Oral candidosis by Candida albicans in normal and xerostomic mice

The aim of this study was to analyze the effect of sialoadenectomy on the development of oral candidosis after one or four inoculations of Candida albicans. Initially, a suspension containing 10 cells/ml of C. albicans ATCC 36801 was prepared. Seventy-eight sialoadenectomized mice and a similar amount of mice with normal salivary flow received a single inoculation of C. albicans suspension. Another group with a similar number of mice received 4 inoculations. The control group consisted of 6 sialoadenectomized mice and 6 mice with normal salivary flow that were not inoculated with C. albicans. Candidosis development was studied histologically in the tongue of the animals 1, 2, 3, 5, and 8 days after inoculation and at 15-day intervals up to 165 days. According to the results obtained, it could be concluded that sialoadenectomy and a higher frequency of yeast inoculation influenced the presence and extension of candidosis lesions. DESCRIPTORS: Candidiasis, oral; Candida albicans; Xerostomia. RESUMO: O objetivo deste estudo foi analisar o efeito da sialoadenectomia sobre o desenvolvimento da candidose oral após uma ou quatro inoculações de Candida albicans. Inicialmente, uma suspensão contendo 10 células/ml de C. albicans ATCC 36801 foi preparada. Setenta e oito camundongos sialoadenectomizados e mesma quantidade de camundongos com fluxo salivar normal receberam uma única inoculação de suspensão de C. albicans. Outro grupo, com o mesmo número de camundongos, recebeu 4 inoculações. O grupo controle consistiu de 6 camundongos sialoadenectomizados e 6 com fluxo salivar normal que não foram inoculados com C. albicans. O desenvolvimento de candidose foi estudado histologicamente na língua dos animais em períodos de 1, 2, 3, 5 e 8 dias após a inoculação e em intervalos de 15 dias até 165 dias. De acordo com os resultados obtidos, conclui-se que a sialoadenectomia e uma maior freqüência de inoculação influenciaram na presença e extensão das lesões de candidose. DESCRITORES: Candidíase bucal; Candida albicans; Xerostomia.


INTRODUCTION
Candidosis is the most common infection of the oral cavity caused by fungi and C. albicans is the main species related to its development.The genus Candida belongs to the indigenous oral flora of 20 to 40% of healthy subjects 7,18 and its pathogenicity is observed in the presence of predisposing conditions such as immunodepression, use of denture or orthodontic appliances and diabetes mellitus.The transformation from the saprophytic to the parasitic form is related to various factors, including not only the virulence of these yeasts, but also host-related variables 19 .Among the hostrelated factors, the modification of the oral microbiota balance is considered a very important factor 6 .
Xerostomia is one of the main factors related to the development of oral candidosis, since the lack of or decrease in salivary flow causes changes in the resident oral microbiota.Also, food and microorganisms are poorly mechanically removed from the oral cavity and subjects lack the protector effect of saliva constituents, such as lactoferrin and lysozyme 20 .
In a previous study, Totti et al. 22,23 (1996, 2002) observed that xerostomia helps in the appearance, proliferation and persistency of five Candida species in the oral cavity of rats.Similar results were observed by Jorge et al. 11,12 (2000, 2002).Xerostomia is also related to the longer permanence of yeasts in the mouth of mice 12 and to a higher Candida albicans transmissibility 11 .(2003) correlated the number of candidosis lesions with the increased number of Candida in the oral cavity.
Rats have been used as an experimental model in studies on oral candidosis and also to evaluate the influence of local and systemic factors 1,4,10 .The use of mice as an experimental model in studies on oral candidosis is relevant, since Candida is not part of their indigenous oral microbiota 14 .
The influence of several inoculations of C. albicans on the development of candidosis in mice has not been discussed yet.The aim of this study was to analyze the effect of sialoadenectomy on the development of oral candidosis in mice after one or four inoculations of Candida albicans suspensions.

MATERIAL AND METHODS
Three-hundred and twenty four male mice (Mus musculus, albinos, Swiss; School of Dentistry of São José dos Campos, University of São Paulo), weighting from 25 to 30 g were included in this study.The animals were divided into three groups as follows: 156 xerostomic mice, 156 mice with normal salivary flow and 12 controls.
Xerostomia was obtained by surgical removal of the major salivary glands, according to Cheyne 4 (1939), with modifications.Before the beginning of the experiment, the presence of Candida in the oral cavity of the normal and sialoadenectomized animals was analyzed by plating the collected material from the oral cavity of the mice with a sterile swab (CB Products, SP, Brazil) in Sabouraud dextrose agar with chloramphenicol (quemicetina succinato, Carlo Erba, 0.1 mg/ml, SP, Brazil).After incubation at 37ºC for 48 h, the presence of Candida ssp. was analyzed.This procedure was repeated two weeks later.All mice were initially negative to Candida.C. albicans ATCC 36801 was subcultured in Sabouraud agar slants (Difco, Detroit, USA) and incubated for 24 h at 35°C.The cells of C. albicans were resuspended in phosphate-buffered saline (PBS, pH 7.4, Sigma, St. Louis, USA) and washed three times.From these cells, a suspension containing 10 8 viable cells/ml of C. albicans was standardized using a Neubauer chamber (Assistent, München, Germany) and 0.05% methylene blue (Dr.Theodor Schuchardt GMBH & Co., München, Germany).The inoculations were performed using this cell suspension as follows: • Single-inoculation group: 78 sialoadenectomized mice and 78 mice with normal salivary flow received a single inoculation of 0.1 ml of the C. albicans suspension containing 10 8 viable cells/ml using a 1 ml syringe and a 30 x 8 mm needle.• Four-inoculation group: 78 sialoadenectomized mice and 78 mice with normal salivary flow received a total of 4 intraoral inoculations of the C. albicans suspension containing 10 8 viable cells/ml, performed during 4 consecutive days.• Control group: 6 sialoadenectomized mice and 6 mice with normal salivary flow that were not inoculated with the C. albicans suspension.All the animals did not receive water for at least 1 h after inoculation.Candidosis was studied in the tongue of normal and sialoadenectomized mice at 1, 2, 3, 5 and 8 days after inoculation and at 15-day intervals up to 165 days.Six animals of each group were sacrificed at each studied time and the tongue was surgically removed, fixed in a 10% formalin solution (Synth, SP, Brazil) and processed for histological examination.For the histological processing, the tongue was divided longitudinally and the two portions were included in paraffin.Semi serial slices of 5 µm were obtained, which allowed the analysis of the central, lateral and edge part of the tongue (designed sites A, B and C, respectively) on the left and right sides.The slides were dyed using PAS (periodic acid -Sigma, St. Louis, USA; Schiff's reagent -Merck, Darmstadt, Germany), H & E (Merck, Darmstadt, Germany) and Gomori-Grocott stains (Sigma, St. Louis, USA).The tongue was thoroughly examined for presence of candidosis, with a total of 18 sections examined for each animal.All the sections were analyzed by one examiner previously trained.The description of the candidosis areas was performed considering the localization, extension, alterations in the tissues, and candidal hyphae presence.

Statistical analysis
Data on the mean and standard deviation of the number of animals with candidosis after one and four inoculations were compared statistically by means of the Student's t-test (α = 5%).

Presence of candidosis
According to the histological analysis of the tongues, it could be observed that the development of candidosis occurred more frequently among sialoadenectomized mice submitted to both one and four inoculations of the yeast suspension.
Table 1 presents the number of sialoadenectomized and normal salivary flow mice that presented candidosis according to the period of time analyzed.Amont the xerostomic animals that received 1 inoculation of C. albicans, candidosis was observed in 33.33% of the animals, after a period of 15 days.This value decreased to 16.6% 75 days after the inoculation.After this period, no animal presented candidosis.
Among the animals that received 4 inoculations of Candida albicans suspension, candidosis was observed in 83.3% to 100% of the xerostomic animals after the period of eight days.This number decreased to 33.33% after 15 days, and 115 days after the last inoculation only 1 (16.6%)animal presented candidosis.
Considering the mice with normal salivary flow, no difference could be observed in relation to the number of animals with candidosis after 1 or 4 inoculations of C. albicans.Eight days after a single inoculation, only one animal presented candidosis in the tongue dorsum.After 4 C. albi-cans inoculations, candidosis was observed in one animal after 45 days.
Candidosis was not observed in animals from the control group, which were not inoculated with C. albicans.
Mean and standard deviations values of the number of candidosis areas observed among normal and xerostomic mice after 1 and 4 inoculations are presented in Table 2. Statistically significant differences were observed between the 4-inoculation normal salivary flow group and the 4-inoculation sialoadenectomized mice group at 2, 3 and 45 days.Considering the single-inoculation group, a statistically significant difference between sialoadenectomized and normal salivary flow mice was observed at day 8.

Histological analysis 8 days after C. albicans inoculation
After this period of time, considering the animals that received only a single inoculation, candidosis was observed in the dorsum of the tongue of 2 sialoadenectomized and of 1 normal mouse.The tongue of sialoadenectomized mice presented flatting of the papillae, acanthosis, parakeratosis, and lymphocytic infiltration.
Observations performed after 4 inoculations of C. albicans, showed necrotic areas on the dorsum of the tongue of normal mice.Sialoadenectomized animals presented extensive areas with intense candidosis, mainly in the papillae regions.Figure 1 shows the histologic examination of a sialoadenectomized animal 3 days after the last inoculation.

to 60 days after the C. albicans inoculation
The epithelium of the dorsum of the tongue of normal salivary flow mice had normal histological aspect 15 days after a single inoculation.Candidosis lesions were observed in 2 sialoadenectomized animals 15 days after the inoculation and in 1 animal 30 days after the inoculation.Forty-five and 60 days after the inoculation, no candidosis lesions were registered.The lesions in the interior of the epithelium's keratine in the dorsum of the tongue presented few candidal hyphae.No alterations in the epithelium were observed.
After 4 inoculations of the C. albicans suspension, one normal and 2 sialoadenectomized mice presented candidosis in the dorsum of the tongue.Fifteen and 30 days after the inoculation, 2 sialoadenectomized animals presented candidosis in the dorsum of the tongue and 60 days after the inoculations just one animal presented signs of candidosis.The observed candidosis areas were extensive, mainly in the lingual papillae region, and were found in all the extension of the dorsum of the tongue.Candidal hyphae could be observed penetrating the epithelium.Among the mice without candidosis, flattening of lingual papillae, hiperkeratosis, acanthosis, lymphocitic infiltrate and areas of necrosis in the epithelium could be observed.

to 165 days after the inoculation
Only one sialoadenectomized mouse presented candidosis in this period.Extensive areas of candidosis with similar histologic aspects to those described previously were observed.In the other animals, the histologic aspect of the dorsum of the tongue was normal.

DISCUSSION
It is well known that infections caused by Candida species have a multifactorial etiology.The transformation from the saprophytic to the parasitic form is related to several factors including not only the virulence of these yeasts but also host-related variables.Among the host-related fac-tors, the salivary anti-Candida immunoglobulins and cellular defenses play an essential role for controlling the infection 13,18 .When these defenses are compromised, such as in cases of xerostomia, candidosis can occur.In fact, Brown et al. 3 (1975) observed an increase in the prevalence of Candida spp., mainly C. albicans, in patients with xerostomia induced by head and neck radiation.In this study, it could be observed that the surgical removal of the major salivary glands produced deep xerostomia in mice.The volume of saliva was reduced by approximately 75%.In human beings, the symptoms of xerostomia can be observed when the salivary flow is reduced by 40 to 50% 5,8 .After surgery, the oral cavity of the mice was dry, presenting only residual viscous saliva and food on mucous tissue and teeth.The residual salivary flow present in the mouth of sialoadenectomized mice was probably produced by the minor salivary glands.
Our results showed that xerostomia induced by sialoadenectomy itself did not promote the appearance of C. albicans in the oral cavity.This fact is probably because these yeasts are not part of their indigenous oral microbiota.Candida was not isolated from the mice's oral cavity before the experiment.The cultures remained negative for Candida 1 and 2 weeks after surgery.
Sialoadenectomy was the technique selected to produce xerostomia in mice because it is a simple method and it causes a permanent reduction in the salivary flow.The use of chemical agents can also produce xerostomia 16 .However, their effects are transient and they can produce side effects that can interfere on the results.
In human beings, xerostomia is associated with atrophy of the oral mucosa, especially in the dorsum of the tongue 24 .In the present study, this alteration was not observed.On the other hand, alterations in the oral microbiota have also been reported in patients with xerostomia 17 .
Mice have been used in studies on the pathogenicity of C. albicans in the oral cavity.These animals do not harbor Candida in their oral cavity and can be considered more adequate for these studies, since other microbial species do not in-terfere with the tested microorganism 1,2,9,14,20 .This way, the infection by Candida spp. is considered as a transient colonization in the oral cavity of these animals.
The development of candidosis, after 1 and 4 inoculations of the C. albicans suspension occurred more frequently in sialoadenectomized mice in comparison with the normal ones.Eight days after the last inoculation, 83.3% of the animals presented extensive candidosis lesions, particularly in the true papillae region.
In relation to the animals with normal salivary flow that received 1 inoculation, only one presented lesions in the dorsum of the tongue.This number was lower than that observed by Lacasse et al. 15 (1990).Among the animals that received 4 inoculations, 33.33% presented candidosis and this number decreased with time.
Candidosis on the dorsum of the tongue was characterized by the presence of pseudohyphaes inside the epithelium, flattening of the lingual papillae, hyperkeratosis, akanthosis and neutrophilic infiltrate, forming intraepithelial micro abscesses.There were no differences among lesions found in the central, lateral or edge regions of the tongue; however, a slightly higher prevalence of the lesions in the true and conical papillae was observed.

CONCLUSION
According to the results obtained, it could be concluded that sialoadenectomy and a higher number of C. albicans inoculations influenced the frequency and extension of candidosis lesions in mice.

FIGURE 1 -
FIGURE 1 -Histologic examination of the tongue of sialoadenectomized animal, 3 days after four inoculations of Candida albicans.A high quantity of pseudohyphae, the presence of inflammatory infiltrate, flattening of lingual papillae, hiperkerathosis and acanthosis can be observed (PAS, 200 X).

TABLE 1 -
Number (n)and percentage of normal and xerostomic mice that developed candidosis in the dorsum of the tongue after a single inoculation of the yeast in the oral cavity.

TABLE 2 -
Mean and standard deviation values of the number of areas of candidosis after one and 4 inoculations of the Candida albicans suspension in the oral cavity of normal and xerostomic mice.Six regions of the tongue were considered: central portion of the left and right sides, lateral portion of the left and right sides and edge of the tongue at the left and right sides.
*Statistical significant difference at the level of significance of 5% (Student's t-test) in relation to normal salivary flow group.