Experimental oral infection with the yeast Candida albicans in mice with or without inherited iron-deficiency anaemia (sla)

The role of iron deficiency in the development of oral candidosis was investigated using the mouse mutant sex-linked anaemia (sla). Susceptibility was assessed in terms of the recovery of organisms, particularly from oral swabs, and histological evidence of infection approximately 10 days after the last exposure to Candida albicans. The influence of three factors was studied in mixed groups of normal and anaemic mice: mode of inoculation, treatment with tetracycline and treatment with hydrocortisone. The most susceptible group had received drinking water containing tetracycline (1 mg/ml), hydrocortisone (0.1 mg/ml) and Candida (5 × 10⁴ c.f.u./ml for 6 days). Anaemic mice showed a rather higher rate of recovery of organisms and more frequent histological evidence of infection that normal mice in certain groups. Neither of these tendencies was statistically significant alone but, taken together, they suggest that some small difference of susceptibility may exist between normal mice and mice with sla. The mouse model could be of value in studying the influence of several other inherited disorders on susceptibility to candidosis.     

Experimental oral candidosis in the mouse: microbiologic and histologic aspects

A model of oral candidosis was developed in order to investigate histologic and microbiologic aspects of this host-parasite interaction under controlled experimental conditions. Normal adult CD-1 mice were inoculated by the topical application of 10(8) Candida albicans blastospores, and oral colonization was monitored by the quantitative culturing of saliva samples and of digested oral mucosa. Tissue sections of the mucosa were examined in a kinetic study ranging from 2 h to 13 days postinoculation. We report here that oral colonization by C. albicans can be induced in normal adult mice without the use of any compromising agent and that the animals recover from this mucosal infection following a reproducible pattern. Temporal analysis of the oral histopathology showed that distinct patterns of inflammation are associated with particular stages in the development of the infectious foci. This experimental model offers a means of further investigating the host-parasite interactions involved in the onset and development of oral candidosis.     

Effect of sialoadenectomy on the carriage of Candida albicans in the mouths of rats

Xerostomia provoked in rats by surgical removal of the major salivary glands significantly increased oral carriage of Candida albicans. Five weeks after 3 oral inoculations of 10⁸ CFU, C. albicans were found in the mouths in 50% of normal animals but after 8 weeks in none. In xerostomic rats, 100% had oral C. albicans after 5 weeks and after 18 weeks, 66.6% still harboured yeasts. Over the whole experimental period the amount of C. albicans was greater in the mouths of sialoadenectomized than in normal rats.     

Effects of chronic Candida albicans in the hamster cheek pouch.

Sixty-four adult male hamsters had a suspension of either C. albicans (UO1) or C. albicans (ATCC 10261) placed in their cheek pouches once a week for up to 9 months. Four hamsters from both experimental groups, along with two untreated control hamsters, were killed at monthly intervals after the initial inoculation. Sections taken from the hamsters and examined in the light microscope showed that all experimental pouches had some or all of the following localized changes: inflammation and increased vascularity of the connective tissue; epithelial inflammation and microabscesses; hyperkeratosis; and isolated rete ridges similar to those in control pouches. C. albicans, usually the yeast form, was present on the exposed surface and between hyperplastic keratin squames. There was no hyphal invasion of the epithelium. Rather than being a true long-term study of chronic infection by C. albicans, the changes seen were probably the result of repetitive, more short-term responses after multiple inoculations.     

The influence of antifungal drugs on virulence properties of Candida albicans in patients with diabetes mellitus

OBJECTIVE: This study investigated the influence of nystatin and fluconazole on virulence properties of Candida albicans. STUDY DESIGN: A total of 108 diabetic patients participated in the study. Eighty-eight patients had clinical oral candidosis. Drug therapy was given at 6 hourly intervals for nystatin or daily with fluconazole for a maximum of 2 weeks. Adhesion of C albicans to buccal epithelial cells was determined by using an autologous adhesion assay prospectively over 6 months. Phospholipase production was estimated by using an agar plate method. The data analysis included a paired Student t test and calculation of correlation coefficients. RESULTS: Unlike nystatin, treatment with fluconazole reduced the ability of C albicans to colonize the buccal mucosa for up to 8 weeks after the treatment. Patients without clinical signs of oral candidosis had significantly fewer C albicans isolates producing phospholipase than did patients with oral candidosis. Treatment with fluconazole, but not nystatin, reduced the production of phospholipase from C albicans oral isolates in patients with diabetes mellitus. CONCLUSIONS: In addition to being antifungal, fluconazole alters phospholipase production, modifies buccal epithelial cells, and reduces adhesion of C albicans to human buccal epithelial cells for up to 8 weeks posttreatment in diabetic patients with oral candidosis.     

Effects of salivary protein flow and indigenous microorganisms on initial colonization of Candida albicans in an in vivo model

ABSTRACT: BACKGROUND: Candida albicans is a dimorphic fungus that is part of the commensal microbial flora of the oral cavity. When the host immune defenses are impaired or when the normal microbial flora is disturbed, C. albicans triggers recurrent infections of the oral mucosa and tongue. Recently, we produced NOD/SCID.e2f1-/- mice that show hyposalivation, decrease of salivary protein flow, lack IgA and IgG in saliva, and have decreased NK cells. Our objective was to characterize C. albicans infection and biofilm formation in mice. METHODS: NOD/SCID.e2f1-/- mice were used as an animal model for C. albicans infection. C. albicans yeast and hyphal forms solutions were introduced in the oral cavity after disinfection by Chlorhexidine. RESULTS: The numbers of C. albicans colonized and decreased in a time-dependent manner in NOD/SCID.e2f1+/+ after inoculation. However, the colonization levels were higher in NOD/SCID.e2f1+/+ than NOD/SCID.e2f1-/- mice. In the mice fed 1% sucrose water before inoculation, C. albicans sample was highly contaminated by indigenous microorganisms in the oral cavity; and was not in the mice fed no sucrose water. The colonization of C. albicans was not influenced by the contamination of indigenous microorganisms. The hyphal form of C. albicans restricted the restoration of indigenous microorganisms. The decreased saliva in NOD/SCID.e2f1-/- did not increase the colonization of C. albicans in comparison to NOD/SCID.e2f1+/+ mice. We suggest that the receptor in saliva to C. albicans may not be sufficiently provided in the oral cavity of NOD/SCID.e2f1-/- mice. CONCLUSION: The saliva protein flow may be very important for C. albicans initial colonization, where the indigenous microorganisms do not affect colonization in the oral cavity.     

白念珠菌口腔黏膜感染小鼠模型的建立及评估

［摘要］ 目的 建立白念珠菌口腔黏膜感染ICR小鼠模型,动态观察口腔黏膜及相应的全身感染情况,并对变化情况进行初步分析。方法 将白念珠菌接种于免疫功能低下小鼠口腔,采用肉眼观察口腔舌背病损改变、口腔内白念珠菌菌量检测、组织病理学等方法评估口腔内黏膜感染情况,检测体重、载菌量（肾、肝、下降结肠内粪便）评估全身感染情况。观察1周,并记录数据。空白对照组为接种生理盐水的免疫功能低下组。结果 ①小鼠接种白念珠菌后第1天口腔白念珠菌菌量较低,随后迅速增长,3~7 d内趋于稳定,稳定于106~107CFU/mL。同时口腔舌背病损也出现类似趋势,第1天未见明显伪膜,3~7 d内可见舌背伪膜存在。②病理切片PAS染色显示接种后3~5 d内白念珠菌形成菌丝侵入并破坏上皮,到接种第7天时,黏膜上皮多见酵母细胞。③小鼠接种白念珠菌后体重逐渐下降。粪便载菌量逐渐上升,第5天达到最大,但肾、肝无白念珠菌感染。空白组未见白念珠菌感染。结论 通过在ICR小鼠口腔内接种白念珠菌可以建立白念珠菌口腔黏膜感染动物模型。接种后白念珠菌感染程度在1周内存在变化。动物实验应根据感染状况选择合适的研究时段。     

Treatment with probiotics in experimental oral colonization by Candida albicans in murine model (DBA/2)

The aim of this study is to evaluate the oral colonization by Candida albicans in experimental murine immunosuppressed DBA/2 and treatment with probiotic bacteria. To achieve these objectives, 152 DBA/2-immunosuppressed mice were orally inoculated with a suspension of C. albicans containing 10(8) viable yeast cells, the animals were treated with nystatin or with the probiotics (Lactobacillus acidophilus and Lactobacillus rhamnosus). Evaluations were performed by Candida count from oral mucosa swabbing. The oral mucosa colonization by C. albicans started at day 1 after inoculation, remained maximal from day 3 until day 7, and then decreased significantly. Probiotics reduced the C. albicans colonization significantly on the oral mucosa in comparison with the untreated animal group. In the group treated with L. rhamnosus, the reduction in yeast colonization was significantly higher compared with that of the group receiving nystatin. Immunosuppressed animal model DBA/2 is a relevant model for experimental Candida oral colonization, and the treatment with probiotics in this model may be an effective alternative to prevent it.     

Effect on experimental palatal candidosis in the Wistar rat of removal and re-insertion of acrylic appliances

Palatal candidosis was produced by inoculating Candida albicans 3091 (serotype A) under an acrylic appliance. Two weeks was allowed for infection to occur. Removal of the appliance for a further period of 2 weeks resulted in complete resolution of the lesion histologically but Candida organisms could still be recovered from the mucosal surface. Re-fitting these appliances at the end of this recovery period, without subsequent inoculation, produced a recrudescence of palatal candidosis after a further 2 weeks. One possible explanation is that C. albicans was encouraged to change from the commensal to the pathogenic form in the presence of the palatal appliance.     

Oral yeast carriage in patients with advanced cancer

The aim of this study was to investigate oral yeast carriage amongst patients with advanced cancer. Oral rinse samples were obtained from 120 subjects. Yeasts were isolated using Sabouraud's dextrose agar and CHROMagar Candida, and were identified using a combination of the API 20 C AUX yeast identification system, species-specific PCR and 26S rDNA gene sequencing. Oral yeast carriage was present in 66% of subjects. The frequency of isolation of individual species was: Candida albicans, 46%; Candida glabrata, 18%; Candida dubliniensis, 5%; others, < 5%. The increasing isolation of non-Candida albicans species is clinically important, since these species are often more resistant to antifungal drugs. Oral yeast carriage was associated with denture wearing (P = 0.006), and low stimulated whole salivary flow rate (P = 0.009). Identification of these risk factors offers new strategies for the prevention of oral candidosis in this group of patients.     

In vitro antifungal susceptibility to six antifungal agents of 229 Candida isolates from patients with diabetes mellitus

The most common antifungal drugs in current clinical use for the treatment of oral candidosis are polyenes and azoles, mainly used topically. Poor glycaemic control in association with other local factors, such as the presence of oral dental prostheses, salivary pH, salivary flow rate and tobacco habits, may lead to the development of oral candidosis. Topical antifungal agents are frequently used to prevent the development of candidal infections in patients with poor metabolic control, particularly in the elderly wearing dentures. The aim of this study was to assess the antifungal susceptibility of Candida isolates to six antifungal agents using a commercially available kit, Fungitest. The isolated were collected from patients affected by diabetes mellitus from two different geographic localities (London, UK, and Parma, Italy) and from a group of healthy non-diabetic subjects. No differences in antifungal susceptibility to the six agents tested were observed between Candida isolates from diabetic and non-diabetic subjects. However, differences were observed between the two geographically different diabetes mellitus populations. Oral yeast isolates from diabetes mellitus patients in the UK more often displayed resistance or intermediate resistance to fluconazole (P=0.02), miconazole (P<0.0001), and ketoconazole (P=0.01) than did isolates from diabetes mellitus patients in Italy. In addition, more C. albicans isolates were found in diabetic and non-diabetic subjects that were susceptible to fluconazole (P=0.0008 and P=0.01, respectively) than non-albicans isolates. The difference in the antifungal resistance of isolates from the two populations of diabetes mellitus patients may be related to differences in the therapeutic management of candidal infections between the two centres.     

Influence of environmental pH on the reactivity of Candida albicans with salivary IgA

Salivary secretory IgA reacts with a group of heat-shock mannoproteins preferentially expressed on Candida albicans yeast cells and germ tubes grown at 37 degrees C. Since other environmental factors can also modulate the expression of those antigens, we have investigated the influence of the pH of the culture medium on the expression of the antigens reacting with human salivary IgA by C. albicans. By indirect immunofluorescence, yeast cells grown in Sabouraud glucose broth at 37 degrees C showed a statistically significant increase in reactivity with salivary IgA (p < 0.0001) when compared with cells grown at 25 degrees C at the 4 pH values studied (3.3, 5.9, 7.5, and 9.5), the highest reactivity and the major heat-shock effect being observed at pH 5.9. The decrease in reactivity with salivary IgA observed in C. albicans cells grown at pH values of 3.3 and 9.5 was confirmed by Western blotting. Salivary IgA reacted with polydispersed materials from the cell walls of molecular masses > 55 kDa, which were more expressed at neutral pH than at acidic or alkaline pH values. A similar reactivity was observed when the antigenic extracts were stained with an antiserum directed against oligosaccharides present in antigen 6 of C. albicans serotype A. The differences in reactivity presented by salivary IgA may be related to a decrease in the expression of polysaccharides present on the surfaces of the yeast cells of C. albicans grown at acidic or alkaline pH values. The low reactivity of salivary IgA with C. albicans cells grown at acidic pH values may help to explain the association between acidic saliva and the carriage of Candida in the oral cavity, as well as with oral candidiasis.     

Sex hormone involvement in the development of experimental virally induced murine salivary gland tumors

Mice were given a single inoculation of polyoma virus at birth and then orchidectomised or oophorectomised. Unoperated groups received polyoma virus alone whilst further males received testosterone, and females received oestradiol thrice-weekly in addition to polyoma virus inoculation. All groups were then observed over the succeeding 370 days for the development of tumors of the submandibular and parotid salivary glands. Polyoma virus inoculation selectively induced salivary gland tumors in 70% of male animals and 30% of female animals given polyoma virus alone. Testosterone therapy increased the salivary tumor frequency from 69-90% whilst orchidectomy reduced the tumor frequency to 50%. Oophorectomy or oestrogen therapy did not significantly alter the salivary gland tumor frequency in females but oestrogen therapy did result in the development of second primary tumors of breast in 60% of female animals bearing salivary gland tumors. The role of androgens in the development of virally induced salivary gland tumors is discussed as are the possible mechanisms responsible for the development of second primary tumor of breast.     

Expression of interleukin-8 and its receptor IL-8RA in chronic hyperplastic candidosis

INTRODUCTION: Neutrophils are the main opponents of Candida albicans in chronic hyperplastic candidosis. They migrate from the circulation to the epithelium where they form microabscesses. We therefore hypothesized that the neutrophil chemokine interleukin-8 (IL-8) might play a role in the neutrophil-Candida interaction. METHODS: Biopsies from patients with chronic hyperplastic candidosis (n = 10) were stained using the avidin-biotin-peroxidase complex protocol for IL-8 and IL-8 receptor A and were compared to healthy control mucosa (n = 3). A set of C. albicans agar sections was similarly analysed. RESULTS: In chronic hyperplastic candidosis lesions IL-8 was strongly expressed in both vascular endothelium and mucosal epithelium. Many resident and immigrant inflammatory cells, including intraepithelial neutrophils, were IL-8 receptor A positive. In addition, IL-8 (or an analogue) was found in the candidal mother cell in chronic hyperplastic candidosis and in agar, whereas the tips of the hyphae expressed IL-8 receptor A (or an analogue). CONCLUSION: IL-8 may play a role in the recruitment of neutrophils from the vascular compartment to the epithelial microabscesses. C. albicans may have developed an ability to sense IL-8. The IL-8 ligand-receptor interaction may help to direct the growth of the IL-8-receptor-containing tips of the hyphae away from the IL-8-producing candidal cell body (a centrifugal growth pattern to facilitate host tissue penetration). Later, this ability might help to keep the vulnerable hyphal tips away from areas with high concentrations of host IL-8 and candidacidal neutrophils. We suggest that this phenomenon, in contrast to chemotropism, is named chemophobia.     

Effect of Sustained Systemic Administration of Ginger (Z officinale) Rhizome Extracts on Salivary Flow in Mice

ObjectiveThis study aimed to evaluate the effect of methanol (70% v/v), ethanol (80% v/v), dimethyl sulfoxide (DMSO; 100% v/v) extracts of ginger rhizome (GR), and 6-shogaol on the pilocarpine-stimulated salivary flow rate in C57BL/6 mice.MethodsThree extracts of ginger (Zingiber officinale) rhizome prepared by maceration using the respective solvents and 6-shogoal were reconstituted in normal saline with 0.2% DMSO. Thirty C57BL/6 15-week-old mice were divided into 5 groups: Group 1, saline; Group 2, 70% methanol extract; Group 3, 80% ethanol extract; Group 4, 100% DMSO extract; and Group 5, 6-shogaol. The baseline pilocarpine-stimulated salivary flow rate was measured at the age of 15 weeks (15th week), and treatment solutions were administered by intraperitoneal injection from the 16th to 18th week. The stimulated salivary flow rate during treatment weeks was recorded for each group, and its difference with baseline was analysed using paired-sample t test. The change in salivary flow rate between the treatment groups and the control group was analysed using one-way analysis of variance.ResultsGroups 2, 3, 4, and 5 showed a significant increase in salivary flow rate when compared to baseline (P < .05). The increase in salivary flow rate in all 4 treatment groups was significant when compared to the control group (P < .05). Group 4 produced the highest increase in salivary flow rate; however, the differences amongst the treatment groups did not reach statistical significance (P > .05).ConclusionsAll GR extracts (70% methanol, 80% ethanol, 100% DMSO) and 6-shogaol were equally effective in increasing the pilocarpine-stimulated salivary flow rate in C57BL/6 mice when administered systemically as a sustained dose for 3 weeks.     

Sex hormone involvement in the development of experimental virally induced murine salivary gland tumors

Mice were given a single inoculation of polyoma virus at birth and then orchideclomised or oopliorectomiscd. Unoperated groups received polyoma virus alone whilst further males received testosterone, and females received oestradiol thrice-weekly in addition to polyoma virus inoculation. All groups were then observed over the succeeding 370 days for the development of tumors of the submandibular and parotid salivary glands. Polyoma virus inoculation selectively induced salivary gland tumors in 70% of male animals and 30% of female animals given polyoma virus alone. Testosterone therapy increased the salivary tumor frequency from 69–90% whilst orchidectomy reduced the tumor frequency to 50%. Oophorectomy or oestrogen therapy did not significantly alter the salivary gland tumor frequency in females but oestrogen therapy did result in the development of second primary tumors of breast in 60% of female animals bearing salivary gland tumors. The role of androgens in the development of virally induced salivary gland tumors is discussed as are the possible mechanisms responsible for the development of second primary tumor of breast.     

In vitro activity of zinc oxide-eugenol and glass ionomer cements on Candida albicans

The aim of this study was to evaluate in vitro the antimicrobial activity of glass ionomer (GIC) and zinc oxide-eugenol (ZOE) cements against Candida albicans. Standardized GIC and ZOE specimens were maintained in contact with C. albicans suspension (1 x 10(6) cells/ml) at 37 degrees C for 24 h, 48 h or 7 days. A control group without any testing cement was included. After the incubation period, aliquots of 0.1 ml were plated on Sabouraud's agar, and then the number of colonies was counted. The results were expressed as values of logarithms of colony-forming units per milliliter (log CFU/mL) and were analyzed statistically by Kruskal-Wallis ANOVA. After 48 h of incubation, the ZOE group presented no growth of C. albicans. GIC and control groups presented similar mean values at all tested periods. According to the results obtained, it could be concluded that, under the experimental conditions, ZOE cement was more effective in vitro against C. albicans than GIC.     

In vitro antifungal resistance of oral Candida albicans strains in non-AIDS Patients

Some cases of oral candidosis are refractory to antifungal treatment. This might be related to development of resistant Candida strains, but susceptibility testing is not standardized and not routinely available, and information related to this problem is scarce in non-AIDS patients. In this study, the in vitro antifungal resistance of oral Candida albicans strains was evaluated. The strains were obtained from a cohort of 72 HIV-negative patients with oral yeast carriage and clinical complaint. Laboratory identification revealed C. albicans in 93% of cases. None of these oral C. albicans isolates showed in vitro resistance to polyenes, but they showed varying resistance levels to fluorocytosine and azoles. This study confirms the usefulness of standardizing susceptibility testing so that it could be routinely available and of realizing a mycological diagnosis including an antifungigram when oral candidosis is suspected, whenever antifungal treatment with azoles is planned.     

Epidermal growth factor concentration in hyperplastic and hypertrophic submandibular salivary glands of mice.

Submandibular salivary glands of mice contain high concentrations of epidermal growth factor (EGF). The EGF content of mouse submandibular salivary glands undergoing hyperplastic and hypertrophic changes was measured and compared to that in the glands of control mice. Salivary gland hyperplasia was induced by giving mice a single injection of isoprenaline and hypertrophy was produced either by repeated, daily injections of isoprenaline, repeated amputation of a lower incisor tooth or by removing the right submandibular salivary gland and thus producing a compensatory hypertrophy of the left submandibular gland. The EGF content of the hyperplastic submandibular salivary glands was not different from that of the control glands. While the EGF content of the hypertrophied glands resulting from either repeated isoprenaline injections or partial sialoadenectomy did not differ from that of the control glands, the concentration of EGF was significantly lower. This reduced concentration is probably a reflection of acinar hypertrophy with a resultant smaller proportionate contribution of the granular tubules to the mass of the gland. Incisor-amputation-induced hypertrophy did not result in a reduced concentration of EGF in the submandibular salivary glands, but the reason for the different response is unknown.The findings provide no evidence for the involvement of EGF in the induced changes of submandibular salivary gland hyperplasia or hypertrophy resulting from either isoprenaline treatment or partial sialoadenectomy. The reason for the higher concentration of EGF in hypertrophied submandibular salivary glands resulting from incisor amputation compared to that measured in hypertrophied glands resulting from the other stimuli used remains unresolved.     

Epidermal growth factor in the submandibular salivary glands of congenitally athymic mice.

The submandibular salivary glands of a group of congenitally athymic (“nude”) mice were assayed for their epidermal growth factor (EGF) content and their histology was examined by light microscopy. The ability of the submandibular salivary glands from athymic mice to respond to an androgenic agent was assessed. The histology of the submandibular salivary glands resembled that of normal mice. Athymic mice had concentrations of EGF in their submandibular salivary glands which were similar to those reported previously for normal mice As in normal mice, male athymic mice had more prominent granular convoluted tubules than female mice, but, as in normal mice, testosterone treatment of female athymic mice resulted in an increase in both the EGF content and in the number of granules within the cells of the granular tubules of the duct system of the submandibular salivary gland.These results provide no evidence for a relationship between the level of submandibular salivary gland EGF and the immunological deficiency of nude mice, and show that the abnormalities in development of nude mice do not extend to their submandibular salivary glands.