introducer, and S. pneumoniae was inoc-
ulated (69). The use of this model greatly helped the under-
standing of several aspects of pneumococcal meningitis, in-
cluding bacterial replication in the CSF, host defenses and
inflammation, BBB alteration, and also the systemic effects of
meningitis on animal respiration and circulation (236). Numer-
ous studies have been undertaken to evaluate antibiotic effi-
cacy (34, 64, 73, 85, 172, 189, 203, 226, 231) and the role of host
and bacterial factors in disease pathogenesis (10, 36, 247).
Interestingly, the effectiveness of a bactericidal but nonbacte-
riolytic antibiotic, such as daptomycin, in clearing pneumococ-
cal infection from the CSF and in reducing inflammation was
proven by means of both this rabbit model (226) and a previ-
ously described rat model (94). Also, of no less importance is
the fact that this rabbit model is a useful tool to analyze
damage in the hippocampus due to meningitis (269). Similarly,
Lindquist et al. described the establishment of a rabbit model
based on the use of a conventional head holder and on the
percutaneous puncture by hand of the cisternae magnae of
rabbits (148). Finally, Bhatt et al. developed a model of me-
ningogenic labyrinthitis via i.cist. infection of New Zealand
rabbits with the aim of assessing hearing loss secondary to the
induction of meningitis. Data obtained from this model indi-
cate that the severity of hearing loss strongly correlates with
the duration of infection (26). The model largely mimics hear-
ing loss due to meningitis in humans and may be instrumental
for testing the efficacies of therapeutic drugs.
ANIMAL MODELS OF OTITIS MEDIA
Otitis media is one of the most common diseases in child-
hood. It manifests in different forms depending on the dura-
tion and type of exudate (e.g., purulent, mucoid, and serous)
and it may lead to sequelae, including hearing loss, learning
disabilities, and speech impediments (65). S. pneumoniae is the
678 CHIAVOLINI ET AL.
main cause of ME disease in children; other common causes
include Haemophilus influenzae and Moraxella catarrhalis. The
pathogenesis of the disease depends on different factors in-
cluding concomitant bacterial and/or viral (e.g., influenza) in-
fection of the upper respiratory tract, impaired immunity, age,
genetic predisposition, and dysfunction of the Eustachian tube
(65). Different models of pneumococcal otitis media have been
developed using the chinchilla, gerbil, guinea pig, rat, and,
more recently, the mouse in order to understand different
aspects of the disease and test novel antimicrobials and poten-
tial vaccine candidates. Description of disease by use of these
animal models has been done by observing different parame-
ters, including the presence of bacteria, polymorphonuclear
cells, and inflammatory molecules in ME fluid as well as his-
tological analysis of Eustachian tube, the ME, and the inner
ear (Table 8).
Chinchilla, Gerbil, and Guinea Pig Models
Historically, the chinchilla has been the preferred model for
mimicking pneumococcal otitis media and studying its charac-
teristics (Table 8). Giebink very elegantly reviewed the impor-
tance of this animal model, emphasizing how infection can be
induced by small bacterial inocula administered either directly
into the ME or i.n. (90). One major advantage of this experi-
mental system is its feasibility for the study of disease patho-
genesis, the immunogenicity of vaccine antigens, and the effi-
cacy of antimicrobials at the mucosal site without causing
disseminated disease (90). In addition, chinchillas have large
bullae, which are easily accessible for both inoculation and
repetitive sampling of ME fluid (211). Two disadvantages re-
late to the unavailability of inbred chinchilla strains and the
high costs of the animal compared to those of other rodents.
The chinchilla model was first described in 1976 by Giebink
and colleagues. Acute otitis media was induced by inoculation
of pneumococci directly into the ME cavity through the dorsal
bulla (89). Pathogenic aspects of otitis media, as well as the
immune response evoked after infection, were analyzed (89).
Both this group and other research teams have employed this
model in several investigations, including characterization of
the inflammatory response to infection (174, 216), analysis of
the role of pneumococcal virulence factors in disease (217,
240) and of the efficacies and kinetics of antimicrobials against
purulent and serous otitis media (48, 107, 108), and vaccine
studies (90, 150). As seen for other pneumococcal disease
agents, the virulence of S. pneumoniae in experimental otitis
media induced in the chinchilla varies largely by serotype (90).
A comparative study was recently conducted to analyze the
virulences of different pneumococcal strains in chinchillas in-
oculated via the transbullar route. Fourteen strains of sero-
types 3, 4, 6A, 9V, 11, 14, 19F, and 23F were tested in the
model, and data showed how even within a single serotype,
strains differ in their capabilities to cause local and systemic
disease (80). Besides the classic intratympanic route, Giebink
et al. also established a chinchilla model of otitis media based
on simultaneous i.n. infection of S. pneumoniae and influenza
A virus: approximately 70% of animals developed the disease,
while only 20% of chinchillas presented with otitis after inoc-
ulation with bacteria only (88). This last model more closely
resembles the clinical manifestations in humans and is valuable
CLIN. MICROBIOL. REV.
for the study of pathological aspects of the disease as well as
the efficacies of drugs and vaccines (90).
The acute otitis media model in the gerbil is also a well-
established system for studying disease pathogenesis and com-
paring the efficacies of different drugs. The anatomies and
histologies of Eustachian tubes and MEs in gerbils and chin-
chillas are similar, with both allowing easy inoculation through
the tympanic membrane or the bulla. The model described by
Fulghum et al. in a comparative study between gerbils and
chinchillas (81) was developed following the method originally
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used by Giebink et al. (89). Advantages of using the gerbil as
a model of ME infection include the availability of inbred
strains, the reasonable cost, and the susceptibility of this ani-
mal to common pathogens of otitis media, including S. pneu-
moniae, H. influenzae, and M. catarrhalis (81). Infection was
carried out by instilling bacteria directly into the ME by trans-
bullar challenge (81). This system was employed in investiga-
tions on the efficacy and pharmacokinetics of ␤-lactams against
otitis media caused by penicillin- and cephalosporin-resistant
S. pneumoniae (17, 18, 54, 190). Intrabullar challenge of gerbils
with virulent pneumococci was also carried out to investigate
the pathogenesis of pneumococcal meningitis secondary to oti-
tis media (160, 170).
Finally, it is also worth mentioning the guinea pig as a model
of pneumococcal otitis media (239). Although less used than
the chinchilla and the gerbil, it represents a reliable and rela-
tively inexpensive otosurgical animal for which inbred strains
and experimental reagents are also available (211). Investiga-
tions on the cytotoxic effects triggered by live pneumococci in
the cochlea (63) and also therapeutic (105) and vaccine (260)
studies were performed.
Rat and Mouse Models
In recent years, interest in both rat and mouse models of
otitis media has increased, since these animals—especially the
mouse—are less expensive and more characterized in terms of
immunological and genetic information than are chinchillas,
gerbils, and guinea pigs.
Otitis media has been induced in the rat by two different
routes of infection: direct inoculation into the ME and i.n.
infection. In a model described in 1988, S. pneumoniae was
directly instilled into the tympanic cavities of rats to investigate
bacterial replication in the ME and blood (101). Inoculation of
rat ME with pneumococci caused otitis media with either pu-
rulent or serous effusion, closely resembling the disease in the
human host (101). This system has been used to evaluate the
efficacy of penicillin for the treatment and prevention of per-
manent mucosal changes due to pneumococcal acute otitis (5,
102) and to assess protection against experimental otitis media
conferred either by anticapsular antibodies (256) or by active
immunizations with PspA (256) or whole killed bacteria (66).
Moreover, the virulences of pneumococcal strains with differ-
ent opacity phenotypes (151) and various levels of susceptibil-
ity to penicillin (154) were also evaluated by intratympanic
challenge of rats. van der Ven et al. described a novel rat
model based on the i.n. instillation of bacteria, thus mimicking
the natural route of infection of otitis media in humans (248).
Histamine was inoculated into Wistar rats [strain Rivm:WU
(CBP)] by the intratympanic route in order to impair ciliary
VOL. 21, 2008
activity and contribute to Eustachian tube dysfunction, and
bacteria were then delivered i.n. with an infusion catheter.
Histamine pretreatment facilitated the ascent of bacteria from
the nasopharynx to the ME, allowing assessment of mucosal
immunity directly in the Eustachian tube and ME. The model
was utilized in a vaccination study to analyze protection against
otitis media following immunization with a tetanus toxoid-
conjugated type 14 capsular polysaccharide (248). The impor-
tance of this model was also revealed in the work of Eriksson
and Hellström, who provided clear evidence of acute otitis
media development in the rat after multiple i.n. inoculations of
pneumococci (77).
Less than 10 years ago, only a few mouse models of pneu-
mococcal otitis media were available (210). Clearly, the small
size of the animal together with the poor accessibility of the
ME for inoculation and sampling makes the mouse a tricky
choice to study otitis media. Two different routes of infection
are used in the mouse model: either direct injection (intrab-
ullar or intratympanic) into the ME or i.n. inoculation. Direct
microbial injection into the ME warrants reproducibility in
disease initiation and development and allows the use of small
bacterial inocula, whereas the i.n. route mimics the natural
mode of ME infection, but induction of experimental otitis is
more sporadic and requires larger doses of bacteria. In 2003,
Melhus and Ryan established a model of otitis media caused by
S. pneumoniae and H. influenzae in BALB/c and C57BL/6 mice
inoculated via the intrabullar route (164). The course of dis-
ease was dependent upon both bacterial and mouse strains,
with BALB/c being the most susceptible mouse strain. The
authors also found that highly virulent pneumococcal serotypes
(types 3 and 6) consistently induced lethal sepsis with or with-
out ME infection, while less virulent strains (types 9 and 19)
produced otitis (164, 210). Direct inoculation of bacteria into
the ME can also be instrumental in assessing inflammation in
the ME following the injection of heat-killed pneumococci
(152, 161). As acute bacterial otitis media is an inflammatory
response to replicating microorganisms, the use of heat-killed
bacteria does not mimic the natural disease but still allows
measurements of inflammatory parameters in the ME, such as
the amount and type of effusions, the thickness of the tympanic
membrane, and the recruitment of polymorphonuclear cells
(152). The mouse intratympanic/intrabullar model of pneumo-
coccal otitis media found applications in both vaccine (211,
212) and therapy (161) research fields. Finally, a recent work
elegantly described the induction and prevention, by means of
phage lysins, of otitis media in BALB/c mice stably colonized
by bioluminescent S. pneumoniae (162). In the model, recur-
rent otitis media was induced by infecting the colonized mice
via the i.n. route with influenza virus (to favor bacterial spread
from the nasopharynx to the ME) and was efficiently treated
with the Cpl-1 lysin (162). This novel model of naturally oc-
curring otitis media mimics infection in children and may
prove useful in studies on the prevention and treatment of this
important disease.
CONCLUSIONS
Pneumococcal disease in humans is varied and multifaceted,
and no experimental animal model is able to completely mimic
human disease. However, despite its limitations, animal exper-
MODELS OF PNEUMOCOCCAL DISEASE 679
imentation remains an unquestionably valuable tool to clarify
the pathogenic mechanisms of disease. This review provides a
perspective on the development and applications of different
animal models used to mimic and study the major diseases
caused by S. pneumoniae. We have highlighted the progress
made over the years, indicating how certain historical models
represented milestones for developing novel and more-effec-
tive methods, while others are still in use largely to investigate
pneumococcal diseases. Some of the techniques for inducing
disease were described to point out how thoroughly these an-
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imal models were set up. Characteristics of pneumococcal
pneumonia, sepsis, and meningitis have successfully been re-
produced in the mouse, the rat, and the rabbit. In the case of
otitis media, the chinchilla, the gerbil, and the rat have been
the preferred animal models throughout the years, although
recently researchers have also reproduced the disease in the
mouse. All these models were and continue to be helpful tools
for elucidating aspects of disease pathogenesis, characterizing
innate and adaptive immunities to S. pneumoniae, and testing
the efficacies of antibiotics and other therapies as well as po-
tential vaccine candidates. Various infection routes and meth-
ods have been employed to induce disease, and we have dis-
cussed the importance of different readouts and emphasized
the main advantages and limitations of the models. The utility
of each animal model is also highlighted to facilitate research-
ers in the choice of the appropriate system. The mouse is
comparatively inexpensive and easy to handle and allows the
screening of drugs and vaccines with high statistical power. The
commercial availability of plentiful murine experimental re-
agents and strains, including transgenic and knockout strains,
makes the mouse an attractive model to address the interplay
between bacteria and host factors. Furthermore, the comple-
tion of the mouse genome sequence and the availability of
technologies to introduce targeted mutations into the genome
provide a powerful and extensive toolkit for studying mouse
susceptibility to pneumococcal disease. In contrast, larger an-
imals present the advantage of obtaining larger samples in
addition to an increased ease in performing inoculation and
surgical procedures, but they are more expensive, and the
results obtained may lack statistical significance due to the use
of smaller animal groups.
The development of pneumococcal disease depends upon
both bacterial (e.g., capsular serotype and other virulence de-
terminants) and host (e.g., genetic background, immune re-
sponse, age, sex) factors. Thus, in addition to the selection of
the appropriate in vivo model, the use of several animal and
pneumococcal strains is highly recommended to test a research
hypothesis. We also wish to emphasize how in vivo experimen-
tation should preferably be combined with in vitro assays (e.g.,
cell culture assays) to understand and reproduce essential
characteristics of the pathogenesis of infectious diseases and to
elucidate key features of host-pathogen interaction. Finally,
worth considering is the fact that certain host-microbe inter-
actions are species specific, thus suggesting additional caution
to scientists who wish to mimic, in an animal model, pathoge-
nicity mechanisms which occur only in the human host.
ACKNOWLEDGMENTS
We thank Tiziana Braccini, Riccardo Parigi, and Velia Braione for
their excellent expertise with animal experiments offered along the
680 CHIAVOLINI ET AL.
years. We also acknowledge Marina Piccinin for secretarial work. We
are grateful to Aras Kadioglu (University of Leicester, United King-
dom) for advice and critical reading of the manuscript.
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