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CLINICAL MICROBIOLOGY REVIEWS,0893-8512/01/$04.0010 DOI: 10.1128/CMR.14.2.398–429.2001
Apr. 2001, p. 398–429 Vol. 14, No. 2
Copyright © 2001, American Society for Microbiology. All Rights Reserved.
Experimental Oral Candidiasis in Animal ModelsYUTHIKA H. SAMARANAYAKE AND LAKSHMAN P. SAMARANAYAKE*
Oral Biosciences, Faculty of Dentistry, The University of Hong Kong, Hong Kong SAR, China
INTRODUCTION .......................................................................................................................................................398CLINICAL EPIDEMIOLOGY OF HUMAN ORAL CANDIDIASIS...................................................................398NEED FOR AND CLINICAL RELEVANCE OF ANIMAL MODELS ...............................................................400EARLY TISSUE CULTURE SYSTEMS AND HISTOPATHOLOGIC STUDIES.............................................401PROS AND CONS OF CURRENT ANIMAL MODELS ......................................................................................401
Monkey Model (Macaca irus) ................................................................................................................................401Rat Model (Wistar and Sprague-Dawley) ...........................................................................................................402Mouse Model ...........................................................................................................................................................402Hamster Model........................................................................................................................................................402
ORAL CANDIDIASIS IN ANIMAL MODELS ......................................................................................................403Monkey Model.........................................................................................................................................................403Wistar Rat Model ...................................................................................................................................................403Sprague-Dawley Rat Model ...................................................................................................................................415Mouse Model ...........................................................................................................................................................420Hamster Model........................................................................................................................................................424
CONCLUSIONS AND FUTURE DIRECTIONS....................................................................................................424REFERENCES ............................................................................................................................................................425
INTRODUCTION
Candida species are ubiquitous, human fungal pathogenscapable of initiating a variety of recurring superficial diseasesespecially in the oral and vaginal mucosae (129, 167). In thelate 1950s there was a steadily increasing number of reports onsuperficial Candida infections associated with the administra-tion of broad-spectrum antibiotics such as tetracycline (91,178). In subsequent years, the extensive use of steroids, immu-nosuppressive agents in organ transplant recipients (158, 192)myeloablative radiation therapy (70, 74, 205), and antineoplas-tics in patients with hematologic malignancies (20, 62, 106)contributed to the increasing morbidity associated with Can-dida. More recently, mucosal Candida infections have receivedprofuse attention due to the advent of the human immunode-ficiency virus (HIV) infection. For instance, it is now knownthat up to 90% of HIV-infected individuals suffer from oro-pharyngeal candidiasis (161). This condition is a key feature instaging HIV disease and was once included as a marker indisease classification (64). Curiously, HIV-infected patientsappear to be more susceptible than immunocompetent indi-viduals to oropharyngeal but not vaginal or disseminated can-didiasis (168, 177). The other general risk factors for oralcandidiasis are age (mainly the very young and the very old),denture prostheses, smoking, diabetes mellitus, iron and vita-min deficiences (159), and salivary gland hypofunction (166).
Candida albicans is the species most often associated withoral lesions, but other, less pathogenic species such as C. gla-brata, C. tropicalis, C. parapsilosis, and C. krusei are also occa-sionally but regularly isolated (105, 170). Recently, a novelspecies, C. dubliniensis, closely related to C. albicans, has been
isolated, particularly from mucosal lesions in HIV-infectedpatients (39).
An important cofactor associated with the pathogenesis oforal candidiasis appears to be the virulence of the infectingorganism (113, 153). The specific features of the fungus thatcontribute to the development of oral candidiasis include itsability to adhere to and colonize the oral mucosa (87), itsability to form cylindrical appendages termed germ tubes (33),and its cell surface hydrophobicity (68). In addition, pheno-typic and genotypic switching (176, 186), extracellular aspartylproteinase secretion (44, 208), and phospholipase production(98) appear to play a subsidiary role in the pathogenicity.Nonetheless, the hierarchy of the importance of these predis-posing attributes is little known, although some animal studiesdescribed here have shed some light on this issue.
CLINICAL EPIDEMIOLOGY OF HUMANORAL CANDIDIASIS
Isolation of Candida from the oral cavity does not implydisease, since its asymptomatic prevalence in healthy personsranges from 3 to 48% (12) and is even higher in healthychildren, 45 to 65% (129). In many epidemiological studies oforal candidiasis, the most commonly isolated yeast species isC. albicans (166). A median carriage rate of 38.1% has beenobserved for C. albicans alone in a number of surveys in com-munity-dwelling outpatients (129), while a higher carriage rate(up to 78%) has been observed in hospitalized elderly patients(41, 204). Yeast carriage is even higher in those who are HIVseropositive and rises as the CD41 T-cell count falls (67, 164,196). Other pathogenic members of the genus Candida oftenisolated from the oral environment are (in descending order ofvirulence) C. glabrata, C. tropicalis, C. parapsilosis, C. pseudo-tropicalis, C. krusei and C. guilliermondi (129). C. dubliniensis isa recently discovered novel species, and its virulence potential
* Corresponding author. Mailing address: Oral Biosciences, Facultyof Dentistry, University of Hong Kong, 34 Hospital Rd., Hong KongSAR, China. Phone: (852) 2859-0480. Fax: (852) 2547-6133. E-mail:[email protected].
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is much like that of C. albicans due to their close genomicrelatedness (190). Despite such diversity among the non-albi-cans species (143, 189), it is the general belief that they are oflow virulence and that disease manifestation is determinedmainly by the health of the host (128, 173, 183). Neither col-onization with Candida species alone nor a significant increasein their salivary concentration (53) is necessarily a precursor ofthe development of oral candidiasis (121). Therefore, otherlocal or systemic factors must be present for the organisms toinitiate infection and cause disease.
Oral candidiasis may present in a variety of clinical forms,and the three main variants are the pseudomembranous type,commonly known as thrush, and the erythematous and hyper-plastic variants (14) (Fig. 1). When two or more of thesevariants appear in unison, the term “multifocal candidiasis” isused (169). Other common lesions include Candida-associateddenture stomatitis, angular cheilitis, and median rhomboidglossitis.
The recognition that Candida is an important pathogen,particularly in the immunocompromised host, has resulted in avast body of in vitro investigations evaluating its virulent at-tributes in an attempt to elucidate the pathogenesis of thedisease. The progress made in understanding some of thesefeatures, such as the mechanisms that result in adherence tohost tissues (88), cell surface hydrophobicity (69), switchingphenomena of the yeast (186, 187), secretion of aspartyl pro-teinases (208), and phospholipase production (98), is very im-pressive. Nonetheless, in vivo studies either in live humans orin animals are essential to elucidate and fully comprehend themechanisms leading to candidal infection.
The host oral defenses against Candida essentially fall intotwo categories: nonspecific immune mechanisms (e.g., integrityof the mucosae, commensal bacteria, polymorphonuclear leu-kocytes, macrophages, and salivary factors) and specific im-
mune mechanisms (e.g., serum antibodies, secretory antibod-ies, and cell-mediated immunity) (38).
The stratified squamous epithelium of the oral mucosaforms a continuous surface that protects the underlying tissuesand functions as an impervious, mechanical barrier. The pro-tection so provided is dependent on the degree of keratiniza-tion and the continuous desquamation or shedding of epithe-lial cells. Indeed, the latter mechanism is considered to play apivotal role in maintaining a healthy oral mucosa and in lim-iting candidal colonization and infection. The interaction be-tween Candida species and the commensal microbial flora isperhaps the next critical mechanism modulating oral candidalcolonization (166). The commensal flora regulates yeast num-bers by inhibiting the adherence of yeasts to oral surfaces bycompeting for sites of adherence as well as for the availablenutrients. A number of studies have also shown, both in vivo ingnotobiotic mice and in vitro, that candidal colonization ofepithelia could be suppressed by streptococci, which are thepredominant resident commensals of oral mucosal surfaces(99, 123, 163).
The human oral cavity is a unique ecological niche becauseit is constantly bathed in saliva, a biological fluid with potentantifungal and antibacterial activity. In addition, the constantsalivary flushing action mechanically inhibits the accumulationof microorganisms in various oral niches. A quantitative re-duction in saliva or salivary flow, for instance in Sjogren’ssyndrome, leads to a xerostomic state with a concomitant in-crease in oral candidal carriage and infection, indicating theimportance of salivary defenses against invading fungi (109,162). Elements in saliva that inhibit the growth of Candidainclude nonspecific factors such as the histidine-rich proteins,the proline-rich proteins, the salivary peroxidase system, lac-toferrin, and lysozyme (142, 171, 174, 193). The anticandidalnature of histidine-rich polypeptides in particular is notewor-
FIG. 1. Simplified schematic diagram comparing the topographic distribution of common lesional sites in human oral candidiasis (A) andexperimental candidal infection in an animal model (rat/mouse/hamster) (B). The most common sites of infection are shaded. The clinical variantsof the disease and their preponderant sites are as follows: 1, erythematous candidiasis; 2, pseudomembranous candidiasis; 3, hyperplasticcandidiasis; 4, Candida-associated denture stomatitis; 5, Candida-associated angular chelitis; 6, Candida-associated median rhomboid glossitis.
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thy. Pollock et al. (142) found that the antifungal activity ofpurified salivary histidine-rich polypeptides is akin to that ofimidazoles (104, 134, 142). Lysozyme and lactoferrin are twofurther nonimmunoglobulin salivary proteins that contributeto the regulation of oral Candida. A number of studies havedocumented the fungicidal effect of apolactoferrin againstCandida (125, 171, 188, 197), while the relative sensitivity ofdifferent Candida species to lysozyme has been demonstratedby Tobgi et al. (193) using six different species.
It is known that two independent systems, the systemic andthe secretory immune systems, are both involved in defendingthe oral cavity against Candida. Lehner (100) was the first tosuggest that salivary (secretory) immunoglobulin A (sIgA) maycontribute to ameliorating the disease process. Individuals withlowered levels of sIgA are more often afflicted with mucosalcandidiasis, and functional sIgA appears to prevent the attach-ment of C. albicans to the mucosal epithelium (200). Polymor-phonuclear leukocytes and macrophages have the ability tophagocytose and kill Candida cells. However, the full expres-sion of their activity is dependent on augmentation by cyto-kines synthesized or induced by T cells (13) and the length oftime they survive in the hostile oral environment bathed insaliva.
Mucocutaneous and systemic candidiasis are both typicallyassociated with defects in the cell-mediated immune response(129). A multiplicity of defects in cell-mediated immunity insubjects with chronic mucocutaneous candidiasis have beenexamined and defined (150). This is further exemplified inpatients infected with the HIV, an agent which causes animpairment of the CD41 T-helper lymphocytes, leading tofrequent recurrences of oropharyngeal candidiasis (73). Theseand other host defenses against Candida have been reviewedrecently by Greenfield (63).
A number of antifungal agents are available for the man-agement of candidal infections (115). The major agents thatare currently used for oropharyngeal candidiasis belong toeither the polyenes (amphotericin B and nystatin), the imida-zoles (clotrimazole, econazole, ketoconazole, and miconazole),or the triazoles (fluconazole and itraconazole) (52). Nystatin isideal for topical treatment of oral infections since it is notabsorbed from the gastrointestinal tract and hence the adverseeffects are minimal. Amphotericin B is less widely used for thispurpose due to its treatment-limiting adverse effects such asnephrotoxicity (86).
The introduction of the imidazole and azole groups of an-tifungals during the last two decades has revolutionized themanagement of fungal infections (86). The approved azoleantifungal agents for the treatment of oral candidiasis aremiconazole, clotrimazole, ketoconazole, fluconazole, and itra-conazole (52). Miconazole is effective for almost all oral man-ifestations of candidiasis including chronic mucocutaneouscandidiasis. Until the introduction of the triazoles (itracon-azole and fluconazole), ketoconazole (an imidazole) waswidely used as an alternative to amphotericin B (85), but itsuffered from the drawbacks of hepatotoxicity and endocrinetoxicity. However the more recently introduced triazole agents,itraconazole and fluconazole, are far superior since they areorally active and water soluble and have a significantly lowertoxicity than do the imidazoles (85). Indeed, fluconazole is thedrug of choice in the treatment of candidiasis in HIV infection.
The euphoria surrounding the efficacy of the azoles has nowbeen tempered by the realization of moderate or high-levelresistance to fluconazole in some species, such as C. glabrata,C. krusei, and C. albicans (148, 170). This phenomenon hasbeen especially common in C. albicans isolated from patients inwhom fluconazole has been extensively used, as in those in-fected with HIV (61, 138). In addition to these topical orsystemic antifungals, antiseptic agents such as chlorhexidinegluconate have been used to supplement the drug regimens,especially in treating Candida-associated denture stomatitis.The animal models described herein have made major contri-butions to the evaluation of these drugs, especially during theirdevelopmental stages.
NEED FOR AND CLINICAL RELEVANCEOF ANIMAL MODELS
Apart from the ethical dilemmas associated with experimen-tation on live humans, humans are notoriously dissimilar interms of their dietary and social habits, immune status, andoral physiology such as salivary function. These factors, plusthe racial, ethnic, and cross-cultural variations in human de-mographics, add to the confounding matrix of factors influenc-ing the etiology and pathogenesis of diseases such as candidi-asis, where the invading organism is not a true parasite but anopportunistic pathogen. Hence, in theory at least, the devel-opment of an ideal animal model for oral candidiasis wouldprovide a standardized tool which can be controlled and ma-nipulated to derive universally comparable data on the etiopa-thology, diagnosis, and management of the disease process.Perhaps it is true that the available animal models have thus farsuccessfully illuminated the pathogenesis of many variants oforal candidiasis from the points of view of both the host andthe yeast. However, the diagnostic and management aspects ofthe disease processes have not been widely addressed, and theresults have been mixed.
The most common form of oral candidiasis is Candida-as-sociated denture stomatitis, seen in 50 to 69% of denturewearers at one time or another (29). Not surprisingly, there-fore, the pathogenesis and management of this condition havebeen studied in a number of models by many investigators.Budtz-Jorgensen, who pioneerd such studies, employed Ma-caca irus monkeys with custom-fitted acrylic plates (26) for thispurpose, while others have used the Wistar rat model to studyCandida-associated denture stomatitis and its histopathology(132, 179, 181). All workers who successfully initiated the dis-ease in animal models have claimed a striking similarity be-tween the human and animal lesions and have stressed theutility of the respective animal model. After reproducing Can-dida-associated denture stomatitis, the next step was to dem-onstrate the cofactors involved and the efficacy of topical an-tiseptics and antifungals in the management of the condition.These therapeutic approaches have included the incorporationof chlorhexidine acetate (97) and azole antifungals (126) todenture base materials, as well as the delivery of systemicimidazoles by this route, using the Wistar rat model (8, 108,201). However, translation of these into human therapeutictrials has met with little success (50).
Thrush, or pseudomembranous candidiasis, is the best-known form of mucosal candidiasis and has currently come to
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the forefront due to HIV infection (161). Efforts to produceoral thrush in rats and mice have been successful to varyingdegrees, and the etiology and therapy of this ailment have beenelucidated. For instance, one recent study has shown that su-perficial candidal invasion and initiation of thrush is favored bytopical application of corticosteroids, which dramatically shiftsthe host-parasite relationship in favor of the yeast (47). Al-though it is possible to obtain mice that are deficient in thequality and quantity of CD41 T cells, thus mimicking HIVinfection, surprisingly little work has been performed to ex-plore this intriguing area (31, 32, 46).
Other predisposing factors for oral candidiasis that havecome under scrutiny in a number of animal models includebroad-spectrum antibiotic therapy (5, 82, 155–157), carbohy-drate-rich diets (66, 154, 155), topical use of corticosteroids(47), corticosteroid inhalation (28), trauma (131), iron defi-ciency (147, 185), diabetes (51), xerostomia (1, 83, 84, 119,133), decrease in CD41 T-cell counts and phagocytic function(31, 32), defective T-cell function (16), and immunosuppres-sive therapy (27, 172). Undoubtedly, these studies have helpedus to understand the etiology of oral candidiasis and the de-velopment of the management protocols that are currentlyprevalent.
From a histopathological and diagnostic point of view, mostof the lesions described in animal models have faithfully re-produced human candidal lesions. For instance, Budtz-Jor-gensen and Bertram (30) and Budtz-Jorgensen (26) experi-mentally induced palatal candidiasis in the monkey model,which closely mimicked the nonspecific inflammatory changesof the oral mucosa seen in Candida-associated denture stoma-titis in humans. The palatal smears from the experimentalinfection yielded yeasts that were almost exclusively in thehyphal form, as in Candida-associated denture stomatitis (30,34). Also, a number of studies by others with the Sprague-Dawley rat model of oral candidiasis have detected coloniza-tion patterns and lesions that were similar to human lesionsboth microscopically and histologically (4, 5). Earlier studies byRussell and Jones (156) and Jones et al. (82) using a rat modelalso demonstrated that Candida carriage and infectivity in thisanimal are similar to those in humans. The recently describedmurine acquired immune deficiency syndrome (MAIDS)mouse model (46) is an exciting new development resemblingearly stages of human HIV infection, which could be harnessedto elucidate the pathogenesis of oral candidiasis. Since 10 to15% of candidal hyperplastic lesions progress to dysplasia andoral carcinoma (166) a few workers have attempted to inves-tigate this relationship in animal models (117). Intriguingly, aputative correlation between specific biotypes of Candida andoral dysplasia has been demonstrated in one experiment (93)and yet no further studies have been conducted as a follow-up.
For all these reasons and more, animal models have servedfor more than five decades to illustrate the enigmatic andtempestuous relationship between this opportunist yeastpathogen and its human host. On perusal of the availableliterature, we were unable to find a comprehensive account ofanimal models in oral candidiasis. The following, therefore, isan attempt to review in detail the microbiological, histopatho-logical, and therapeutic approaches and potential caveats per-taining to experimentally induced oral Candida infections in
animal models described in the English language literatureduring the last half century.
EARLY TISSUE CULTURE SYSTEMS ANDHISTOPATHOLOGIC STUDIES
In vitro tissue culture systems derived from nonhumansources were used by a few investigators to study the patho-logical processes in candidal infection much earlier than theintroduction of the in vivo experimental animal models. Par-tridge (136) was the earliest to confront this problem and usedthe chick chorioallantoic membrane to culture pathogenicfungi. Subsequently, Cawson (35) used the same assay to eval-uate the hyperplastic response of the ectoderm to candidalinvasion while Hurley and Stanley (77) experimented with cul-tured mucosal cells from the lingual dorsum of neonatal ratsfor the same purpose. They also assessed the yeast-inducedcytopathic effect and the association between the growth phaseof yeasts and the lethal effect on tissues.
As opposed to these animal systems, cultured human ex-plants and tissues have been used by a few investigators. Pem-berton and Turner (137) used cultured human gingival epithe-lium to investigate C. albicans invasion, while cultured explantsfrom the lingual dorsum were used in ultrastructural studies byMiles (120) and Howlett (75) to compare the invasive potentialof different Candida species. The findings by these workerswere very similar to those for clinical (oral) candidiasis, sup-porting the notion that in vitro cell culture systems were anappropriate model for the study of the disease.
At about the same time, Montes and Wilborn (122) demon-strated in clinical histopathologic studies that Candida pene-trates the human oral epithelium in both acute and chronicphases of the infection and essentially behaves as an intracel-lular parasite. These findings gave impetus for more detailedstudies on oral mucosal invasion of Candida. Subsequent elec-tron microscopic studies by Cawson and Rajasingham (36),with biopsy tissues from patients, also demonstrated clearly theinvasion of the hyperplastic oral epithelium by candidal hy-phae. The results of these investigations were barely adequateto unravel the complexities of the disease process, and animalmodels (e.g., monkeys, rabbits, rats, and mice) have been con-tinually used since then to study the genesis of oral Candidainfections. We review below the advantages and disadvantagesof these animal models and then the experimental details andoutcomes of investigations related to each model.
PROS AND CONS OF CURRENT ANIMAL MODELS
Monkey Model (Macaca irus)
Primates appear to be the ideal animal model for experi-mental Candida infections because of their relatively closekinship to humans. The composition of the oral microflora ofmonkeys, especially M. irus, is both qualitatively and quantita-tively very similar to that of humans (23, 24), and C. albicans isa frequent oral saprophyte in monkeys (23, 152). In addition,monkeys are able to retain acrylic plates resembling dentureprostheses in place, a prerequisite for experimental studies onCandida-associated denture stomatitis. However, primates arerelatively expensive and difficult to maintain, especially forlarge-scale experiments. Some workers have also reported that
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artificial oral infestation of monkeys with Candida is difficultand unreliable (133). Hence, the monkey model has beenlargely replaced by smaller mammals such as rats and mice,which have gained popularity.
Rat Model (Wistar and Sprague-Dawley)
Two species of rats—Sprague-Dawley (SD) and Wistar—have been widely used in experimental oral Candida infections.The two main advantages of the rat model are the low main-tenance cost and the sufficient size of the oral cavity, whicheasily permits inoculation and sample collection. Furthermore,the tongue of this animal is fairly easily colonized by Candida,demonstrating conditions such as median rhomboid glossitisand atrophic candidiasis (2) (Fig. 1).
Candida infections in SD rats can be experimentally inducedwithin a few weeks without traumatizing the mucosal epithe-lium, and a number of investigators claim this model to besatisfactory since it is known to yield consistently reproducibledata (3–5, 54, 55, 82, 155, 156). However, the vast majority ofthese workers (with the exception of perhaps one group) hadto provide antibiotic (e.g., tetracycline)-laced food to initiatethe lesions. The clinical and histologic findings in experimentaldisease in SD rats are similar to those of humans. Clinically,small white patches of “thrush” can be visualized on the kera-tinized lingual mucosa and sometimes on the cheek mucosa.
According to some workers, rats are likely to harbor C.albicans in the oral cavity, albeit to a lesser extent than humansdo (80). However, we were unable to find quantitative esti-mates of candidal colonization of the oral mucosa in wild-typerats. Therefore, one disadvantage of the rat model could bethat the animal may harbor C. albicans as a low-level transientcommensal (130, 206) and therefore the contribution of theinnate immune response to the disease process would be dif-ficult to fathom. However, it could be argued that such naturalprevalance of oral Candida mimicks the human ecosystemsince 30 to 50% of humans carry oral yeasts (166). Workersusing this model for future studies should therefore bear inmind the critical importance of ruling out natural oral coloni-zation by Candida prior to artificial inoculation.
Mouse Model
As opposed to the rat, Candida is not a constituent residentoral microbe of the conventional laboratory mouse (96, 139).This appears to be a major advantage of the experimentalmouse model of oral candidiasis. In addition, since the murinebacterial flora has been well characterized and recognized toconsist of fewer than 20 species, (194), this model permitsevaluation of the role of oral commensal bacteria in initiatingor suppressing candidal infection. Moreover, the immunobiol-ogy of the healthy murine oral mucosa has also been fairly wellcharacterized by a number of workers (48, 49, 94), making itideal for unraveling adaptive immune responses of the mucosaltissues to candidal infection. Furthermore, mice are easily ob-tained in large numbers and their maintenance is cheap. Con-ventional infant mice can be readily colonized by topical inoc-ulation of the oral mucosal surfaces with 108 pelleted C.albicans blastospores per ml (95). Their small size could beconsidered an added advantage as it facilitates routine dailymonitoring, especially when large numbers are used. Never-
theless, the size of the murine oral cavity can also be consid-ered a distinct drawback due to the difficulty in monitoringmucosal changes by naked-eye examination. Hence, someworkers have cultured the tissues or organs of the whole ani-mal to ascertain infestation or infection (15).
Mouse mutants are also extremely useful for experimentalstudies. The sex-linked anemia (sla) mutant, for instance, isideally suited for experimental candidiasis since it shows aconsistent and a prolonged degree of anemia without artificialdietary restriction or bleeding (18, 65). Another mutant, aninbred strain with a metabolic disease resembling diabetesmellitus in humans, has been reported (76). Since uncontrolleddiabetes mellitus is well known to predispose individuals tooral candidiasis, this model could be of potential value inunderstanding diabetes-related oral candidiasis.
Other mutants of this model appear useful for studying theeffect of inherited disorders on the development of oral can-didiasis. For instance, an autosomal recessive mutation respon-sible for severe combined immune deficiency (SCID syn-drome) has been reported in mice (22). SCID is a rarecongenital syndrome of humans that results in loss of both B-and T-cell immunity, and SCID mice are also severely deficientin these lymphocytes. Interestingly, the SCID-hu model hasbeen used to study the infection of human lymphoid cells withHIV-1 a condition which is well known to initiate and aggra-vate mucosal candidiasis (114, 124). Perhaps the SCID mousemodel may be used in future for studying oral candidiasis inHIV infection, together with the very recently describedMAIDS model (see below).
Other immune disorders such as the athymic state, X-linkedB-lymphocyte defects, and candidiasis related to these syn-dromes have been investigated using the mouse model (71,175). A mutant mouse strain called the beige mouse, with alysosomal defect resulting in deficient phagocytosis (60, 151) aswell as deficient NK-cell activity (11, 57, 107, 149), has beenused for additional studies. Indeed, beige mice handled thrush-like lesions less well than their littermates did. These mice, asexpected from their lysosomal defect, which impairs phagocy-tosis, are also susceptible to systemic candidiasis. The forego-ing mouse mutant variants have given us a fresh insight into thehost defense mechanisms operational in superficial forms oforal candidiasis. Further details of oral Candida infections inthese models are provided later in this review.
Hamster Model
Although not as popular as the preceding animal models,the cheek pouch of the hamster has been used by some workersto investigate experimental oral candidiasis (116). McMillanand Cowell, (116) found that a single inoculation of the organ-ism (107 CFU per ml) was adequate to cause infection orinfestation of the hamster cheek pouch mucosa. Artificial liga-tion of the cheek pouch with sutures after Candida inoculationwas also noted to be a simple manouvere to retain the inocu-lum within the cheek pouch. These workers used the lattertechnique to study candidal infection in the hamster cheekpouch after induction of epithelial hyperplasia by turpentine(in liquid paraffin) application. The disadvantages of the ham-ster cheek pouch are its low oxygen tension and the lack of anatural salivary flow, which only poorly mimic the oral milieu.
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Hence, this model, for all intents and purposes is rarely used(118).
In the next section we sequentially review in detail the re-ports in the English literature on experimental oral Candidainfections conducted in five different animal models, namely,monkey, rat (Wistar and SD), mouse, and hamster. Furtherexperimental details of these studies are tabulated in chrono-logical order in Table 1 for ease of reference.
ORAL CANDIDIASIS IN ANIMAL MODELS
Monkey Model
Denture stomatitis is the most common condition whichaffects the palatal mucosa of denture wearers, about 69% ofwhom are infected (29). The main etiologic agent responsiblefor the condition is Candida, which proliferates at the interfacebetween the denture (almost always the upper prosthesis) andthe mucosa (43). Budtz-Jorgensen (26), in an elegant series ofstudies using the monkey model, demonstrated that the palatalinflammatory changes observed in these animals resembledthose of human denture wearers.
Early experiments were conducted by inoculating C. albicansunder custom-made acrylic plates fitted to the palatal surfaceof monkeys (26). Candidal inoculation of control groups ofmonkeys without an acrylic plate, or those fitted with an uni-noculated plate, did not reveal clinical or histologic changes inthe palatal epithelium. However, when C. albicans was inocu-lated under the acrylic appliance, acanthosis and hyperplasia ofthe epithelium, together with a cellular infiltrate of the laminapropria, were noted. A diffuse erythema confined to the mu-cosa in contact with the acrylic plate was also observed, rem-iniscent of Candida-associated denture stomatitis (26, 30). Al-though all animals demonstrated Candida carriage, the yeastload on the palatal mucosa was not quantified by these work-ers. The authors also observed that (i) it was essential to coverthe mucosa to produce the experimental infection and (ii)topical treatment with tetracycline enhanced candidal prolif-eration and the severity of the infection. The primary inflam-matory lesion showed spontaneous healing 2 to 3 weeks afterinfection, clinically and histologically. Interestingly, repeat in-oculation leading to reinfection of the healed mucosa resultedin a more intense erythema in comparison with the primarylesion. These primary and secondary inflammatory responsesappeared to indicate that delayed hypersensitivity may be in-volved in Candida infections of the oral mucosa, although toofew animals were investigated to give statistically valid infor-mation.
In a subsequent study, the same author showed that thenatural healing process after candidal infection can be tempo-rarily suppressed by systemic immunosuppressive therapy withazathioprine (27). Experimental Candida infection of the pal-atal mucosa was induced in 14 adult M. irus monkeys by inoc-ulating C. albicans (serotype A) under acrylic plates. Sevenanimals were given azathioprine, and the remainder acted ascontrols. The control animals demonstrated an atrophic anderythematous epithelium which resolved within 2 to 3 weeks.Cellular hypersensitivity to C. albicans was measured by an invitro leukocyte migration test (27). In the normal animals, themigration inhibition was significant from 1 week to 5 months
after infection. Cellular hypersensitivity developed concomi-tantly with clearing of the infection, while antibody was not yetdetectable as assessed by an agglutination reaction. On theother hand, in azathioprine-treated animals, the infection per-sisted and cellular hypersensitivity did not develop until 1 to 3weeks after the drug treatment was discontinued. The antibodytiter also rose consistently after 4 weeks, reaching a maximumat 8 months. In immunosuppressed monkeys, a depressed mi-gration inhibition reaction, together with a delayed cellularimmune response (up to 2 to 3 weeks), was seen; the humoralimmune response was early and of shorter duration. Thesemonkeys also developed thrush-like lesions on the palatal mu-cosa and a mild inflammatory response in the lamina propria;Candida hyphae were visible in the stratum corneum, as inhuman lesions. This study demonstrated that cellular hyper-sensitivity to Candida plays a critical role in host resistance toexperimentally induced candidiasis.
Oral thrush is relatively common in those using steroid in-halers for asthma and other allergic conditions. To investigatethis condition, Budtz-Jorgensen (28) used monkeys injectedwith the corticosteroid triamcinolone acetonide. Of 13 mon-keys in this study, 6 were injected with the steroid triamcino-lone acetonide intramuscularly 2 weeks before and 2 weeksafter inoculation. In the control group, acute atrophic candi-diasis without hyphal invasion was noted and healed within aperiod of 2 to 3 weeks, while in the steroid-treated group,thrush was seen in all animals together with hyphal invasion ofthe ortho- and parakeratinized epithelium. A depressed in-flammatory response also persisted in 50% of the animals inthe steroid group for 5 to 6 weeks until the plates were re-moved. The inflammatory response was marked under the areacovered by the acrylic plate. These studies confirmed that thelocal environmental conditions such as restriction of salivaryflow due to the acrylic prosthesis, as well as the systemic im-munity, are important in the initiation and aggravation of oralCandida infections.
Another group of workers used the same model but a dif-ferent monkey species, Cercopithecus aethiops, to induce thrushusing maxillary acrylic plates and inoculations of C. albicans(133). They investigated the effect of a reduced salivary flowinduced by systemic oxyphencyclimine chloride on pseudo-membranous lesions under a maxillary plate. They reportedthat monkeys with reduced salivary flow developed largerlesions while reaffirming that the acrylic plate is a prerequisitefor initiation of oral candidiasis.
To conclude, due to reasons such as the purchase and main-tenance cost cited above, the monkey model has fallen intodisfavor. Nonetheless, the pioneering work of Budtz-Jorgensen(26–28) and Olsen and Haanaes (133) using this model wasinstrumental in defining the basic pathological processes in-volved, especially in Candida-associated denture stomatitis. Itis noteworthy that the monkey model served as the “goldstandard” for subsequent animal models—namely the rat, themouse, and the hamster.
Wistar Rat Model
At almost the same time as the experimental studies with themonkey model were being conducted in Scandinavia, Jonesand Adams (78) were investigating an alternative, the rat
VOL. 14, 2001 ORAL CANDIDIASIS IN ANIMAL MODELS 403
on March 28, 2019 by guest
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.org/D
ownloaded from
TA
BL
E1.
Exp
erim
enta
lstu
dies
onor
alca
ndid
iasi
sin
anim
alm
odel
s
Mod
el,y
r(r
efer
ence
)A
utho
rsN
o.of
anim
als/
age/
sex/
avg
wta
Die
taO
ther
cond
ition
saC
andi
dast
rain
,in
ocul
umsi
zea
Len
gth
ofst
udy
Can
dida
carr
iage
aR
emar
ksa
Mon
key
1971
(26)
Bud
tz-J
orge
nsen
6/ad
ult/m
and
f/N
RN
RT
onto
acry
licpl
ate
wee
kly/
biw
eekl
y
CA
type
A(H
asen
clev
er)/
SDA
/48
h/37
°C;i
nocu
lum
,10
0m
g(w
etw
t)of
CA
grow
th
12w
kN
RT
hepa
lata
lcan
dida
linf
ectio
nde
mon
stra
ted
feat
ures
sim
ilar
toch
ange
sob
serv
edin
Can
dida
-indu
ced
dent
ure
stom
atiti
s.T
reat
men
tw
ithT
enha
nced
cand
idal
grow
than
dsu
stai
ned
anin
tens
ein
flam
mat
ory
reac
tion.
1973
(27)
Bud
tz-J
orge
nsen
14/a
dult/
man
df/N
RN
RA
nim
als
wer
etr
eate
dw
ithA
ZA
CA
type
A;i
nocu
lum
:100
mg
(wet
wt)
ofC
Agr
owth
8m
oC
andi
dabl
asto
spor
esan
dhy
phae
obse
rved
Spon
tane
ous
heal
ing
ofth
eat
ropi
cty
peof
cand
idal
infe
ctio
nw
asob
serv
edin
cont
rola
nim
als.
Ani
mal
sim
mun
osup
pres
sed
with
azat
hiop
rine
deve
lope
dth
rush
like
cand
idal
lesi
ons
inth
epa
lata
lmuc
osa.
1975
(28)
Bud
tz-J
orge
nsen
13/a
dult/
man
df/2
.5–5
.4kg
NR
Ani
mal
sw
ere
trea
ted
with
TR
I
CA
type
A(H
asen
clev
er)/
SDA
/48
h/37
°C;i
nocu
lum
:10
0m
g(w
etw
t)of
CA
grow
th
5m
oC
ontr
ol:m
ucos
alsm
ears
show
edC
andi
dain
smal
lnu
mbe
rs;t
est:
larg
enu
mbe
rsof
Can
dida
and
hyph
aew
ere
seen
inm
ucos
alsm
ears
Con
trol
:an
acut
eat
roph
icca
ndid
iasi
sde
velo
ped
inth
egr
oup
ofno
n-st
eroi
d-tr
eate
dm
onke
ys,t
hat
heal
edin
2–5
wk;
test
:an
acut
eps
eudo
mem
bran
eous
cand
idia
sis
was
indu
ced
inth
est
eroi
d-tr
eate
dm
onke
ys,w
hich
heal
edsl
owly
.
1977
(133
)O
lsen
and
Haa
naes
10/N
R/m
and
f/N
RC
MF
OX
Yw
asgi
ven
tosu
ppre
sssa
liva
flow
CA
type
A(H
asen
clev
er)/
SDA
/48
h/37
°C;i
nocu
lum
:10
0m
g(w
etw
t)of
CA
grow
th
14w
kC
ontr
ol:f
ewye
asts
wer
ese
enin
pala
tal-s
mea
rs;
test
:bla
stas
pore
san
dhy
phae
wer
eab
unda
nt
Sust
aine
dde
pres
sion
ofsa
liva
flow
with
ahi
gher
dose
ofth
edr
ugca
used
larg
erth
rush
lesi
ons.
The
lesi
ons
did
not
exte
ndbe
yond
the
max
illar
yac
rylic
plat
es,s
how
ing
the
acry
licpl
ate
isa
prer
equi
site
for
oral
cand
idia
sis.
Wis
tar
rat
1970
(78)
Jone
san
dA
dam
s26
/NR
/man
df/
350
gF
ood
and
wat
erad
libitu
mH
Hin
ject
edC
Aty
peA
/SD
A/4
8h/
37°C
;in
ocul
um:0
.25
mlo
f10
8
cells
/mli
nsa
line
10da
ysC
Afo
und
inth
em
outh
sof
anim
als;
hyos
cine
did
not
chan
geth
efr
eque
ncy
ofre
cove
ryof
CA
50%
ofra
tsha
dle
sion
sth
atre
sem
bled
oral
cand
idia
sis.
Hys
ocin
ead
min
istr
atio
ndi
dno
tpr
oduc
em
easu
rabl
ech
ange
inth
era
teof
infe
ctio
n.
1971
(1)
Ada
ms
and
Jone
s42
/NR
/NR
/NR
NR
CA
type
A/S
DA
/48
h/37
°C;
inoc
ulum
:0.2
5m
lof
108
cells
/mli
nsa
line
6w
kN
R5
of30
rats
show
edhi
stol
ogic
evid
ence
ofca
ndid
alin
fect
ion.
The
Wis
tar
rat
isa
suita
ble
anim
alm
odel
for
the
stud
yof
oral
cand
idia
sis.
404 SAMARANAYAKE AND SAMARANAYAKE CLIN. MICROBIOL. REV.
on March 28, 2019 by guest
http://cmr.asm
.org/D
ownloaded from
1978
(132
)O
lsen
and
Bon
devi
k38
/NR
/m/N
RSR
Pan
dT
-w
ater
CA
from
dent
ure
stom
atiti
spa
tient
/SD
A/4
8h/
37°C
;in
ocul
um:3
0m
g
2w
kN
RA
nim
als
cont
ract
eda
gene
ral-
ized
,sim
ple
type
ofC
andi
dain
fect
ion
ofth
epa
late
.
1981
(179
)Sh
akir
etal
.77
/NR
/m/3
50g
Foo
dan
dw
ater
adlib
itum
CA
3091
/ser
otyp
eA
/SD
A/
36h/
30°C
;ino
culu
m:3
0m
g(w
etw
t)of
CA
susp
ensi
on
6w
kN
RA
nim
als
fitte
dw
ithac
rylic
appl
ianc
esan
din
ocul
ated
with
CA
show
edin
fect
ion
and
infla
mm
atio
n.
1982
(54)
Fis
ker
etal
.50
/5–8
mo/
man
df/2
00–3
70g
SRP
and
T-
wat
erC
AH
asen
clev
erst
rain
A/
SDA
/20
h/35
°C;i
nocu
lum
:0.
1m
lof
63
108
cells
insa
line
6w
k10
0%of
swab
sw
ere
1ve
for
Can
dida
,w
k1;
33%
Ca
1ve
,wk
5
Aco
rrel
atio
nbe
twee
nth
esi
teof
Can
dida
infe
ctio
nan
dth
ear
eas
ofth
eor
alm
ucos
aw
itha
less
dens
ely
kera
tiniz
edsu
rfac
ew
ases
tabl
ishe
d.
1983
(180
)Sh
akir
etal
.35
/NR
/NR
/NR
Foo
dan
dw
ater
adlib
itum
CA
(ser
otyp
esA
and
B),
CT
,CG
2w
kN
RC
Ase
roty
peA
ism
ore
path
ogen
icth
anse
roty
peB
.A
lthou
ghC
Tan
dC
Gco
loni
zed
the
muc
osa,
both
faile
dto
indu
cepa
thol
ogic
alch
ange
sin
the
rat.
1983
(97)
Lam
ban
dM
artin
10/N
R/m
/350
gF
ood
and
wat
erad
libitu
mA
utop
olym
eriz
-in
gac
rylic
plat
esus
ed
CA
3091
18
wk
Can
dida
was
obse
rved
inan
imal
sfit
ted
with
acry
licpl
ates
unsu
pple
men
ted
with
chlo
rhex
idin
e
Ala
rge
num
ber
ofye
asts
and
heav
yin
fect
ion
wer
ese
enin
rats
infe
cted
with
untr
eate
dC
andi
da.N
ogr
owth
was
obse
rved
inra
tsin
ocul
ated
with
yeas
tsbu
ttr
eate
dw
ithch
lorh
exid
ine.
1984
(111
)M
artin
etal
.24
/NR
/m/3
50g
Foo
dan
dw
ater
adlib
itum
CA
(GT
1ve
and
2ve
stra
ins)
,CA
3091
sero
-ty
peA
/SD
A/2
4h/
30°C
;in
ocul
um:3
0m
g(w
etw
t)of
CA
susp
ensi
on
2w
kC
Aw
asre
cove
red
from
alla
nim
als
Ger
mtu
befo
rmat
ion
was
nece
ssar
yto
indu
cepa
lata
lca
ndid
iasi
sin
the
rat.
1985
(126
)N
orri
set
al.
Exp
t1,
26/N
R/m
/32
4g;
expt
2,37
/NR
/m/3
15g
Foo
dan
dw
ater
adlib
itum
Aut
opol
ymer
iz-
ing
acry
licre
sin
was
used
CA
3091
/SD
A/2
4h/
37°C
;in
ocul
um:3
0m
g(w
etw
t)of
CA
susp
ensi
on
Exp
t1,
4w
k;ex
pt2,
8w
k
Swab
sta
ken
from
anim
als
wea
ring
MS-
trea
ted
plat
esw
ere
2ve
for
CA
Exp
t1
(pre
vent
ive
effe
ct):
Rat
sfit
ted
with
anap
plia
nce
supp
lem
ente
dw
ith10
%(w
t/w
t)m
icon
azol
ein
the
poly
mer
pow
der
did
not
deve
lop
pala
tal
cand
idia
sis.
Exp
t2
(cur
ativ
eef
fect
):Pr
evio
usly
infe
cted
anim
als
coul
dbe
cure
dby
fittin
gm
icon
azol
esu
pple
men
ted
appl
ianc
es.
1986
(181
)Sh
akir
etal
.20
/NR
/NR
/NR
Foo
dan
dw
ater
adlib
itum
CA
3091
/ser
otyp
eA
;in
ocul
um:3
0m
g(w
etw
t)of
CA
susp
ensi
on
6w
kC
Aw
asre
cove
red
from
inoc
ulat
edan
imal
s
Ani
mal
sfit
ted
with
acry
licap
plia
nces
and
inoc
ulat
edw
ithC
Ash
owed
infe
ctio
nan
din
flam
mat
ion.
Rem
oval
ofth
eap
plia
nce
reso
lved
the
infe
ctio
nco
mpl
etel
y.R
efitt
ing
the
appl
ianc
een
cour
aged
the
chan
gefr
omco
mm
ensa
lto
path
ogen
icfo
rm.
VOL. 14, 2001 ORAL CANDIDIASIS IN ANIMAL MODELS 405
on March 28, 2019 by guest
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.org/D
ownloaded from
1986
(182
)Sh
akir
etal
.35
/NR
/m/N
RF
ood
and
wat
erad
libitu
mT
hera
tsw
ere
sequ
entia
llyex
pose
dto
12-h
dark
and
12-h
light
peri
ods
tost
imul
ate
diur
nalv
aria
-tio
n
CA
3091
/ser
otyp
eA
;in
ocul
um:3
0m
g(w
etw
t)of
CA
susp
ensi
on
4w
kN
RIn
itial
depr
essi
onin
thic
knes
sof
epith
eliu
man
dre
duct
ion
inm
itotic
activ
ityin
rats
may
bedu
eto
loss
ofbo
dyw
eigh
t.T
hepa
lata
lepi
thel
ium
ofan
imal
sin
ocul
ated
with
CA
and
fitte
dw
ithap
plia
nces
had
asi
gnifi
cant
rise
inth
em
itotic
inde
xan
dth
eth
ickn
ess
ofnu
clea
ted
epith
elia
llay
ers
than
did
that
ofno
rmal
cont
rol
anim
als.
1987
(110
)M
artin
etal
.34
/NR
/m/N
R;
expt
1,18
;exp
t2,
16
SER
Dan
dw
ater
adlib
itum
CA
NC
PF30
91se
roty
peA
/SD
A/4
8h/
37°C
;in
ocul
um:3
0m
g(w
etw
t)of
CA
susp
ensi
on
Exp
t1,
10w
k;ex
pt2,
4w
k
NR
Exp
t1:
The
pala
tala
cryl
icap
plia
nce
and/
orin
fect
ion
affe
cted
the
sele
ctiv
epe
rmea
bilit
yof
the
pala
tal
epith
elia
lbar
rier
.Rem
oval
ofth
epr
osth
esis
resu
ltsin
heal
ing
ofth
eor
alep
ithel
ium
.Exp
t2:
The
pres
ence
ofan
oral
appl
ianc
eco
uld
affe
ctth
eul
tras
truc
tura
lapp
eara
nce
ofth
eep
ithel
ium
.
1987
(51)
Dou
rov
and
Cor
eman
s-Pe
lsen
eer
48/N
R/m
and
f/30
0g
Dia
bete
sm
ellit
usw
asin
duce
din
rats
CA
stra
in40
19;i
nocu
lum
:12
310
6ce
lls/m
lin
H2O
10m
oC
andi
dalc
arri
age
was
1ve
for
diab
etic
rats
afte
rm
ycot
icin
fect
ion
Rat
sw
ithst
rept
ozot
ocin
-indu
ced
diab
etes
mel
litus
wer
ehi
ghly
susc
eptib
leto
Can
dida
infe
ctio
nan
dpr
oved
tobe
afa
vora
ble
mod
elfo
rth
est
udy
oflo
ng-t
erm
oral
cand
idia
sis.
1989
(108
)M
artin
224/
NR
/m/2
30–
290
gN
RF
Lan
dK
Eus
edC
AN
CPF
3091
/SD
A/4
8h/
37°C
;ino
culu
m:3
0m
g(w
etw
t)of
CA
susp
ensi
on
42da
ysC
andi
daw
asre
cove
red
from
rats
trea
ted
with
.7.
0m
gof
KE
kg2
1an
d.
0.5
mg
ofF
Lkg
21
Flu
cona
zole
isef
fect
ive
ata
low
erdo
seth
anke
toco
nazo
lein
reso
lvin
gra
tpa
lata
lca
ndid
iasi
s.
1993
(83)
Jorg
eet
al.
20/N
R/m
/170
–20
0g
Maj
orsa
livar
ygl
ands
surg
ical
lyre
mov
ed
CA
/SD
A/2
4h/
37°C
;in
ocul
um:0
.2m
lof
108
CF
U/m
lin
salin
e
32w
kC
andi
daw
asob
serv
edin
40%
ofco
ntro
lani
mal
san
d10
0%of
xero
stom
ican
imal
s
70%
ofxe
rost
omic
rats
deve
lope
dor
alca
ndid
iasi
s,w
here
ason
ly20
%of
norm
alra
tssh
owed
cand
idal
infe
ctio
n.
1993
(84)
Jorg
eet
al.
12/N
R/m
/170
–20
0g
Foo
dan
dT
-w
ater
adlib
itum
Tre
duce
dto
0.00
1m
g/m
lC
Ais
olat
edfr
ompa
tient
with
chro
nic
oral
cand
idia
sis;
inoc
ulum
:0.2
mlo
f10
8C
FU
/mli
nsa
line
18w
kN
umbe
rsof
CA
isol
ates
wer
esi
gnifi
cant
lyla
rger
insi
aloa
dene
cto-
miz
edra
ts(P
,0.
05)
than
inno
r-m
alco
ntro
ls
NR
406 SAMARANAYAKE AND SAMARANAYAKE CLIN. MICROBIOL. REV.
on March 28, 2019 by guest
http://cmr.asm
.org/D
ownloaded from
Spra
gue-
Daw
ley
rat
1973
(154
)R
usse
llan
dJo
nes
Exp
t1,
60;E
xpt
2,20
CR
D,S
MP,
and
wat
erM
ycel
iala
ndye
ast
form
sw
ere
used
CA
;ino
culu
m:0
.1m
lof
63
108
cells
/mli
nsa
line
Exp
t1,
36da
ys;
expt
2,39
days
Exp
t1:
cand
idal
carr
iage
was
grea
ter
inth
egr
oup
give
nC
RD
Exp
t1:
Infe
ctio
nw
asgr
eate
rin
CR
Dan
imal
sbu
tno
tsi
gnifi
cant
.Exp
t2:
Sim
ilar
resu
ltsto
expt
1.T
his
expt
did
not
perm
ita
com
pari
son
betw
een
the
path
ogen
icity
ofye
ast
and
myc
elia
lpha
ses
ofC
A.
1973
(155
)R
usse
llan
dJo
nes
60/N
R/m
and
f/20
0gC
RD
,SM
P,an
dT
-wat
erC
Ais
olat
edfr
omhu
man
carr
ier/
SDA
/35°
C/4
8h;
inoc
ulum
:0.1
mlo
f6
310
8ce
lls/m
lin
salin
e
34da
ysN
SDbe
twee
nno
rmal
and
CR
Dor
yeas
t/hyp
hal
phas
eC
Ace
lls
Myc
elia
lpen
etra
tion
ofto
ngue
in26
of30
rats
give
nno
rmal
diet
and
19of
30gi
ven
CR
D(b
utP
.0.
05).
1973
(79)
Jone
san
dR
usse
ll36
/12
days
/NR
/N
R/
NR
Myc
elia
land
yeas
tfo
rms
wer
eus
ed
CA
;ino
culu
m:0
.1m
lof
108
cells
/mli
nsa
line
15da
ysR
ats
harb
ored
the
myc
elia
lfor
mw
hen
CA
was
inoc
ulat
ed
Som
era
tsde
mon
stra
ted
hist
olog
icev
iden
ceof
infe
ctio
nw
hen
the
myc
elia
lfor
mof
CA
was
inoc
ulat
ed.
1975
(156
)R
usse
llan
dJo
nes
60/N
R/m
and
f/N
RSR
Dan
dT
-w
ater
CA
;ino
culu
m:0
.1m
lof
63
107
cells
/mli
nsa
line
12m
oM
uch
vari
atio
nin
cand
idal
carr
iage
;th
era
tse
ems
tobe
com
ead
apte
dto
the
pres
ence
ofth
eye
ast
Sign
ifica
ntch
ange
sin
the
tong
uesu
rfac
ean
dth
esu
perfi
cial
laye
rsof
the
lingu
alm
uscl
ew
asse
en.E
pith
elia
laty
pia
note
d,bu
tth
ere
was
nopr
ogre
ssto
carc
inom
a.
1975
(157
)R
usse
llet
al.
120/
NR
/man
df/2
00g
SRD
and
T-
wat
erC
A;i
nocu
lum
:0.1
mlo
f5
310
7ce
lls/m
lin
salin
e22
wk
Initi
alsh
ort-
term
orlo
ng-t
erm
Ttr
eat-
men
tdi
dno
taf
-fe
ctco
loni
zatio
nby
CA
Tre
atm
ent
with
T(s
hort
orlo
ngte
rm)
did
not
affe
cthi
stol
ogic
chan
ges
ofth
era
tto
ngue
due
toC
Ain
fect
ion.
1976
(82)
Jone
set
al.
Exp
t1,
80/N
R/
NR
/80–
100
g;E
xpt
2,39
/NR
/N
R/N
R
Oxo
idir
radi
-at
eddi
et,
vita
min
Kw
ithor
with
-ou
tT
-wat
er
CA
;ino
culu
m:0
.1m
lof
63
108
cells
/mli
nsa
line
Exp
t1,
9w
k;ex
pt2,
27w
k
Ant
ibio
tics
and
GF
stat
efa
vore
dor
alca
ndid
alca
rria
ge
The
GF
stat
efa
vore
din
fect
ivity
toa
sign
ifica
ntle
vel(
P,
0.05
)in
com
pari
son
toco
nven
tiona
lra
ts,T
trea
ted
orno
ntre
ated
.
1982
(6)
Alle
net
al.
10/N
R/f/
200
gSL
Can
dT
-w
ater
CA
/myc
olog
ical
agar
;in
ocul
um:0
.1m
lof
53
107
cells
/mli
nsa
line
40w
kV
aria
tion
inca
n-di
dalc
arri
age
was
evid
ent
with
inra
tsin
apa
rtic
ular
wee
kan
din
suc-
cess
ive
cultu
res
from
apa
rtic
ular
rat
The
lesi
ons
indu
ced
inth
era
tto
ngue
rese
mbl
edhi
stol
ogic
feat
ures
ofhu
man
med
ian
rhom
boid
glos
sitis
.
1982
(55)
Fis
ker
etal
.10
4/6
wk/
man
df/1
20–1
50g
SRP
and
T-
wat
erSP
Fra
tsC
A-H
asen
clev
erst
rain
A;
inoc
ulum
:0.1
mlo
f6
310
8ce
lls/m
lin
salin
e
34w
k50
%of
anim
als
wer
e1
vefo
rC
andi
dadu
ring
34w
k25
%of
ani-
mal
ssh
owed
hy-
phal
pene
trat
ion
15of
60ra
tsde
mon
stra
ted
cand
idal
infe
ctio
n.T
opog
raph
ical
dist
ribu
tions
ofin
fect
ive
foci
wer
esi
mila
rto
resu
ltsob
tain
edin
anea
rlie
rin
vest
igat
ion
(50)
.
VOL. 14, 2001 ORAL CANDIDIASIS IN ANIMAL MODELS 407
on March 28, 2019 by guest
http://cmr.asm
.org/D
ownloaded from
1983
(3)
Alle
nan
dB
eck
50/N
R/f/
200
gSL
Can
dT
-w
ater
Fou
rst
rain
sof
CA
from
clin
ical
lesi
ons;
inoc
ulum
:0.
1m
lof
53
107
cells
/ml
insa
line
25w
kT
hree
ofth
efo
urC
Ast
rain
sw
ere
reco
vere
don
cultu
reof
swab
s
Tw
ost
rain
spr
oduc
edca
ndid
alle
sion
s,w
hile
the
othe
rtw
ow
ere
unab
leto
indu
cein
fect
ion.
Stra
in-r
elat
eddi
ffere
nces
inm
ucos
alpa
thog
enic
ityfo
rth
elin
gual
muc
osa
ofth
era
tw
ere
prop
osed
.
1983
(147
)R
enni
eet
al.
62/N
R/m
/NR
Nor
mal
rat
diet
and
TF
e-fr
eevi
tam
in-r
ich
diet
CA
MR
L31
53/S
DA
/48
h/37
°C;i
nocu
lum
:0.2
mlo
f10
7–1
08C
FU
/mli
nsa
line
14w
kT
was
effe
ctiv
ein
redu
cing
oral
cand
idal
carr
iage
Can
dida
linf
ectio
nw
aspr
omot
edin
rats
rece
ivin
gbo
thC
RD
and
Tco
mpa
red
toan
imal
sre
ceiv
ing
the
diet
ordr
ugal
one.
1985
(66)
Has
san
etal
.12
0/35
days
/man
df/1
00–1
10g
SRP
and
T-
wat
er;C
RD
and
T-w
ater
CA
isol
ated
from
hum
anca
rrie
r/SD
A/3
5°C
/48
h;in
ocul
um:0
.1m
lof
63
108
cells
/mli
nsa
line
14w
kT
and
CR
Den
-ha
nced
cand
idal
carr
iage
rega
rdle
ssof
bein
gin
ocu-
late
don
ceor
onse
vera
locc
asio
ns
Can
dida
linf
ectio
nin
mor
esi
tes
inra
tstr
eate
dw
ithT
and
CR
Dth
anin
rats
give
nT
orC
RD
alon
e.
1985
(5)
Alle
net
al.
40/N
R/f/
200
gSL
C;g
roup
1,T
-wat
er;
grou
p2,
DD
DH
CA
/SD
A/2
3°C
/72
h;in
ocul
um:0
.1m
lof
53
107
cells
/mli
nsa
line
20w
kN
odi
ffere
nce
inca
ndid
alca
rria
gebe
twee
nth
e2
grou
ps
No
sign
ifica
ntdi
ffere
nce
was
note
din
the
num
ber
ofle
sion
sbe
twee
nth
e2
grou
ps.
How
ever
,the
size
ofth
ele
sion
alar
eado
esse
emto
bein
fluen
ced
byT
indr
inki
ngw
ater
.
1986
(abs
trac
t)W
alra
than
dB
lozi
s10
7/N
R/f/
NR
LR
Fan
dT
-w
ater
Foo
dpe
llets
soak
edin
anet
hano
l/cl
otri
maz
ole
solu
tion
and
drie
d
CA
8m
oH
yper
kera
tosi
spr
oduc
edby
CA
can
bere
solv
edby
inco
rpor
atin
gcl
otri
maz
ole
into
the
labo
rato
ryfo
od.H
owev
er,
diffe
rent
stra
ins
ofC
Am
ayre
spon
ddi
ffere
ntly
.
1987
(4)
Alle
nan
dB
eck
320/
NR
/f/15
0–17
5g
SLC
and
T-
wat
erC
Ais
olat
esw
ere
from
diffe
rent
patie
nts
16is
olat
esof
CA
/SD
A/2
3°C
/72
h;in
ocul
um:0
.1m
lof
53
107
cells
/mli
nsa
line
16w
kV
aria
ble
reco
very
rate
sw
ere
note
dfo
rth
e16
isol
ates
Aw
ide
vari
ety
ofcl
inic
albe
havi
ors
wer
ede
mon
stra
ted
for
the
16is
olat
esw
ithre
spec
tto
thei
rab
ility
toin
duce
muc
osal
lesi
ons.
1987
(199
)V
anW
yket
al.
46/5
–8w
k/N
R/
NR
CA
/BH
Ibr
oth/
37°C
/96
h;in
ocul
um:a
bove
susp
en-
sion
in25
0m
lof
drin
king
wat
er(1
06C
FU
/ml)
36da
ysC
andi
daor
gani
sms
appe
ared
inth
eor
alep
ithel
ium
afte
r48
han
dco
loni
zatio
nbe
-ca
me
exte
nsiv
eaf
ter
3–7
days
;ye
asts
wer
eel
imi-
nate
daf
ter
8da
ys
GF
rats
deve
lope
dca
ndid
iasi
sfr
om48
hon
war
ds.T
hele
sion
sw
ere
pron
ounc
edfr
om72
hto
6da
ysan
dth
enre
solv
edaf
ter
day
15.T
hede
velo
pmen
tan
dth
eex
tent
ofle
sion
sva
ried
amon
gth
era
ts.
408 SAMARANAYAKE AND SAMARANAYAKE CLIN. MICROBIOL. REV.
on March 28, 2019 by guest
http://cmr.asm
.org/D
ownloaded from
1988
(8)
Alle
net
al.
60/N
R/f/
200
gSL
Can
dw
ater
Inoc
ulat
edra
tsw
ere
trea
ted
with
KE
CA
;ino
culu
m:0
.1m
lof
53
107
CF
U/m
l27
wk
Eig
htan
imal
s(1
00%
)sh
owed
clin
ical
reso
lutio
nin
the
anti-
myc
otic
-tre
ated
grou
p,an
d2
of9
(22%
)sh
owed
clin
ical
res-
olut
ion
inth
eun
trea
ted
grou
p.T
hus
muc
osal
lesi
ons
indu
ced
byC
Aco
uld
besp
onta
neou
sly
elim
inat
ed,a
ndth
eep
ithel
ial
chan
ges
prod
uced
wer
ere
vers
-ib
lein
som
ean
imal
s.
1989
(7)
Alle
net
al.
210/
NR
/f/17
5g
SLC
and
tap
wat
erSE
Mw
asco
nduc
ted
CA
/SD
A/2
5°C
/48
h;in
ocul
um:0
.1m
lof
33
108
yeas
ts/m
l
20w
kN
RH
isto
logi
cev
alua
tion,
SEM
&cl
inic
alph
otog
raph
sde
mon
-st
rate
dth
ata
sing
leor
alin
ocu-
latio
nw
itha
viru
lent
CA
stra
inw
assu
ffici
ent
topr
oduc
eth
ecl
assi
cep
ithel
ialc
hang
es.M
ost
chan
ges
occu
red
duri
ng2-
3w
ksof
infe
ctio
n.A
fter
18w
ksan
imal
sap
pear
edto
deve
lop
resi
stan
ceto
cand
idal
infe
ctio
n.
1990
(145
)R
eed
etal
.80
/NR
/m/1
50–
250
gR
RD
CA
was
grow
nin
chem
ical
lyde
fined
med
ium
CA
/SD
A/3
7°C
/24
h;in
ocul
um:5
-an
d23
-hC
Acu
lture
supe
rnat
ants
31h
NR
Can
dida
cultu
resu
pern
atan
tsw
hich
cont
ain
unkn
own
fact
ors
may
indu
ceep
ithel
ialp
rolif
era-
tion.
1990
(119
)M
eitn
eret
al.
Exp
t1,
18/2
7da
ys/N
R/N
R;
expt
2,40
/26
days
/NR
/NR
;ex
pt3,
40/2
6da
ys/N
R/N
R
Die
t20
00(5
6%su
cros
e)an
dsu
cros
ein
wat
er
PSG
ligat
ed;
SMan
dSL
glan
dssu
rgic
ally
rem
oved
CA
stra
ins
613
and
623-
ml
(aco
lony
mor
phol
ogy
mut
ant)
3–4
wk
CA
carr
iage
ofH
SRw
ere
30fo
ldm
ore
than
norm
alra
ts
Can
dida
linf
ectio
nw
asin
duce
dw
itha
smal
lcha
lleng
ein
ocu-
lum
.Muc
osal
lesi
ons
deve
l-op
edin
oral
cavi
ties
inH
SRm
uch
fast
erth
anin
the
inta
ctre
cipi
ent
anim
als.
Infe
ctio
nfr
omon
ede
saliv
ated
anim
alto
anot
her
desa
livat
edan
imal
oc-
curr
edra
pidl
y.In
cont
rast
,the
mor
phol
ogic
alm
utan
tto
oklo
nger
totr
ansm
itor
alin
fec-
tion
toun
inoc
ulat
edca
gem
ates
.
1993
(131
)O
’Gra
dyan
dR
eade
63/N
R/f/
NR
SRD
and
T-
wat
erT
raum
aw
asin
duce
dby
appl
ying
heat
onth
eto
ngue
CA
/SD
A;i
nocu
lum
:0.1
ml
of6
310
8ye
asts
/ml
35da
ysN
RT
hede
gree
ofin
fect
ion
was
far
grea
ter
inra
tssu
bjec
ted
totr
aum
aan
din
ocul
atio
nof
CA
than
inth
eco
ntro
lrat
s(t
rau-
ma
with
out
inoc
ulat
ion)
.
1994
(9)
Alle
net
al.
79/N
R/f/
200
gSL
Can
dw
ater
Cyc
losp
orin
was
give
nto
som
era
ts
Tw
ois
olat
esof
CA
(les
ion-
indu
cing
and
non-
lesi
on-
indu
cing
isol
ates
);in
ocul
um:0
.1m
lof
108
yeas
ts/m
l
8w
kT
heno
n-le
sion
-indu
cing
isol
ate
show
edno
sign
ifica
ntin
crea
sein
itsab
ility
topr
oduc
em
uco-
sali
nfec
tion
inth
ese
ttin
gof
redu
ced
host
imm
une
stat
us,i
nco
ntra
stto
the
lesi
on-in
duci
ngis
olat
e,w
hich
dem
onst
rate
da
sign
ifica
ntin
crea
sein
itsab
ility
topr
oduc
ele
sion
s.
VOL. 14, 2001 ORAL CANDIDIASIS IN ANIMAL MODELS 409
on March 28, 2019 by guest
http://cmr.asm
.org/D
ownloaded from
1998
(172
)Sa
mar
anay
ake
etal
.15
/4w
k/m
/200
gC
RD
,SR
P,an
dT
-wat
erR
ats
wer
egi
ven
CY
CA
/2C
Kis
olat
es/S
DA
/37°
C/2
4h;
inoc
ulum
:0.1
mlo
f10
8
yeas
t/ml
29w
kC
Ade
mon
stra
ted
ahi
gher
oral
carr
iage
rate
inco
mpa
riso
nto
the
2C
Kis
olat
es
Und
erno
rmal
cond
ition
s,al
lCA
and
CK
isol
ates
faile
dto
indu
celin
gual
infe
ctio
n.H
ow-
ever
,und
erim
mun
osup
pres
sed
cond
ition
s,C
Apr
oduc
ed10
0%in
fect
ion
whi
letw
oC
Kis
olat
espr
oduc
ed25
and
50%
lingu
alin
fect
ion.
Mou
se19
82(1
85)
Sofa
eret
al.
150/
NR
/m/N
RN
RC
A/M
B/3
7°C
/48
h;In
ocul
um:e
xpt
1,a
drop
ofa
108-C
FU
/mly
east
susp
ensi
on;e
xpt
2,5
310
4C
FU
/mla
dded
todr
inki
ngw
ater
23da
ysE
xpt
1:ap
prox
imat
eco
mpa
riso
nof
CF
Uw
asm
ade;
expt
2:gr
eate
rnu
mbe
rsof
Can
dida
wer
eob
serv
ed
Exp
t1:
No
hist
olog
icev
iden
ceof
Can
dida
infe
ctio
nw
asob
-se
rved
inan
yof
the
rats
.Exp
t2:
Tw
oty
pes
ofC
andi
dain
fec-
tion
wer
eob
serv
ed:(
i)la
rge
num
bers
ofC
andi
dahy
phae
pene
trat
edth
eep
ithel
ium
,and
noas
soci
ated
infla
mm
ator
yre
actio
nw
asno
ted;
(ii)
few
yeas
tsan
dhy
phae
inke
ratin
-iz
edtis
sue
lesi
ons,
and
ade
nse
neut
roph
ille
ukoc
yte
infil
trat
e.
1983
(72)
Hol
broo
ket
al.
80/N
R/N
R/N
RN
RV
irul
ent
stra
in19
321
and
atte
nuat
edst
rain
2211
4;in
ocul
um:1
04C
FU
/ml
adde
dto
drin
king
wat
er
3w
kA
ppro
xim
ate
colo
nyco
unts
wer
eta
ken
The
viru
lent
stra
inco
loni
zed
and
caus
edm
ore
disr
uptio
nof
the
kera
tinan
dal
sode
mon
stra
ted
ahi
gher
infla
mm
ator
yre
spon
seth
anth
eat
tenu
ated
stra
in.
1984
(15)
Bal
ish
etal
.N
R/N
R/N
R/N
RN
RC
AB
311
(typ
eA
)SD
A/
37°C
/24
h;in
ocul
um:1
05
cells
/mli
ndr
inki
ngw
ater
for
2–24
h
GF
mic
ew
ere
colo
nize
dw
ithC
AW
hen
CA
mon
oass
ocia
ted
mic
e(n
u/nu
or1
/nu)
wer
ein
fect
edw
ithC
A,e
xten
sive
muc
osal
infe
ctio
non
the
tong
uean
dch
eeks
was
obse
rved
.
1989
(92)
Kra
use
and
Scha
ffner
NR
/NR
/NR
/NR
Rat
pelle
tsan
dac
idifi
edw
ater
adlib
itum
Cyc
losp
orin
Aw
asgi
ven
CA
#1;
inoc
ulum
:106
cells
insa
line
15da
ysN
RM
acro
scop
icth
rush
like
lesi
ons
deve
lope
dw
ithin
4–6
days
ofin
fect
ion.
1990
(16)
Bal
ish
etal
.N
R/6
–8w
k/N
R/
NR
NR
CA
B31
1ty
peA
/SD
A/3
7°C
/24
h;in
ocul
um:d
ippi
nga
swab
into
anin
ocul
um,
106ce
lls/m
lin
wat
er
24w
kC
Ain
vade
dth
edo
r-sa
lton
gues
ofbo
thnu
/nu
and
nu/1
mic
e;in
fect
ion
pers
iste
dfo
r24
wk
innu
/nu
mic
ebu
tre
solv
edin
nu/1
mic
eby
10w
k
The
nu/n
uan
dnu
/1m
urin
em
odel
sof
cand
idia
sis
desc
ribe
dhe
rem
imic
muc
osal
cand
idia
sis
obse
rved
inpa
tient
sw
ithde
-fe
cts
inT
-cel
l-med
iate
dim
mu-
nity
.
1990
(96)
Lac
asss
eet
al.
NR
/17–
23w
k/m
/N
RN
RC
A/L
ee’s
med
ium
/25°
C/1
8h;
inoc
ulum
size
:50
ml
(108
cells
/mli
nPB
S)
13da
ysC
andi
dare
cove
red
duri
ng1–
7da
yspo
stin
ocul
atio
nfr
omor
alm
ucos
aan
dsa
liva
sam
ples
Prog
ress
ive
cand
idal
infe
ctio
nw
asob
serv
edin
earl
yst
ages
upto
48h.
Dur
ing
the
latt
erst
ages
(7–1
3da
ys),
the
oral
muc
osa
retu
rned
tono
rmal
.
410 SAMARANAYAKE AND SAMARANAYAKE CLIN. MICROBIOL. REV.
on March 28, 2019 by guest
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1991
(32)
Can
tom
aan
dB
alis
hN
R/N
R/N
R/N
RN
RC
AB
311
type
A/S
DA
/37°
C/
24h;
inoc
ulum
:105
cells
/m
lmix
edin
drin
king
wat
er
20w
kY
east
san
dhy
phae
was
obse
rved
onth
eto
ngue
surf
ace
bg/b
gnu
/nu
mic
ew
ere
extr
emel
ysu
scep
tible
toor
alca
ndid
iasi
s1–
4w
kaf
ter
colo
niza
tion,
whi
chpe
rsis
ted
thro
ugho
uta
20-w
kpe
riod
,but
the
infe
ctio
ndi
min
ishe
dov
ertim
e.T
heor
alca
vitie
sof
bg/b
gnu
/1m
ice
beca
me
Can
dida
infe
cted
mai
nly
inw
k1,
but
the
infe
ctio
nw
asqu
ickl
ycl
eare
d.
1993
(95)
Lac
asse
etal
.N
R/1
7–23
wk/
m/
NR
NR
CA
/Lee
’sm
ediu
m/2
5°C
/18
h;in
ocul
um:1
08ce
lls/m
lin
salin
e
33–9
2da
ysF
ollo
win
gpr
imar
yin
ocul
atio
n,C
andi
dash
owed
peak
CF
Uon
days
3–4;
thes
eco
unts
decl
ined
afte
ra
seco
ndin
ocul
atio
n
The
prim
ary
infe
ctio
nre
solv
edun
der
8da
ysof
stim
ulat
ing
cellu
lar
imm
unity
inth
ean
imal
s.A
seco
ndch
alle
nge
inoc
ulum
ofC
andi
da30
days
afte
rpr
imar
ych
alle
nge
faile
dto
prod
uce
ast
rong
reac
tion
inth
eor
alm
ucos
a.
1993
(17)
Bal
ish
etal
.N
R/N
R/N
R/N
RN
RC
Ygi
ven
CA
B31
1ty
peA
/SD
A/3
7°C
/24
h;in
ocul
um:1
05ce
lls/
mlm
ixed
indr
inki
ngw
ater
16w
kN
RC
Yen
hanc
edth
eto
ngue
cand
idia
sis
inSC
IDm
ice.
1994
(37)
Cha
kir
etal
.N
R/8
–10
wk/
m/
NR
C.a
lbic
ans
(LA
M-1
)/IM
DA
;in
ocul
um:1
08ce
lls/m
lin
salin
e
25da
ysA
t5
hpo
stin
ocul
a-tio
n,C
Aw
asse
enin
dige
sted
muc
o-sa
ltis
sue
ofbo
thB
AL
B/c
and
DB
A/2
mic
e;a
carr
ier
stat
eof
the
yeas
tw
asm
ain-
tain
edfo
llow
ing
reso
lutio
nof
can-
dial
infe
ctio
n;st
a-tis
tical
anal
ysis
indi
cate
dth
atth
evi
able
Can
dida
carr
ier
patt
ern
for
DB
A/2
was
sign
if-ic
antly
diffe
rent
from
the
BA
LB
/cpa
tter
non
days
3–6.
The
Can
dida
carr
ier
stat
eis
asso
ciat
edw
ithth
epe
rsis
tenc
eof
intr
aepi
thel
ialC
D41
Tce
lls.
The
clea
ranc
eof
viab
leC
andi
dafr
omm
ucos
altis
sue
isas
soci
ated
with
the
diffe
rent
ial
recr
uitm
ent
ofgd
Tce
lls.
The
reis
evid
ence
that
the
diffe
rent
kine
tics
ofC
andi
dacl
eara
nce
may
invo
lve
the
diffe
rent
ialp
rim
ing
ofT
-cel
lsu
bset
sin
the
two
stra
ins
ofm
ice
that
are
not
asso
ciat
edw
ithth
ehi
stoc
ompa
tibili
tyco
mpl
ex.
1995
(47)
Des
laur
iers
etal
.75
/8–1
0w
k/m
/NR
NR
Top
ical
appl
ica-
tion
ofco
rti-
cost
eroi
d
CA
(LA
M-1
)IM
DM
/27°
C/
48h;
inoc
ulum
:108
cells
/m
lin
salin
e
Can
dida
carr
ier
stat
ew
ases
tabl
ishe
dw
ithin
10da
ysan
dpe
rsis
ted
for
atle
ast
3m
o
NR
VOL. 14, 2001 ORAL CANDIDIASIS IN ANIMAL MODELS 411
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.org/D
ownloaded from
1997
(46)
Des
laur
iers
etal
.6/
adul
t/f/3
–4w
kN
RM
ice
wer
ein
-je
cted
with
the
Du5
H(G
6T
2)
viru
sm
ixtu
reof
mur
ine
leu-
kem
iavi
ruse
sto
indu
ceM
AID
S
C.a
lbic
ans
(LA
M-1
);in
ocul
um:1
08ce
lls/m
lin
salin
e
210
days
Can
dida
carr
ier
stat
ew
ases
tabl
ishe
din
cont
rolm
ice
in,
10da
ysan
dre
-m
aine
dst
able
at,
100
CFU
for
mor
eth
an6
mo;
sim
ilar
colo
niza
tion
patt
erns
was
seen
for
70%
ofre
trov
i-ru
s-in
fect
edm
ice
onda
y10
afte
rC
.alb
ican
sin
ocul
a-tio
n;th
eca
rrie
rst
ate
fluct
uate
din
30%
ofin
fect
edm
ice
from
day
100
post
inoc
ulat
ion,
with
high
leve
lsof
Can
dida
prol
ifera
-tio
nfo
r2–
3-w
kep
isod
es,s
epar
ated
bytr
ansi
ent
reco
v-er
ies
toth
eca
rrie
rst
ate
MA
IDS
synd
rom
esh
ows
man
ysi
mila
ritie
sto
hum
anA
IDS,
alth
ough
the
depl
etio
nof
CD
41ce
llsis
not
obse
rved
inth
isdi
seas
e.It
has
been
view
edas
am
odel
for
the
earl
yst
ages
ofA
IDS.
Ham
ster
1985
(116
)M
cMill
anan
dC
owel
l64
/NR
/NR
/NR
NR
CA
(AT
CC
1026
1),C
A(U
OI)
,CT
(310
0)/M
EA
;in
ocul
um:1
mlo
f10
7
cells
/mli
nw
ater
6w
kN
ohy
phal
inva
sion
ofth
esu
perfi
cial
epith
eliu
m
All
anim
als
trea
ted
with
CA
(UO
I)ex
hibi
ted
visu
alch
ange
sin
som
eor
alll
evel
sof
the
chee
kpo
uch
epith
eliu
m.O
nly
half
ofan
imal
str
eate
dw
ithC
A(A
TC
C10
261)
orC
T(3
100)
show
edch
ange
sin
som
ear
eas
ofth
ech
eek
pouc
h.
1986
(58)
Fra
nklin
and
Mar
tin35
/4–6
wk/
m/N
RF
ood
and
wat
erad
libitu
mE
pith
elia
lhy
perp
lasi
ain
duce
dw
ithT
LP5
0
CA
NC
PF30
91se
roty
peA
/SD
A/3
6h/
37°C
;in
ocul
um:6
0m
gof
aque
ous
susp
ensi
onof
CA
20w
k6
anim
als
reta
ined
CA
for
3w
kC
Aca
used
chan
ges
tohy
perp
last
icep
ithel
ium
whi
chre
sem
bled
thos
ese
enin
Can
dida
leuk
opla
kia.
1992
(117
)M
cMill
anan
dC
owel
l80
/adu
lt/m
/NR
NR
CA
(AT
CC
1026
1),C
A(U
OI)
/ME
A;i
nocu
lum
:1m
lof
107
cells
inw
ater
CA
inth
eye
ast
form
was
seen
scat
tere
don
the
entir
em
ucos
a;no
epith
elia
linv
asio
nby
CA
was
foun
d
Are
asof
hype
rort
hoke
rato
sis
orhy
perp
arak
erat
osis
was
seen
asso
ciat
edw
ithm
icro
absc
esse
s.T
heaf
fect
edar
eas
vari
edin
size
and
appe
ared
toco
rre-
spon
dto
“whi
te”
plaq
ues
seen
gros
sly.
aA
bbre
viat
ions
:aw
,ave
rage
wei
ght;
AZ
A,a
zath
iopr
ine;
BH
I,br
ain
hear
tin
fusi
on;C
A,C
andi
daal
bica
ns;C
G,C
andi
dagl
abra
ta;C
K,C
andi
dakr
usei
;CT
,Can
dida
trop
ical
is;C
MF
,com
mer
cial
mon
key
fodd
er;C
Y,
cycl
opho
spha
mid
e;D
DD
H,
doub
le-d
istil
led
dem
iner
aliz
edw
ater
;E
RC
,E
pol
rat
cube
s;f,
fem
ale;
FL
,flu
cona
zole
;G
F,
germ
free
;G
T,
germ
tube
s;H
H,
hyos
cine
hydr
obro
mid
e;H
SR,
hypo
saliv
ator
yra
ts;
IC,
imm
unoc
ompe
tent
mic
e;IM
DM
,Isc
ove’
sm
odifi
edD
ulbe
cco’
sm
ediu
m;
KE
,ket
ocon
azol
e;L
RF
,lab
orat
ory
rat
food
;m
,mal
e;M
B,m
alt
brot
h;M
S,m
icon
azol
esu
pple
men
ted;
NR
,not
reco
rded
;N
SD,n
osi
gnifi
cant
diffe
renc
e;O
XY
,oxy
phec
yclim
ine;
RR
D,r
outin
era
tdie
t;SD
A,S
abou
raud
’sde
xtro
seag
ar;S
ER
D;S
prot
tsex
pand
edro
dent
diet
,SG
,sal
ivar
ygl
ands
;SL
,sub
lingu
al;S
LC
,sta
ndar
dla
bora
tory
chow
;SM
,sub
man
dibu
lar;
SMP,
stan
dard
mou
sepe
llets
;SPF
,spe
cific
path
ogen
free
;SR
D,s
tand
ard
ratd
iet;
SRP,
stan
dard
ratp
elle
ts;T
,tet
racy
clin
e;T
RI,
tria
mci
nalo
neac
eton
ide;
TSB
,try
ptic
soy
brot
h;T
LP5
0,50
%tu
rpen
tine
and
liqui
dpa
raffi
n.
412 SAMARANAYAKE AND SAMARANAYAKE CLIN. MICROBIOL. REV.
on March 28, 2019 by guest
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.org/D
ownloaded from
model, in the United Kingdom. These workers found that theWistar rat was a simple and less expensive alternative to themonkey model for experimental oral fungal infections. In theirfirst investigation, which was done with 26 Wistar rats andlasted 10 days, Jones and Adams (78) demonstrated asymp-tomatic colonization of the mouths of all the animals andhistologic evidence of candidiasis in some 50% of the ratsorally inoculated with C. albicans. To demonstrate asymptom-atic colonization, they sampled the oral cavities of the rats withsterile paper points (1 cm in length), which were then incu-bated in Sabouraud’s broth for 24 h at 37°C to check forcandidal growth. Histologically, infection occurred on the dor-sal lingual surface, the buccal mucosae, and the free and at-tached gingivae (Fig. 1). Both the histology and the clinicalappearance of the lesions closely resembled acute oral candi-diasis in humans.
The authors subsequently extended these experiments todemonstrate the effect of xerostomia on oral candidiasis bydesalivating the rats with hyoscine hydrobromide (1). A total of42 Wistar rats were subjected to similar experimental condi-tions as before but for an extended period of 6 weeks. Todetermine oral candidal infection, one rat from each of the sixgroups was sacrificed weekly up to 6 weeks. The decapitatedheads were fixed in 10% formol saline and decalcified, andsections were prepared for hematoxylin and eosin and periodicacid-Schiff staining, which showed evidence of candidiasis in 5of 30 rats inoculated with C. albicans. The investigators ob-served epithelial abnormalities such as parakeratosis and thick-ening of the stratum corneum in lesional tissues, indicating thatthe model faithfully mimics the chronic hyperplastic variant ofcandidal infection.
Subsequently, Olsen and Bondevik (132) used the Wistar ratas an alternative model to study Candida-associated denturestomatitis. They used 38 Wistar rats in two experiments, eachwith an observation period of 2 weeks. The rats in the controland test groups were fitted with uninoculated or Candida-inoculated acrylic plates, respectively. After 1 week, a gener-alized simple palatal inflammation similar to that of humanswas seen in the test group, and its histopathology resembledthat of palatal inflammatory lesions in humans.
A similar but more extensive study, conducted by Shakir etal. (179) using 77 male albino Wistar rats, lasted for 6 weeks,in comparison to the 2-week observation period of Olsen andBondevik (132). The results were similar since they observedthat both an acrylic appliance and C. albicans inoculation wereprerequisites for inducing palatal inflammation. The epithelialchanges intensified with the duration of the experimental pe-riod, and after 6 weeks focal areas of the palatal mucosa wereatrophic and markedly hyperplastic with hyphal penetration,resembling the later stages of Candida-associated denture sto-matitis (Newton’s type III) seen in humans. This study alsohelped dispel the theory that trauma alone from ill-fitting den-tures can induce palatal inflammation since the presence ofC. albicans was essential to the induction of inflammatorychanges. Using a similar experimental design, Shakir et al.(179) further observed that C. albicans serotype A is morepathogenic than serotype B in inducing palatal candidiasis.Also, a single strain each of C. tropicalis and C. glabrata failedto induce pathologic changes (180), implying a heirarchy ofvirulence in Candida species. Although this simple experiment
is indicative of the relative pathogenicity of Candida species,more comprehensive animal studies to illustrate this phenom-enon are needed.
In another investigation with the Wistar rat, the same groupobserved that after inducing palatal candidiasis (with C. albi-cans CA 3091 serotype A) by using an acrylic appliance, re-moval of the appliance resulted in complete resolution of thelesion, although Candida still persisted as a commensal for upto 2 weeks (181). Nonetheless, the organisms transformed intothe pathogenic form when the appliance was refitted withoutfurther inoculation. Microbiological sampling was conductedby swabbing the palatal mucosa immediately after killing andobserving the resultant growth on Sabouraud’s agar. To con-firm whether the recovered yeasts were C. albicans 3091 sero-type A, the isolated colonies were serotyped. This experiment,which parallels the clinical experience in denture wearers, con-firms the critical role of the denture in initiating Candida-associated denture stomatitis and the importance of good den-ture hygiene in the management of the disease.
The association between filament formation in yeasts andoral candidiasis is still unclear. The superior virulence of bothforms of Candida, i.e., Candida blastospores and hyphal forms(129), in human tissue has been reported. Germ tube forma-tion, which precedes hyphal growth in C. albicans, is generallyassociated with increased adherence to epithelial cells (89) andresistance to phagocytosis by virtue of their large physical di-mensions. Some studies also suggest that the hyphal structuresare better than the individual yeast cells of Candida at gaininga foothold during the primary invasion process of the host(111, 184). These views have generally led to the belief thatgerm tube and hyphal formation in C. albicans accentuatesdisease induction in humans (127). To investigate this phenom-enon, Martin et al. (111) compared the pathogenic potential oftwo germ tube-negative strains and a single germ tube-positivestrain of C. albicans. When inoculated into three groups of ratsfitted with an appliance covering the palatal mucosa, the germtube-negative strains (MS997 and XTM2) did not producepalatal histologic changes; no changes were observed in ratsnot fitted with an appliance. In contrast, the germ tube-positivestrain (C. albicans 3091 serotype A) elicited a chronic inflam-matory response together with hyphal invasion and epithelialhyperplasia. Nonetheless, in the absence of an appliance, nopathologic changes were noted. These results reinforced thecontention that hyphal formation or filamentation is an impor-tant pathogenic attribute of Candida species (42, 184, 209).
Candida species have a predilection for specific anatomicalsites of the oral cavity. They commonly reside on both thenonkeratinized and keratinized oral mucosae of humans, par-ticularly the lingual dorsum and the buccolingual surfaces,while gingivae are not normally favored. The oral colonizationprofile of Candida was determined by Fisker et al., followingshort-term oral inoculation of C. albicans in Wistar rats on atetracycline-laced diet (54). This experiment revealed prefer-ential C. albicans colonization of four main areas of the oralmucosa. Almost 98.8% of infective foci evidenced by pseudo-hyphal penetration of mucosal epithelium were found on thebuccal mucosa, the buccal and lingual sulci, and the crest of themolar gingivae, and in the interpapillary areas of the dorsum ofthe tongue (Fig. 1). The remaining foci (1.2%) were in themucosa of the hard palate and the attached gingivae. Associ-
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ated ultrastructural studies clearly revealed that the lingualsurfaces which show preferential yeast colonization, particu-larly the interpapillary areas, were characterized by an unevenirregular epithelium with a loosely structured stratum corneum(54). The investigators surmised that the loss of cell cohesionand the abundant intercellular clefts between keratinized cellshaving a microplicated surface facilitated the colonization andinitiation of hyphal penetration (140). The contention that theprofile of oral candidal colonization and hyphal penetration isrelated to the degree of keratinization and/ or surface mor-phology of the mucosa was well supported by these findings.
The Wistar rat palatal candidiasis model has also been usedto evaluate the therapeutic efficacy of topical oral antisepticsand antifungals used to treat this condition. Lamb and Martin(97) incorporated chlorhexidine acetate into an autopolymer-izing resin appliance at a sufficient concentration to preventpalatal candidiasis in the Wistar rat and proposed that theeffect was due to the slow release of the antiseptic. Similarly,Norris et al. (126) examined the therapeutic efficacy of theazole antifungal miconazole incorporated into autopolymeriz-ing acrylic resin and observed that palatal candidiasis could beprevented by fitting Wistar rats with appliances supplementedwith 10% (wt/wt) miconazole in acrylic polymer powder. Theappliances were well tolerated, since the rats remained healthyduring the experimental period, and indeed the rats wearingdrug-laced appliances gained weight more rapidly than didtheir drug-free counterparts. In contrast, in the investigationwith chlorhexidine acetate, the test animals lost weight, prob-ably due to the adverse effect of chlorhexidine (97).
The efficacy of imidazole and triazole antifungals (ketocon-azole and fluconazole) in denture stomatitis has also beenstudied in Wistar rats using palatal acrylic appliances inocu-lated with C. albicans (108). The authors observed that a ke-toconazole dose of 7.0 mg/kg of body weight21 and a flucon-azole dose of 0.75 to 1.0 mg/kg of body weight21 for 14 dayswas necessary to prevent the recrudescence of palatal candidi-asis. Although human trials of drug-laced palatal applianceshave not been conducted to our knowledge, some workershave used this principle and incorporated antifungal agentsinto denture-lining materials with some degree of clinical suc-cess in Candida-associated denture stomatitis patients (50).
The oral mucosa serves as a rugged, impenetrable barrieragainst a multitude of physiological and pathological insults.This primary host defense mechanism is highly effective due tothe prolific and incessant epithelial cell turnover, and it hasbeen postulated that this activity may accelerate under slowlyprogressing chronic disease conditions. However, histologicinvestigations of Candida-associated denture stomatitis pa-tients have revealed that the mitotic activity of the palatalepithelium is similar to that of the healthy palatal mucosa (198,203). Nonetheless, experiments by Shakir et al. (182) withWistar rats indicate that Candida infection results in a signif-icant increase in the mean numbers of mitotic figures per unitlength of basement membrane in the palatal epithelium of theinoculated animals fitted with appliances. Since this increasedepithelial proliferation and desquamation could be considereda protective measure that wards off systemic fungal invasion,Van Mens et al. (198) have suggested that hyperplastic lesionsin Candida-associated denture stomatitis are defense mecha-nisms of the host. Indeed, the exuberant granulomas of chronic
mucocutaneous candidiasis and similar syndromes could beconsidered an extreme evasive reaction of the body to fungalinvasion (129).
Since the presence of an oral prosthesis traumatizes thepalatal epithelium (19, 29), some workers have conductedWistar rat studies to investigate the effect of candidal infectionon the barrier properties and permeability of the palatal epi-thelium (110). They observed that in the healthy rat, the pal-atal epithelial barrier was impermeable to the passage of lan-thanum, whereas in the presence of candidal infection, thepermeability barrier was selectively operational, with a pre-dominant leakage of low-molecular-weight proteins and selec-tive permeability of macromolecules. Furthermore, an elec-tron-dense material was noted throughout the subepithelialtissue. Removal of the prosthesis resulted in healing of theepithelium and a reversal of the barrier properties to its orig-inal state, implying that permeability changes are intimatelyassociated with palatal inflammation in Candida-associateddenture stomatitis. The pathological effects, if any, of the lossof permeability of the diseased human palatal epithelium areunknown.
As stated above, diabetes mellitus is a common disease thatpredisposes to oral candidiasis (160). Dourov and Coremans-Pelseneer (51) conducted experimental studies with streptozo-tocin-treated diabetic rats to investigate the oral candidal car-riage and histopathology induced over a 40-week experimentalperiod. The oral flora was quantified before and after inocu-lation. Tongue swabs were taken and cultured on Sabouraud’sagar for candidal growth. Results were scored according to theyield of CFU. Diabetic rats given a single lingual inoculation ofC. albicans remained positive for the yeast throughout the 40weeks, in contrast to three other control groups, namely, non-diabetic rats inoculated with C. albicans, normal rats, and di-abetic rats without C. albicans inoculation. Moreover, thesecontrols were devoid of the histologic changes seen in the testgroup that were consistent with long-term mycotic lesions ofthe lingual mucosa, such as loss of filiform papillae, paraker-atosis, irregular thickening, and a diffuse lymphocytic infiltra-tion of the deeper layers of the epithelium. Although thismodel appears useful for investigating diabetes-induced oralcandidiasis, no other researcher to our knowledge has ex-ploited in full the etiopathology of this condition using theWistar rat.
Pathologic changes in salivary glands due to diseases such asSjogren’s syndrome, cytotoxic therapy, and irradiation maylead to reduced salivary flow and xerostomia (102, 103, 109,162, 191). Oral candidiasis is a common manifestation of xe-rostomia, which also promotes chronic candidal colonization(101). The relationship between xerostomia and oral candidi-asis was investigated in the monkey model (133), as describedearlier in this review. Jorge et al. (83) used Wistar rats tofurther study this phenomenon. They rendered 20 Wistar ratsxerostomic by surgical removal of the major salivary glands(parotid, sublingual, and submandibular) and orally inoculatedthem with C. albicans three times a week for 32 weeks. Whenthe rats were sacrificed and examined, candidiasis and hyphalinfiltration of the lingual mucosa were found in 70% of thesialoadenectomized animals compared with 20% of the con-trols, confirming the critical importance of saliva and salivaryflow in preventing oral candidiasis.
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The vast majority of workers to date have resorted to theunnatural use of antimicrobials to eradicate the antagonisticpopulation pressure of the commensal oral flora and thus ini-tiate oral candidal colonization (Table 1). Since the broad-spectrum antibiotics used, such as tetracycline, adversely affectthe immune response, a model that obviates the use of anti-microbials is a desirable alternative to mimic the clinical status.Jorge et al. (84) claim that the sialoadenectomized Wistar ratfits this requirement, since they noted 100% oral Candidacarriage in xerostomic rats (after consecutive once-weekly oralinoculation for 5 weeks) compared with 50% carriage in con-trols. Candida was totally eradicated from the latter groupwithin 18 weeks, whereas 66.6% of sialoadenectomized ratscontinued to harbor the yeasts. This model therefore appearssuitable for the investigation of oral candidiasis since it main-tains the normal oral flora with its competitive, colonizationpressure akin to the clinical conditions in humans. However,since no researcher thus far has substantiated the claims ofthese authors, further studies with the sialoadenectomizedWistar rat models are urgently warranted.
Sprague-Dawley Rat Model
As can be seen above, the Wistar rat model has been used bya number of workers to induce experimental oral candidiasis.However another species, the SD rat, has been more exten-sively used by others and appears to be the most popular modelby far for the study of mucosal candidiasis (2–5). Jones andRussell, who pioneered Candida studies with the SD model,demonstrated that animals that succumb to infection showhistologic changes similar to chronic candidiasis of the poste-rior dorsum of the human tongue (80). Furthermore, they havealso shown ultrastructurally that C. albicans changes into themycelial phase and penetrates the cornified layer of the ratlingual epithelium as in humans (81). The following studieswith the SD model have contributed significantly to our un-derstanding of the host-fungus interactions in oral candidiasis.
In early investigations, Bowen and Cornick (25) demon-strated that a carbohydrate-rich diet (CRD) positively encour-ages the oral carriage of C. albicans in SD rats. A number ofworkers have confirmed this finding using both in vitro and invivo studies and have elucidated the role of dietary carbohy-drates in the pathogenesis of oral candidiasis (66, 91, 154, 165).Therefore, Russell and Jones (154) tested the effect of a CRDcontaining 42% powdered icing sugar and 30% starch againstthe “standard 3/8” mouse and rat diet on the oral carriage of C.albicans. In experiments with both the mycelial- and yeast-phase C. albicans, the organism was recovered more frequentlyfrom the CRD-fed animals than from the controls on a normaldiet. It was also observed that oral infection, noted as mycelialpenetration of the superficial epithelial layers, was more fre-quent in CRD-fed animals.
Armed with this information and the effect of broad-spec-trum antibiotics on the genesis of candidal infection, Russelland Jones (155) further studied the effect of both tetracycline-laced drinking water and a CRD on disease progression. Thepersistence of Candida in the mouth of each rat was examinedby swabbing the tongue and mucosal surfaces throughout theexperimental period. The oral yeast carriage was monitoredsemiquantitatively by counting the number of oral swabs pos-
itive for C. albicans (and not by quantifying the yeast growth).Tetracycline administration resulted in oral persistence of C.albicans in all rats over a period of 24 days. The prolongedcarriage induced by a tetracycline-laced diet was far superior tothat achieved by feeding a CRD alone. Furthermore, increasedfrequency and severity of lingual infection were seen in tetra-cycline-fed rats compared with the controls. For instance, incontrast to controls, the tetracycline-fed rats sacrificed on day13 demonstrated lingual, gingival, and buccal mucosal infec-tions. The posterior of the oral cavity was affected more thanthe anterior (Fig. 2), and candidal infection was seen withpseudomembranes, mimicking human oral candidiasis. Histo-logically, mycelia penetrated the orthokeratotic epithelium,which also demonstrated inter- and intracellular edema, andthere was a marked inflammatory cell infiltration of the co-rium. A concomitant increase in the severity of infection wasalso seen with prolongation of the experiment.
The onset of thrush in neonates is usually seen 4 days afterbirth (129), and the predisposing conditions are thought to beimmature immune defenses, antibiotic therapy, maternalcross-infection, and cross-infection from nursery staff (166).Jones and Russell (79) explored the importance of these hostfactors, including infancy, leading to the transition of C. albi-cans from saprophytism to parasitism. When infant (12-day-old) SD rats were inoculated with the yeast form of C. albicans(without tetracycline and a CRD), they were unable to dem-onstrate either candidal carriage or infection. However, inoc-ulations of the mycelial form produced histologically demon-strable infection after 15 days. This study suggests that infancyper se may not be a predisposing factor in the initiation ofcandidal infection and reaffirmed the generally held belief thatthe mycelial form of C. albicans is more pathogenic than theyeast form. Since the authors did not include a control adultgroup of rats in this study, the question of high oral carriage ofCandida due to infancy per se remains unresolved.
After these preliminary experiments, Russell and Jones(156) studied the effect of prolonged candidal inoculation andtetracycline treatment on murine oral infection. This experi-ment is perhaps the most extensive animal study to date, con-ducted over a period of 12 months with 60 rats to identify oralhistologic changes that were wholly due to candidal infection.The oral carriage of C. albicans after fortnightly inoculationand recorded at 1-, 3-, 6-, 9-, and 12-month intervals was 58.6,48.3, 38.3, 40.0, and 45.0% respectively. These results wererather disappointing since the animals were inoculated regu-larly. Nonetheless, the authors recorded in detail the lingualhistologic changes and, after 21 weeks, observed a loss ofpapillae together with flat-surfaced hyper- or parakeratoticstratified squamous epithelium. Changes in the deeper layersincluded a mononuclear cell infiltrate of the corium, degener-ative changes of superficial muscle cells with a giant cell reac-tion, and sarcolemmal proliferation and perivascular inflam-matory infiltrate in deep muscle layers. These observationstended to suggest that the candidal infestation, though re-stricted to the superficial cornified epithelium, may also pro-duce pronounced histologic changes in the deeper corium andthe underlying muscle. This was the first observation in ananimal model that Candida may elicit pathologic effects insubjacent tissues in addition to the immediate viscinity of hy-phal infiltration. Other studies have now confirmed that can-
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didal extracellular enzymes, such as secreted aspartyl protein-ases and phospholipases, may account for such effects (21,144).
Since the administration of tetracycline encourages the oralcarriage of C. albicans in SD rats, it was postulated that agerm-free gnotobiote would be ideal for the study of oralcandidiasis. To test this hypothesis, Jones et al. (82) comparedthe oral carriage of C. albicans in germ-free and conventional(specific-pathogen-free) SD rats with and without tetracyclinetreatment. The mouth and the rectum of the rats were swabbedand the swabs were cultured for Candida in Sabouraud’s agarat the beginning of the experiment, before inoculation, and atregular intervals afterwards, and the number of positive swabswas recorded. The authors observed that the oral cavity of allgerm-free animals, whether treated with tetracycline or not,remained colonized with C. albicans until the end of theexperimental period. In contrast, only 50% of the tetracycline-free, as opposed to 85% of the tetracycline-treated, conven-tional rats harbored C. albicans, reconfirming that the antibi-otic does favor oral yeast carriage (P , 0.05). Infection wasclearly evident in both the germ-free and conventional rats asmycelial penetration of the cornified epithelium, particularlythe dorsal lingual surface.
Further experiments by Jones et al. (82) reconfirmed that (i)germ-free animals can remain colonized for up to 19 weekswith or without receiving tetracycline and (ii) colonization inconventional rats receiving tetracycline is longer lasting than inthose without the antibiotic (P , 0.01). Interestingly, theyfound no evidence of oral infection, as opposed to superficialinfestation, in any of the conventional rats whereas they sawhistologic evidence of infection in gnotobiotes. Contradictoryfindings on infectivity have been reported by others using con-ventional rats (157). The latter group tested the effect of dif-ferent schedules of tetracycline administration in two groups of
SD rats (60 in each group) that were either maintained ontetracycline throughout the experimental period of 22 weeks orgiven the drug only during the first fortnight. All animals wereinoculated with C. albicans orally on three alternate days in thesecond week. The results showed that initial administration oftetracycline fosters long-term oral candidal colonization withno significant difference in the incidence of infection.
Further studies by Hassan et al. (66) have shown that oralcarriage of C. albicans of SD rats rapidly diminished whenanimals were fed a normal diet free of tetracycline and givenonly an initial inoculum of the yeast at the beginning of theexperiment, compared with the following combinations (i)CRD, (ii) normal diet and tetracycline, and (iii) CRD andtetracycline. Significant differences in the recovery of C. albi-cans between the last three groups of rats were maintainedirrespective of whether the inoculum was continuous or givenonly once at the beginning of the experiment.
To conclude, the foregoing studies indicate that a combina-tion of tetracycline treatment and a CRD favor the oral car-riage of C. albicans in SD rats regardless of whether an ade-quate inoculum of the challenge strain is administered once oron several occasions. It should, however, be noted that at leastone group has found that tetracycline exposure is not prereq-uisite for oral Candida colonization provided that the SD ratsare infected with a mucosally virulent strain of C. albicans.Allen et al. (5) studied the development of oral candidiasis intest and control groups of 20 SD rats each, receiving tetracy-cline—laced and drug-free water, respectively. The animalswere all inoculated with a mucosally pathogenic strain of C.albicans that was noted to produce infection in 80% of theanimals in an earlier study (141). There were no significantdifferences in C. albicans carriage rate in the two groups, andafter 20 weeks grossly visible lesions were seen in 50 to 55% ofboth the test and control groups. Nonetheless, the lesions in
FIG. 2. Macroscopic appearance of typical lesions observed on the dorsal surface of SD rat tongue infected with C. albicans after tetracyclineand cyclophosphamide administration. Note the areas of hyperplasia or leukoplakia (arrows) and the conical papillae appearing as a crescent inbetween.
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the tetracycline-treated group were significantly larger thanthose in the controls (P , 0.05). These results suggested thatthe establishment of yeast infection is not necessarily dictatedby antibiotic exposure. However, the degree and severity ofinfection are likely to be related to the synergistic effect oftetracycline and the virulence of the infecting strain.
Van Wyk et al. (199) also investigated the possibility of usinggerm-free SD rats as a model for oral candidiasis. They ob-served that the daily inoculation (106 CFU) of C. albicans indrinking water for 14 days was adequate to infect the oralepithelium of the gnotobiotes sufficiently to produce epithelialchanges such as acanthosis, loss of papillae, and a chronicinflammatory infiltrate of the lamina propria. In a second ex-periment, they investigated the chronological events leading tooral candidiasis in germ-free animals supplied with C. albicans-laced drinking water, over a period of 36 days. Invading yeastswere seen in the superficial lingual, palatal, and cheek epithe-lium within 72 h but were scanty after day 8. The resultinglesions were pronounced from 72 h to 6 days and resolved afterday 15. This implied that host immunity to the invading patho-gen was the major force in eliminating the infection in the SDmodel. They also proposed that the genetic differences amongthe rats may result in variant oral lesions and that geneticallyhomogeneous inbred animals should be used to reproducesimilar lesions.
An animal model that resembles human oral candidiasis is ofvalue not only for studying the pathogenesis of the disease andthe virulence of the organism but also for evaluating newantifungals which are introduced from time to time. The SDrat model has therefore been evaluated for antifungal drugtesting by a few workers. Walrath and Blozis (J. Dent. Res.Spec. Issue, abstr. 975, 1986) produced clearly visible hyper-keratotic tongue lesions in female SD rats using tetracycline-laced drinking water and oral inoculation of C. albicans. (Theauthors observed continuous oral carriage for up to 8 monthsand tongue lesions for 7 months.) The rats were then treatedwith food pellets laced with the antifungal clotrimazole, andthe lingual lesions resolved within 1 week. Hence, the authorssuggested that the SD rat model was a satisfactory in vivo toolto study the effect of antifungals in the management of chronicoral candidiasis.
A study was also designed by Allen et al. (8) to investigatethe effect of ketoconazole on lingual candidal infection in SDrats. Two control and two test groups of animals were orallyinoculated with a C. albicans isolate known to produce mucosallesions. Several animals in the test and control groups devel-oped lingual candidal lesions (9 of 20 and 8 of 20) respectively.All lesions in the test group of animals treated with ketocon-azole resolved, while 2 of 9 animals in the untreated controlgroup also showed spontaneous resolution of lesions. Thesefindings confirmed that the observed leukoplakic lesions wereindeed caused by the Candida inoculum and that the SD modelwas suitable for testing antifungal therapy. However, it shouldbe borne in mind that natural resolution of the lesions iscommon with this model and that appropriate controls need tobe used to obviate spurious results. Despite the availability ofthis satisfactory model, very few workers appeared to haveventured into studies of the efficacy of newer antifungals usingthe SD rat model.
There is a comprehensive body of data on the oral histopa-
thology of Candida infection in SD rats, and these are dis-cussed below. A number of workers have reported that mostcandidal lesions are concentrated on the posterior midlinedorsum of the tongue (6). In studies by Allen et al. (6), hyphaewere present in the parakeratotic layer, together with chronicinflammation of the underlying connective tissues. The authorsspeculated that the topography of the conical papilla regionmight favor the retention of yeasts in the interpapillary crevicesand thus provide them with an increased opportunity to invadethe epithelium. Hence, it would seem that the surface archi-tecture of the mucosa plays a role in selective candidal colo-nization, a view that has been echoed by Fisker et al. (54) andPhilipsen et al. (140). Further investigations were performedby Fisker et al. (55), who subjected SD rats to prolonged oralcandidiasis to localize the infection foci and evaluate the mu-cosal response. Candidal infection confirmed by histologic ex-amination was observed in 15 of 60 animals; the majority of theinfective foci were localized in the buccal sulcular folds, thegingival margin, the cheek, and the interpapillary area of thetongue. These areas accounted for 92.2% of the infective foci,and the remainder were in densely keratinized attached gingi-vae and palatal epithelium. A noteworthy observation was theapparent similarity of the tongue lesions and the histologicfeatures of human median rhomboid glossitis (40, 207). Themucosal response in median rhomboid glossitis comprises aninflammatory reaction without degenerative changes in thesubepithelial tissues.
Other histopathologic features of experimental oral candi-diasis in animal models that have been documented thus farinclude increased epithelial mitotic activity, epithelial prolifer-ation leading to hyperplasia (Fig. 3), and rapid desquamationof the oral mucosa. The factors causing enhanced epithelialproliferation are not clear, although they could involve eitherhost immune mediators or enzymes or metabolites released bythe organism. To determine the impact of the latter attributesof Candida on epithelial cell turnover, Reed et al. (145) in-jected yeast-free culture supernatants into the buccal epithe-lium of young adult SD rats and assessed the mitotic activity byusing a metaphase arrest technique at 11 and 31 h. Theyobserved a significant rise in mitotic activity 31 h after injectionof a 5-h culture supernatant of C. albicans, indicating thatextracellular products of the yeast may induce proliferation ofthe buccal epithelium. Although they postulated that a rangeof candidal products such as hydrolytic enzymes and cell wallpolysaccharides may adversely affect the epithelial and connec-tive tissue cell turnover, they have performed no further ex-periments to substantiate these assertions.
It has been stated that the different incidences of infectionseen in different animal models, and even within the samemodel, could be due to strain variations and the related viru-lent attributes of C. albicans. Hence, studies have been con-ducted to elicit differences in pathogenic traits among C. albi-cans isolates (3). In one study, four groups of SD rats (with 10animals per group) were orally inoculated weekly for 25 weekswith four disparate strains of C. albicans. Of these, oral can-didal carriage of various degrees was seen in three groupswhile the fourth group was completely devoid of infection.Further, it was observed that some strains exhibited consis-tently high colony counts while others invariably produced lowcolony counts. In addition, histologic evidence of infection was
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observed only in two of the three groups exhibiting candidalcarriage and only 4 of 10 (40%) and 2 of 10 (20%) rats in eachsuch group exhibited characteristic candidal lesions of the lin-gual mucosa. Allen and Beck (4) extended their experimentswith 16 strains of C. albicans and an experimental period of 16weeks without tetracycline supplements. They demonstratedsignificant variations in the oral recovery of Candida, rangingfrom 0 to 65%, and intraspecies differences in pathogenicity interms of lingual infection, yielding results consistent with theirearlier study (3).
Allen et al. (7) also observed that a single oral inoculationwith a mucosally virulent strain of C. albicans without the helpof any antibiotics or immunosuppressive agents was adequateto induce dorsal tongue lesions in SD rats. In a study of 210animals sequentially killed over a 20-week period to follow upthe clinical evolution of the lesions, they observed the mostextensive epithelial changes, such as papillary atrophy and thedestruction of dorsal lingual papillae, during the weeks 2 and 3of infection (Fig. 4). Between 4 and 20 weeks, the percentageof animals with clinically evident lesions ranged from 10 to30% although after week 18 all tongue lesions had been re-solved. These observations and the histopathology concurredwell with those seen in human mucosal lesions in candidalinfections while reaffirming that Candida is an opportunisticpathogen easily overcome by innate body defenses. It shouldbe noted that this is one of the few experiments described inthe literature where the authors were able to induce infectionwithout antibiotic or carbohydrate supplements in the food.
The same workers noted the distinct strain-related patternsof C. albicans infections on the dorsal lingual mucosa of im-munocompetent rats (3, 4). They showed that while some iso-lates produced lesions particularly on the posterior-dorsal lin-gual mucosa accompanied by flattening of the normal papillaryarchitecture of the epithelium, another group failed to produceany mucosal lesions. Allen et al. (9) further evaluated a lesion-
inducing isolate and a nonpathogenic isolate by using bothnormal and cyclosporin-immunosuppressed rats. The lesion-inducing isolate showed a significantly increased rate of infec-tion in normal as well as cyclosporin-treated rats comparedwith the nonpathogenic strains.
An association between oral candidiasis and iron deficiencyhas been documented by a number of investigators (34, 56,159). The SD rat model has an added advantage for use instudies of this relationship, since there are several dietarymethods for producing iron deficiency in these rats (10, 112).In one such study, Rennie et al. (147) demonstrated that irondeficiency may not necessarily predispose SD rats to oral can-didiasis since some malnourished animals did not acquire theinfection. Further, they observed a reduced capacity of anemicrats to recover from candidal infection. Similar results havebeen reported by Sofaer et al. (185) using anemic mice (seebelow).
The protective role of saliva in preventing oral candidiasishas been studied previously in the Wistar rat model (83, 84).To further investigate this in SD models, hyposalivatory ratshave been used (119). The latter authors conducted a series ofstudies and observed that all desalivated rats were susceptibleto C. albicans infection and that the oral carriage in the in-fected animals was 30-fold greater than that in the normalcontrol animals (i.e., 3.8 3 105 and 1.1 3 104 CFU, respec-tively) (P , 0.05). The importance of an intact salivary re-sponse in preventing C. albicans infection was clearly shownwhen transmission of infection from one desalivated animal toits counterpart occurred in 1.2 days while transfer from anormal donor to a recipient took 4.3 days. Interestingly, in thehyposalivatory model of oral candidiasis, pretreatment withtetracycline was unnecessary to initiate infection. The authorstherefore concluded that the hyposalivatory-rat model is usefulto assess the infectivity, pathogenesis, and virulence of differ-ent Candida strains in both qualitative and quantitative terms.
FIG. 3. Photomicrograph of a biopsy specimen illustrating the hyperplastic epithelial response to candidal invasion of the rat oral mucosa. Notethe hyphal elements in the superficial layers. Periodic acid-Schiff stain. Magnification, 340.
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A relationship between Candida infection and mucosaltrauma has been addressed by other workers (30, 131, 195).When the role of thermal trauma to the oral mucosa wasinvestigated, it was noted that thermal ulceration facilitated
candidal invasion of the dorsal lingual mucosa (131). Theseresults also suggested that mild, long-term trauma due tochronic irritation from unstable dentures may contribute to theinitiation or aggravation of Candida-associated denture stoma-
FIG. 4. Scanning electron micrographs of a depapillated lingual lesion observed in experimental candidiasis in a rat, resembling erythematouscandidiasis of humans (magnification, 338) (A) and the lesion showing a hyphal element of C. albicans penetrating the epithelium, together witha multitude of commensal bacteria (magnification, 32,400) (B). Photographs provided courtesy of Carl M. Allen, College of Dentistry, Ohio StateUniversity.
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titis. The authors further postulated that the inflammatoryexudate consequential to trauma might enhance the adhesionof the yeasts and thus facilitate infection.
One major host factor preventing fungal infections in gen-eral and oral candidiasis in particular is the cell-mediated limbof the immune system. The number of patients with immuno-logical problems and hence susceptible to candidiasis is in-creasing in the community and includes those undergoing or-gan transplantation, those undergoing cancer therapy, andHIV-infected individuals (38). Recent experimental studies toinvestigate the impact of immunosuppression on mucosal can-didiasis have been carried out by Samaranayake et al. (172),using both C. albicans and C. krusei. They noted that oralcolonization by C. albicans was 12-fold greater than that of C.krusei prior to immunosuppression during an initial experi-mental period of 21 weeks with weekly inoculation of organ-isms. However, none of the animals succumbed to candidalinfection. This was confirmed by histopathologic studies of afew selected animals within each group. However, when theanimals were immunosuppressed with cyclophosphamide, theleukocyte counts of all the animals were significantly depressedand both Candida species produced histopathologic changeson the lingual mucosa characteristic of mucosal candidiasis(Fig. 5). C. albicans produced 100% infection in animals (threeof three), while only 25 to 30% infection was observed with twodifferent C. krusei isolates. Both species produced fungal hy-phae that penetrated the lingual epithelium and stopped shortof the prickle cell layer. However, the C. albicans hyphaepenetrating the lingual mucosa were longer than C. kruseihyphae (17 and 8 mm, respectively) and tended to be relativelymore profuse (Fig. 6). The results of this study substantiatedthat (i) immunosuppresion facilitates candidal infection and(ii) C. krusei is capable of transformation into an invasivepathogen in the setting of immunosuppression. The latter find-ing is consistent with recent clinical epidemiologic data whichindicate a reemergence of C. krusei infections among debili-tated persons (168).
Mouse Model
One of the earliest investigations with the mouse model wasa study by Sofaer et al. (185) to define the role of iron defi-ciency in oral candidiasis. They used the mouse mutant sex-linked anemia (sla) model and conducted two experimentswith normal and anemic mice. In the first experiment, threegroups of mice were tested: (i) untreated mice, (ii) mice re-ceiving Candida by oral inoculation, and (iii) mice receivingCandida by oral inoculation together with tetracycline in thedrinking water. In the second experiment, the effect of com-bined tetracycline and cortisone administration was tested,where one group of animals received hydrocortisone in thedrinking water in addition to receiving Candida and tetracy-cline. At different stages of the experiments, Candida was re-covered by oral swabs and immediately plated on malt agar foryeast growth. A standard technique was used throughout sothat an approximate comparison of colony counts betweendifferent groups of animals could be made. In the first exper-iment, there was no significant difference in Candida isolationbetween normal and anemic mice and no histologic evidence ofcandidal infection. In the second experiment, all mice in the
cortisone group yielded significantly higher Candida counts,indicating enhanced yeast colonization potentiated by hydro-cortisone. Furthermore, hydrocortisone- and tetracycline-treated mice showed increased histologic evidence of lingualinfection.
Corticosteroids are commonly used for their anti-inflamma-tory and immunosuppresive properties. A major side effectassociated with their use is oral and pharyngeal candidiasis(160). Apart from the foregoing study, others have used themouse model to evaluate the effect of corticosteroids on oralcandidiasis. Holbrook et al. (72) determined the colonizationpotential and infectivity of a pathogenic and a nonpathogenicstrain of C. albicans in the mouse model. Two groups of 40male mice (inbred strain CBA/CA) were given chlortetracy-cline and hydrocortisone; one of the groups was orally inocu-lated with a known virulent strain of C. albicans, and the otherwas inoculated with a known attenuated strain. The authorsfound significantly pronounced lingual colonization, disruptionof keratin, and inflammatory response after administration ofa virulent strain compared to an attenuated strain.
More recently, the effect of topical local corticosteroids wasexperimentally demonstrated in the mouse model by Deslau-riers et al. (47). In this study, two groups of mice (34 and 41mice per group) were inoculated with a C. albicans strain thatestablished a low-level, long-term carrier state. Quantificationof the longitudinal yeast carriage in the oral cavity was carriedout from days 1 through 49 postinoculation, using Calgiswabs.The alginate tips were immersed in 2 ml of Ringer’s citratebuffer, and the CFU were enumerated on selective agar me-dium. The carrier state was established in less than 10 days andpersisted for at least 3 months at oscillating recovery levels.Subsequent administration of topical corticosteroids resultedin increased carriage of up to 40-fold higher than the controls;by day 21, this rose to 400-fold. The carrier state was restoredto normal 10 to 15 days after cessation of drug administration.Immunological investigations revealed three- to fourfoldgreater persistence of intraepithelial CD41 T cells in infectedanimals than in control animals. However, in animals treatedwith the topical corticosteroid, these cells virtually disappearedfrom the epithelium. Within 24 h after cessation of treatment,CD41 T cells were massively recruited (7 to 10 times thenumber seen in control carrier mice), first in the subepithelialcapillary beds and then in the epithelium. The authors alsonoted that a significant reduction of the local CD41 T cellslevel paralleled an increase in the oral carriage of C. albicans.This elegant study with an animal model clearly demonstratesthe critical relationship between cellular immunity and oralcandidiasis—a relationship all too frequent in HIV infection.
The mouse model has also been used to observe candidalcolonization patterns and the inflammatory response inchronic recurrent candidal infection. When a group of micewas orally challenged with topical application of a C. albicansstrain isolated from a patient with systemic candidiasis, theyeast population plateaued to a constant titer (approximately300 6 100 CFU per g of excised mucosal tissue) after 7 dayspostinoculation (95). However, this primary infection stimu-lated the cellular immunity, and a secondary topical challenge30 to 43 days later failed to produce a mucosal reaction com-parable to the first. The authors also observed a 10-fold dif-ference between colony counts 24 h after the first and second
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inoculations. These results confirmed the findings of a numberof previous studies (55, 82, 96) on the ability of mice and otheranimals to acquire resistance to the pathogen, which led toonly a transient carrier state. This response in healthy animalshas frustrated the attempts of many workers to produce afaithful model reproducing chronic oral candidiasis. Indeed,debilitation of the animal via artificial means (e.g., drugs andradiation) is essential to elicit such a response in normal inbredmice.
In another study, normal adult CD-1 mice were orally inoc-ulated with C. albicans (108 cells/ml in sterile phosphate-buff-ered saline) or by topical application. The yeast could be re-covered from both the digested oral mucosa and saliva samples
for up to a week. Although maximal colonization was noted at48 h, the candidal infection greatly decreased after 2 to 3 days.Histologically, yeasts were seen attached to the oral epithelium3 h after inoculation and hyphal penetration reached a maxi-mum around 48 h. After 2 days of microbial challenge, a stronginflammatory reaction characterized by polymorphonuclear in-filtrates was seen in the epithelium, together with parakerato-sis. The infection involved a large area of the cheek mucosaand sometimes reached the muscle layer. However, the oralmucosa returned to normal after 7 to 13 days, indicating aself-limiting infection. The most important observation wasthat oral Candida colonization could be induced in normaladult mice without the aid of compromising agents, as previ-
FIG. 5. (A) Histopathologic section of leukoplakia on the dorsal tongue of an SD rat infected with C. albicans. Note the loss of filiform papillaeand the flat-surfaced, parakeratotic, edematous and acanthotic lingual epithelium. (B) Photomicrograph of the lingual mucosa from the posteriordorsal tongue of an SD rat demonstrating uninfected epithelium. Note the normal filiform papillae covered by a layer of thick acellularorthokeratin. Hematoxylin and eosin stain. Magnifications, 3200.
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ously shown by a few studies with Wistar and SD rat models (5,82). Further, the microbiological and histopathologic data sug-gested that the mouse model is suitable to unravel the adaptiveimmune responses of the oral immune system. However, it isdisappointing that none of these models mimics the humanoral carriage of Candida, where chronic carrier states persist inup to 40 to 50% of healthy adults. Thus, an animal modelwhich resembles human oral candidal carriage is needed tofurther clarify the host-parasite interactions in oral candidiasis.
Since a defective cellular immune response is a precursor ofmucosal candidiasis, Balish et al. (15, 16) conducted extensiveinvestigations of this aspect of the disease using the mousemodel. For this purpose, they used adult athymic and euthymicnude mice bearing nu/nu and nu/1 genotypes, respectively.When C. albicans was given in the drinking water (105 cells/perml), the yeasts colonized both groups of mice in large numberswith minimal hyphal invasion of the oral mucosa. Scanningelectron microscopy revealed superficial yeast infestation ofthe ventral surface of the tongue and the cheek mucosa with novisible candidal infection. Therefore, these investigators con-cluded that both genotypes of the mice manifested resistanceto extensive mucocutaneous candidiasis and that thymus-ma-tured T cells may not be obligatory for resisting mucosal can-didiasis in this particular murine model (15).
They extended this experiment with mice (nu/nu or nu/1)that were intraperitoneally injected with cyclophosphamideand noted extensive infection and hyphal penetration of thetongue and cheek mucosa within 5 to 7 days. Hence, it appearsthat nu/nu or nu/1 mice that are artificially debilitated mayserve as a model that can be manipulated to study the role ofnutrition, endocrine function, and immunosuppression in mu-cosal candidiasis.
Balish et al. (16) also used athymic and euthymic gnotobioticmice to investigate the immune response to Candida in theabsence of colonization pressure by oral bacteria. In one studywhere adult mice were orally inoculated with C. albicans, suchhyphal penetration of the dorsal lingual surface was seen after14 days in both nu/nu and nu/1 gnotobiotes and remained forperiod of 24 weeks in the nu/nu mice, while nu/1 mice clearedcandidal hyphae within 10 weeks. Furthermore, spleen cellsfrom nu/1 mice showed a positive in vitro lymphocyte prolif-erative response from 3 to 22 weeks after colonization andinfection with C. albicans while nu/nu mice could not mountsuch a blastogenic response. This study with the gnotobioticmouse model indicated that although nu/nu and nu/1 mice areboth apparently equally susceptible to oral candidiasis, theformer cannot mount a lymphoproliferative response to Can-dida. This result is not surprising since nu/nu mice lack func-
FIG. 6. Histopathologic sections of depapillated areas on the dorsal tongue surface of two SD rats, showing epithelial invasion by C. albicans(5.00 to 17.71 mm) (A) and C. krusei (5.01 to 8.34 mm) (B) hyphal elements. Periodic acid-Schiff stain. Magnifications, 3400. Note the longer hyphalelements of C. albicans compared with C. krusei.
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tional T cells. However, the authors clearly demonstrated thatthe nu/nu and nu/1 murine models could serve to mimic mu-cosal candidiasis observed in individuals with defective T-cellfunction.
Mucosal candidiasis is also the commonest opportunisticinfection in HIV-infected persons, and a decrease in the CD41
T-cell count is a hallmark of this disease (45, 59, 135). It is alsoknown that patients with defective phagocytic function oftenbecome infected with C. albicans (90, 202). A mouse modelthat is claimed to exemplify both these conditions was intro-duced by Cantorna and Balish (31). This, the congenitallyimmunodeficient germfree mouse model (bg/bg nu/nu mouse),appears to be naturally susceptible to oral (hard palate andtongue) and esophageal candidiasis even without supplementsof antibiotics, immunosuppressives, or cytotoxic drugs. Can-torna and Balish (31) investigated the ability of athymic (nu/nu), euthymic (nu/1), beige (bg/bg), black (bg/1), beige athy-mic (bg/bg nu/nu), and beige euthymic (bg/bg nu/1) germ-freemice to contract systemic as well as superficial candidiasis.Adult germ-free mice were orally inoculated by administeringC. albicans-laced drinking water (105 yeasts per ml). On his-topathologic testing, the hard palate and esophagus of theimmunocompetent mice and the singly immunodeficient micedid not show signs of infection, although small numbers of C.albicans organisms colonizing the outer keratinized layers ofthe tongue were observed. However, yeasts and hyphae wereseen in large numbers attached to and penetrating the kera-tinized portions of the tongue and hard palate of bg/bg nu/numice. Interestingly, colonization with C. albicans was accom-panied by death in these doubly immunodeficient (bg/bg nu/nu)mice (22 of 73) within 4 weeks. The dead mice had macro-scopic, plaquelike hyperkeratotic lesions on the dorsal lingualsurface and the hard palate. As expected, little inflammationwas observed in the infected tissues of bg/bg nu/nu mice whichsurvived C. albicans colonization, and they had fewer yeast andhyphae on the palate, tongue, and esophageal surfaces as wellas less hyperkeratosis. This shows that although bg/bg nu/numice were susceptible to mucosal candidiasis, a proportionwere able to mount an inflammatory response, primarily ofpolymorphonuclear leukocytic origin.
Cantorna and Balish (32) extended their immunologicalstudies by using bg/bg nu/nu and bg/bg nu/1 mice to evaluatethe role of CD41 T cells in mucosal candidiasis. When themice were orally inoculated with C. albicans (B311 type A)-laced drinking water, bg/bg nu/nu mice were highly susceptibleto oral (mainly lingual) and esophagal candidiasis during thefirst 4 weeks of the experiment, and this susceptibility dimin-ished over a period of 20 weeks. In contrast, although the bg/bgnu/1 mice showed oral infection during the first week, theyeast was cleared promptly, since no infected tissue was ob-served in mice sacrificed after 2 and 4 weeks into the experi-ment. This clearly indicated that the CD41 T cells play acritical role in protecting against mucosal candidiasis.
SCID mice have provided valuable information on the rolesof both the B and T cells in mucosal candidiasis (15, 16, 32).For this purpose, Balish et al. (17) used adult immunodeficientSCID mice that were orally inoculated with C. albicans (105
cells/ml)-laced drinking water. Although the mice were chron-ically colonized with large populations of yeast cells (106 to108/g), no oral mucosal lesions were apparent. Thus, in the
absence of T- and B-cell function, SCID mice seem to com-pensate by using innate mechanisms such as phagocytic cellfunction to resist extensive mucosal candidiasis. Nevertheless,when SCID mice were given cyclophosphamide (100 mg per kgof body weight at 11 weeks after oral colonization with C.albicans), they showed more severe lingual candidiasis thandid saline-injected controls. Thus, cyclophosphamide, whichcauses severe neutropenia and further impairment of innateimmune mechanisms, enhanced the susceptibility of SCIDmice to mucosal candidiasis. Future workers studying innateand acquired immunity in C. albicans infection could usefullyemploy the gnotobiotic SCID mouse model as a tool to exploreimmune events involved in oral candidiasis, especially thoseassociated with immunosuppressive therapy.
It is known that the carrier state of Candida is associatedwith an intrinsic cellular immune response, as described above.To further demonstrate the association of different T-cell sub-sets and the histocompatibility complex (H-2) in Candida in-fection, Chakir et al. (37) conducted studies with DBA/2 andBALB/c mice. These two strains of mice with the same majorhistocompatibility complex haplotype (H-2d) were inoculatedby topical application of pelletted blastospores with C. albicans(LAM-1). Oral Candida populations were established in allmice postinoculation. Both DBA/2 and BALB/c mice demon-strated a peak of infection and a residual Candida populationduring the early period of the experiment. The infection was ofshorter duration in BALB/c mice than in DBA/2 mice. Fur-thermore, in the latter, the infection was sustained, with areproducible second peak of proliferation on day 5 postinocu-lation, after which the Candida counts dropped sharply (a60-fold-lower residual level) but were present for at least 25days. The comparative kinetics of the oral Candida infection inDBA/2 mice was significantly different from that in BALB/cmice on days 3, 4, 5, and 6 after yeast inoculation.
These workers also quantified Candida organisms in di-gested cheek, palatal and lingual mucosal tissues. Analysis ofthese data revealed that the DBA/2 and BALB/c patterns weresignificantly different from days 3 through 6 postinoculation.Furthermore, the cheek and palatal mucosal tissues of infectedmice showed that a first peak of proliferation occurred on day2 at this site whereas it occurred on day 3 on the tongue. Asimilar recruitment of CD41 and CD81 T cells and of MAC-1cells in mucosal tissue of both strains of mice was noted duringcandidal infection. The carrier state of Candida was associatedwith the persistence of intraepithelial CD41 T cells. However,there was a time-specific recruitment of gd T cells that coin-cided with a dramatic decrease in viable Candida organisms inthe mucosal tissue, which occurs on day 3 in BALB/c mice andon day 6 in DBA/2 mice. Taken together, these results indicatethat the two strains of mice sharing the same major histocom-patibility complex (H-2d) display different kinetics of Candidaclearance and primary oral infection after topical applicationof a standardized inoculum. Further, it appears that the prim-ing modalities of T cell subsets in the oral mucosa are notassociated with the H-2 complex in the mouse model.
Oral candidiasis is a criterion in most, if not all, stagingsystems for HIV infection (12, 34, 51). HIV infection leadsslowly but inexorably to a loss of immune competence, themost striking feature of which is a depletion of CD41 T cells(23, 45). Mice infected with the Du5H(G6T2) mixture of
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mouse leukemia viruses develop a syndrome, MAIDS, that hasmany of the immune abnormalities found in HIV infection.Using this model, Deslauriers et al. (46) studied retrovirus-infected C57BL/6 mice and examined them for their ability toresist the development of oral candidiasis, as indicated by de-velopment of the carrier state after a self-limiting acute infec-tion, and to clear a subsequent, secondary oral inoculum of C.albicans. After oral inoculation with Candida, the carrier statewas established in both control and test mice in less than 10days and remained stable at ,100 CFU for more than 6months in both the control and 70% of virus-infected animals.The carrier state fluctuated in the remainder (30%) of theretrovirus-infected mice especially from day 100 after virusinoculation, sometimes reaching acute-infection values and re-maining at these high levels for 2- to 3-week periods inter-spersed by episodic transient returns to the carrier state, rem-iniscent of recurrent oral candidiasis in human HIV infection.The isolation frequencies of CD41 and CD81 lymphocyteswere unchanged and significantly decreased (P , 0.05), respec-tively, in both the cervical lymph nodes and spleens of coin-fected mice compared with the C. albicans-carrying, virus-free,age-matched control animals. Furthermore, the secretion ofgamma interferon by concanavalin A-stimulated spleen cellsfrom retrovirus-infected mice was significantly decreased (P ,0.05) compared to that from virus-free mice, in parallel withknown abnormalities associated with MAIDS. These data areconsistent with a role for Th1 cells in host resistance to muco-sal candidiasis but suggest that CD81 and/or gd T cells, or NKcells, may also contribute either through the production ofgamma interferon (15, 24, 55) or through the recently de-scribed direct antifungal activity of CD81 cells against C. al-bicans (39).
Although not directly relevant to oral candidiasis, a modelwhich demonstrates thrushlike candidal lesions has been de-scribed in artificial pneumatized cysts in mice (92). To producethese candidal lesions, subcutaneous cysts were formed by in-jecting 3 to 3.5 ml of air through a hypodermic needle into thesubcutis of the back of a mouse. The cysts, which were lined byan epitheliod cell layer of mesenchymal origin within a 5- to7-day period, were challenged with 106 cells/ml of yeast sus-pension in saline. The mice were immunosuppressed with cy-closporin A, which selectively impairs T-cell immunity andNK-cell activity without affecting nonspecific phagocytic activ-ity. Mice thus immunosuppressed developed distinct, macro-scopic thrushlike lesions in the cysts within 4 to 6 days, while nolesions were visible in control animals. These experiments havenot been substantiated, and it is doubtful whether they providea useful alternative to the less cumbersome models describedabove.
The foregoing illustrates the multifaceted exploitation of themouse model by various researchers to elucidate clinical, ther-apeutic, and immunological features of oral candidiasis. Takentogether, the many variants of the mouse model appear espe-cially suited to study the short-term yet complex humoral andcellular immune responses associated with the disease. Therecently described MAIDS mouse model, in particular, is wor-thy of special mention since it offers promising scope for work-ers interested in the immunobiology of oral candidiasis in HIVinfection.
Hamster Model
The cheek pouch of hamsters has been a favorite site forstudies of oral carcinomas. Hence, a few investigators haveexperimented with this model to evaluate oral Candida infec-tion but without much success. McMillan and Cowell (116)carried out an investigation by inoculating Candida into thecheek pouch of 64 adult hamsters. A single strain each of C.albicans and C. tropicalis were used. While only one-third ofthe animals treated with either organism showed pathologiclesions in the pouch mucosa, microabscesses reminiscent ofcandidal infection were found only in animals treated with C.albicans. Other histopathologic changes included inflamed ep-ithelium with neutrophils, and a lymphocyte and macrophageinfiltrate in the connective tissue. Epithelial thickening withincreased thickness of the stratum corneum and random dis-tributions of yeast and hyphae were also noted, with no hyphalinvasion.
Chronic hyperplastic candidiasis is characterized by invasionof a thickened epithelium by candidal hyphae. However, it isnot clear whether the hyperplasia is caused by C. albicans orwhether the organism invades an already hyperplastic epithe-lium (146). To investigate this rather enigmatic phenomenonFranklin and Martin (58) induced epithelial hyperplasia in thehamster cheek pouch mucosa by application of 50% (vol/vol)turpentine in liquid paraffin. Afterward, the pouches were in-oculated with C. albicans and the inoculum was retained bysutures. In six animals which satisfactorily retained Candida forup to 1 month, the investigators observed increased mitoticactivity and both a hyperplastic and an atrophic epithelium.The pathologic features of the hyperplastic epithelium resem-bled human candidal leukoplakia. These preliminary resultsindicate that C. albicans is a prime agonist in initiating hyper-plastic epithelial changes associated with candidal leukoplakia.
This model was once again used by McMillan and Cowell(117) in the hope of clarifying the role of C. albicans in leu-koplakia, dysplasia, and neoplasia. They inoculated the cheekpouch of 80 hamsters with C. albicans (either CA UOI or CAATCC 10261) once a week for 9 months. During this pro-longed period of inoculation, hamsters were sacrificed atmonthly intervals and examined for abnormalities. Althoughthe histopathologic changes were similar at all ages, the num-ber of abnormal foci was greater in hamsters inoculated for aminimum of 6 months. These changes were said to be similarto those observed by the same investigators (over a 6-weekperiod after a single inoculation of C. albicans) in the cheekpouch of adult hamsters (116). However, a heavy chronic in-flammatory response was more extensive than in the lattershort-term study. Since the majority of candidal organismswere in the yeast form, it was apparent that hyphal penetrationinto the epithelium was not essential for the induction ofpathologic changes observed in chronic hyperplastic candidia-sis. Since invasion of the epithelium is probably an importantcontributory factor for dysplasia, these studies should be fol-lowed up over a much longer period to determine if epithelialhyperplasia does indeed lead to oral malignancy.
CONCLUSIONS AND FUTURE DIRECTIONS
The diverse attributes of Candida species as opportunisticpathogens and the multiple oral diseases they cause are well
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recognized. Therefore, carefully designed animal studies arecritical to provide new insights into the complex biological andpathologic behavior of C. albicans, as well as non-albicansspecies of Candida. Further, a number of Candida species areeither emerging or reemerging as agents of increasing morbid-ity and mortality in compromised patient cohorts. This repre-sents another important arena in which animal models cancontribute to unraveling the pathogenic mechanisms of thisubiquitous opportunist.
A number of animal models have been described to studyoral candidiasis over the past half a century or so. These in-clude the monkey, rat, mouse, and hamster models and theirvariants. Most of these studies have been performed underdifferent experimental conditions, as a result of which the out-comes are difficult to compare. Other variables that confounddata interpretation include differences in the numbers of ani-mals, the Candida strains used, and the duration of the studies.In the foregoing review, we have attempted to evaluate theanimal models (that are described in the English languageliterature) in order to objectively redefine the criteria for theiruse.
Most animal studies of experimental oral candidiasis havebeen conducted following alteration or manipulation of theoral environment by administration of antibiotics, special diets,or mechanical trauma, exclusion of the protective effect of anormal salivary flow, or accumulation of unwanted metabolitesunder an artificially inserted appliance. Furthermore, germ-free gnotobiotes have been used by some workers, since it hasbeen difficult to study the interactions between the artificiallyintroduced exogenous yeasts and the host in the presence ofthe population pressure exerted by commensal bacteria. A casehas also been made for the use of inbred animals since outbredanimals are genetically heterogeneous.
On the pathogen level, it has been evident that differentstrains show different potentials to cause infection. Althoughthis strain disparity in the pathogenic attributes of Candida,even within the same species, is well established, only a fewinvestigators have attempted to use a reference or a uniformcontrol strain of Candida between experiments to make theresults from different centers or experiments globally compa-rable. With the advances in genomics, molecular typing meth-ods have become the most precise method of identifying ayeast isolate. Therefore, the use of one or more genotypedreference Candida strains in future animal studies wouldgreatly facilitate the comparison of global data from differentgroups of researchers or different experiments within the sameresearch group.
Notwithstanding these drawbacks of the studies describedabove, the following could be deciphered from the availabledata. (i) The primate model appears ideal for experimentalinvestigations of Candida-associated denture stomatitis, mainlydue to the need for fabrication of a close-fitting acrylic appli-ance analogous to human denture prostheses. Both erythem-atous candidiasis and pseudomembranous candidiasis havebeen produced in monkeys fitted with such acrylic plates. How-ever, monkeys are difficult to obtain and expensive to maintain,and hence their use is highly resource sensitive. (ii) The ratmodel (both SD and Wistar) is the most well proven for ob-serving clinical oral candidiasis in vivo, especially because of itsrelatively large oral compartment, ease of breeding and han-
dling, and ready availability. Consequently, rats are the animalsof choice for studying long-term candidal colonization andchronic infection. (iii) The mouse model and its variants withimmune abnormalities are undoubtedly suited to evaluate hu-moral and cellular immune response in oral candidiasis. Manywell-characterized variants of the mouse model with baselineinformation on their immunologic and genetic constitution areavailable, including gnotobiotes, athymic (nu/nu), euthymic(nu/1), beige (bg/bg), black (bg/1), beige athymic (bg/bg nu/nu), beige euthymic (bg/bg nu/1), and SCID mice, as well asthe recently developed MAIDS model. Furthermore, mice arealso widely available, easy to handle, and relatively inexpen-sive. Hence, it is not surprising that the mouse model hasproved popular, especially for short-term studies of the hu-moral and cellular immune response in oral candidiasis. (iv)The hamster cheek pouch epithelium, although used as amodel for oral infection, is not a good surrogate for the humanoral mucosa due to the absence of a salivary component, theartificiality of tying off cheek pouches to initiate the lesion, andthe unique oral anatomy.
A number of other general conclusions can be drawn fromthese studies. (i) In almost all models, the infection could beinitiated with antibiotic and/ or dietary supplements. (ii) Theyeasts preferentially colonize different sites of the oral cavity(dorsal surface of tongue, buccal mucosa, and gingival muco-sa), but the mid-posterior lingual dorsum appears to be themost favored site of infestation, as in humans. (iii) There isinter- and intraspecies variations in the infectivity of Candidain animal models, although this area has been very poorlyresearched. (iv) The histopathologic changes of the mucosa arehighly consistent with those of human lesions. Nonetheless, anideal model which is relatively inexpensive and representativeof the human oral environment in ecological and microbiolog-ical terms has yet to be described.
Our knowledge of the pathogenesis and management of oralcandidiasis has been considerably expanded by the use of thesevarious animal models. However, there is much more to beelucidated. For instance, the chronic recurrences of oral Can-dida infection in HIV-infected patients and the increasinglyfrequent emergence of Candida resistance to the newer anti-fungals pose new challenges, which may be resolved by animalexperimentation. Unfortunately, the enthusiasm and interestshown in animal experiments in the 1970s and 1980s is grad-ually diminishing, with only a handful of investigations con-ducted during the last decade. Although the reasons for thismay be obscure, the time-consuming and intrinsic difficultiesassociated with animal experimentation, which is not alwaysrewarding, may have played a contributory role; the politics ofvivisection may be another relevant factor. Notwithstandingthese drawbacks, animal experiments play a definitive role inour understanding and the management of this all too commonmucosal disease. Prudent choices of models to suit the inves-tigational aims, as reviewed above, and appropriate standard-ization of experimental protocols to obtain broadly compara-ble and meaningful data, which need to be analyzed andinterpreted cautiously, should be rewarding for researcherswho venture into this arena.
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