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Antimicrobial Agents and Chemotherapy, November 2001, p. 3195-3197, Vol. 45, No. 11
Biological Research
Laboratories1 and Laboratory Animal
Science and Toxicology Research
Laboratories,2 Sankyo Co., Ltd.,
Shinagawa-ku, Tokyo 140-8710, Japan, and Harbor-UCLA
Research and Education Institute, Torrance, California
905023
Received 9 February 2001/Returned for modification 6 June
2001/Accepted 3 August 2001
We established a straightforward murine model of oropharyngeal
candidiasis. Mice were immunosuppressed with cortisone acetate, anesthetized, and then inoculated by placing cotton wool balls saturated with Candida albicans sublingually for 2 h. A prolonged, reproducible infection was induced. This model may be
useful for antifungal screening or pathogenesis studies.
Oropharyngeal candidiasis (OPC) is
the most common fungal infection in patients with AIDS (3, 7,
9). Animal models of OPC are useful for evaluating the efficacy
of antifungal agents and investigating the pathogenesis of this
infection (8). In this study, we established and
characterized a new model of OPC in mice.
Specific pathogen-free male ddY mice (5 weeks old; Japan SLC, Inc.,
Shizuoka, Japan) were used. All animal experiments were carried out
according to the guidelines of the Institutional Animal Care and Use
Committee of Sankyo Co., Ltd. Mice were immunosuppressed with 4 mg of
cortisone acetate (Sigma Chemical Co., St. Louis, Mo.) administered
subcutaneously on the day before and 1 and 3 days after inoculation.
The mice were also given tetracycline hydrochloride (Achromycin V;
Lederle Japan, Ltd., Tokyo, Japan) in their drinking water (0.5 mg/ml),
starting the day before infection. All experiments used at least five
mice per treatment group.
Candida albicans SANK51486 from our laboratory was used for
this study. This strain is susceptible to fluconazole (FLC) with an MIC of 0.25 µg/ml, as determined by the NCCLS M27A broth
microdilution method (5). Before inoculation, the mice
were anesthetized by intraperitoneal injection with 26 µg of
dimorpholamine (Theraptique; Eisai Co., Ltd., Tokyo, Japan), 207 µg
of xylazine (Bayer Yakuhin, Ltd., Osaka, Japan), and 1.04 mg of sodium
pentobarbital (Nembutal; Dainabot Co., Ltd., Tokyo, Japan). Next,
3-mm-diameter cotton wool balls (Hakujuji Inc., Tokyo, Japan) were
saturated with 100 µl of a suspension containing
108 blastospores per ml and then placed
sublingually in the oral cavity for 2 h.
To determine the number of viable organisms in the tissues, each mouse
was sacrificed, and the mandible with attached tissue and the esophagus
were excised. After removing the bone and teeth, the tissue was
homogenized and serial dilutions were plated onto Sabouraud dextrose
agar plates containing 10 µg of chloramphenicol per ml for colony counting.
For histopathological analysis, the excised tissue was fixed with
formalin and embedded in paraffin, after which thin sections were
prepared and stained with periodic acid Schiff (PAS). To study the
effects of FLC on the course of OPC, the drug (Diflucan; Pfizer
Pharmaceuticals Inc., Tokyo, Japan) was dissolved in 0.5% sodium
carboxymethyl cellulose (Wako Pure Chemical Industries, Osaka, Japan)
and administered orally once daily, starting on day 3 postinoculation.
The control mice received 0.2 ml of the vehicle.
Initially, we investigated the effects of cortisone acetate and
tetracycline on the organism burden in the oral tissues on day 6. Groups of mice were treated as follows: cortisone acetate plus
tetracycline; cortisone acetate alone; tetracycline alone; and no
treatment (Fig. 1A). The number of
organisms in the oral tissues of mice receiving cortisone acetate was
significantly greater than in mice that did not receive this drug,
regardless of whether they received tetracycline. These results
indicate that cortisone acetate is necessary for inducing and
maintaining a high level of infection. Although tetracycline had no
effect on the number of fungi in the oral tissues, we used it to
prevent bacterial superinfection.
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.11.3195-3197.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
New Model of Oropharyngeal Candidiasis in
Mice
ABSTRACT
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FIG. 1.
Development of OPC in mice. (A) Effect of cortisone
acetate and tetracycline on the numbers of viable C.
albicans cells in the oral tissue on day 6. Data are the mean
plus standard deviation for five mice per group. ***,
P < 0.001 by the Tukey test. CA, cortisone
acetate; TET, tetracycline. (B) Time course of OPC in mice treated with
cortisone acetate and tetracycline. Data are the mean ± standard
deviation for five mice per group. Open circles, tongue; closed
circles, nonlingual oral tissue; open triangles,
esophagus.
Next, we investigated the time course of infection in the tongue, the
nonlingual oral tissue, and the esophagus. In these tissues, the number
of viable cells progressively increased over time and then remained at
5 to 6 log10 CFU/tissue from day 3 to at least
day 9 (Fig. 1B). In this experiment, white patches, which are a common
clinical feature in human OPC, were observed to cover virtually the
entire dorsum of the tongue beginning on day 2 or 3 and remaining
throughout the experiment (Fig. 2). PAS
staining of the tongue and esophagus demonstrated that the C. albicans cells had invaded the superficial epithelial layer of the
mucosa and that both mycelial and blastoconidial forms of the organism were present (Fig. 3). These histological
findings are similar to those for humans with OPC.
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We also investigated the virulence of another C. albicans isolate, SC5314 (2), in this model of OPC and observed that it induced a level of infection similar to that induced by strain SANK51486 (data not shown). Therefore, our findings are reproducible with two different strains of C. albicans.
To determine if this model could be used to assess the efficacy of
antifungal agents for the treatment of OPC, we evaluated the efficacy
of FLC. When FLC was administered at 10 mg/kg of body
weight/dose starting on day 3, the number of CFU in the oral tissues progressively decreased from day 4 to day 7. In contrast, the
oral fungal burden of the control mice remained constant over this time
(Fig. 4). Also, 2 days of therapy with
FLC at doses of 2 and 10 mg/kg/dose reduced the oral fungal burden on
day 5 by 1.3 log10 and 2.7 log10, respectively, compared to that of control
mice (P < 0.001 for each comparison, Dunnett's test). The oral fungal burden of mice receiving FLC at 0.4 mg/kg/dose was
similar to that of control mice (data not shown). These results demonstrate the dose-dependent efficacy of FLC in this model.
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To date, a number of animal models have been developed for studying OPC (1, 4, 6, 8, 10). Some of these models, such as the one presented here, are best suited for evaluating the therapeutic efficacy of selected antifungal agents (1, 4, 6, 10). However, these antifungal agents must have favorable pharmacokinetics in the animal model that is being used. Simple animal models of OPC that are used for antifungal screening may also be appropriate for screening C. albicans mutants for loss of virulence. Those mutants with diminished virulence can be evaluated further in more sophisticated (and expensive) animal models, such as the CD4+-depleted mouse or the sialadenectomized rat.
In summary, we have established a new technique for inducing sustained OPC in mice. The inoculation procedure using a cotton wool ball is rapid and simple. The infection induced by this method is reproducible, and the animal-to-animal variability in the tissue fungal burden is low. These characteristics suggest that this model may be useful for basic antifungal screening.
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ACKNOWLEDGMENTS |
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S.G.F. was supported by Public Health Service grant R01 DE13974.
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FOOTNOTES |
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* Corresponding author. Mailing address: Biological Research Laboratories, Sankyo Co., Ltd., 2-58 Hiromachi 1-chome, Shinagawa-ku, Tokyo, 140-8710, Japan. Phone: 81-3-3492-3131. Fax: 81-3-5436-8565. E-mail: ykamai{at}shina.sankyo.co.jp.
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Antimicrobial Agents and Chemotherapy, November 2001, p. 3195-3197, Vol. 45, No. 11
0066-4804/01/$04.00+0 DOI: 10.1128/AAC.45.11.3195-3197.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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