Establishment Of A Mouse Model Of Candidiasis With Concurrent Oral And Vaginal Mucosal Infection&Virulence Comparison Of Candida Albicans Isolated From Different Human Candidiasis

Chapter Ⅰ Establishment and application of a mouse model of candidiasis with concurrent oral and vaginal mucosal infectionSection Ⅰ Establishment of a mouse model of candidiasis with concurrent oral and vaginal mucosal infectionObjective To establish a mouse model of candidiasis with concurrent oral and vaginal mucosal infection. Methods Three groups were set as prednisolone alone, estrogen alone and combinational scheme. These groups were compared and the best one was selected based on clinical manifestation, fungal direct KOH examination, tissue fungal burden and histopathological analysis. Results Mice from the combination group developed oral thrush and vaginal candidiasis at day4(relative to day of inoculation (Day0)). Direct fungal examination showed plenty of hyphae and fungal burden of oral and vaginal tissue reached a high level of105～106cfu/g. Histopathological examination also revealed mucosal epithelium was covered with thick pseudomembranous substance mainly consisting of hyphae accompanied by deep epithelial penetration by hyphae and PMN infiltration. However, these characterizations had a remission at day7. Outcomes from groups of prednisolone alone and estrogen alone were not as good as the combination group. Conclusion (1) Prednisolone combined with estrogen could successfully establish a mouse model of candidiasis with concurrent oral and vaginal mucosal infection;(2) The combinational scheme was superior than single treatment scheme.Section Ⅱ Dynamic investigation of mouse model with concurrent oral and vaginal mucosal candida albicans infection.Objective To observe the changes of mouse model with concurrent oral and vaginal mucosal candida albicans infection at different time points. Methods Establishment the mouse model with concurrent oral and vaginal mucosal candida albicans infection using the combinational scheme and observe the changes such as weight, clinical manifestations, tissue fungal burden and histopathological examination at day1,3,5and7. Results The weight of mice gradually descended after inoculation, especially from day1to3. Pseudomembranous lesions appeared on dorsal mucosa of tongue at day2-3and had a more prominent manifestation between day3and day5that covering nearly90%of the whole mucosal surface. Tissue fungal burden had a upward tendency from day1and reached a maximum between day3and5(about105～106cfu/g), while the values descended after day5. Histopathological examination showed yeast cells existing on the mucosal surface of tongue and in the vaginal lumen with rare hyphae invading epithelium. Then thick layer of hyphae had formed on the mucosal surface since day3and hyphae invaded deeply into the local tissue accompanied by destruction of epithelium and inflammatory infiltration. The epithelium of both anatomical tissues began to recover after day5and had a prominent recession at day7. Conclusion The mouse model with concurrent oral and vaginal mucosal candida albicans infection had the most typical manifestation from day3to5after infection using the method of combinational scheme.Section Ⅲ Establishment of a mouse model of candidiasis with concurrent oral and vaginal mucosal infection using different glucocorticoids with estrogen.Objective To explore the best regimen among different glucocorticoids to establish mouse model with concurrent oral and vaginal mucosal candida albicans infection. Methods Two kinds of mice were used and separately divided into three groups of prednisolone with estrogen, cortisone with estrogen and dexamethasone with estrogen. The best one was chosen mainly based on results of weight loss and fugal burden at day3. Results The weight had a persistent loss from day1to7during group of ICR mice treated with prednisolone and estrogen, while the weight loss of the other two groups stopped and rebounded from day3-5. Both kinds of mice treated with prednisolone and estrogen had the highest fungal burden and the results of ICR mice were ATCC62342(oral,1.05×106cfu/g; vagina,5.50×105cfu/g) Vs SC5314(oral,4.79×105cfu/g; vagina 4.07×105cfu/g). As to BALB/c mice, the outcomes were ATCC62342(oral,1.92×106cfu/g; vagina,1.33×106cfu/g) Vs SC5314(oral,7.57×105cfu/g; vagina,2.58x105cfu/g). Conclusion (1) Prednisolone was most suitable for model establishment;(2) ATCC62342had a more favorable result than SC5314;(3) Both ICR mice and BALB/c mice were suitable for model establishment.Section IV Establishment of a mouse model of candidiasis with concurrent oral and vaginal mucosal infection using cyclophosphamide and (or) prednisolone combined with estrogen.Objective To explore the best regimen using cyclophosphamide (CTX) and (or) prednisolone combined with estrogen to establish mouse model with concurrent oral and vaginal mucosal candida albicans infection. Methods Two kinds of mice were used and separately divided into three groups of prednisolone with estrogen, CTX with estrogen and CTX combined with prednisolone and estrogen. The best one was chosen mainly based on results of weight loss and fugal burden at day3. Results The weight had a prominent loss at day3among all groups of mice, especially during BALB/c mice. All mice had a weight loss than10%refer to the weight before infection and BALB/c mice treated with the combinational scheme were75percent of initial weight. The effects of combinational scheme were the most striking in two strains of mice and candida albicans. As to fungal burden, prednisolone combined with estrogen and the combinational scheme had the most values, while the latter also had a high mortality of mice. Conclusion (1) Prednisolone was more suitable for model establishment than CTX;(2) The scheme of prednisolone combined with CTX and estrogen had a prominent fatal effect on mice though the fungal burden was synchronously favorable.Section V Application of the mouse model of candidiasis with concurrent oral and vaginal mucosal infection using prednisolone with estrogen. Objective To explore the probable applications of mouse model with concurrent oral and vaginal mucosal candida albicans infection. Methods ICR mice were first used to establish the model under the treatment of prednisolone with estrogen. Then (1) Fluconazole was used for the therapeutic assessment to candida albicans infection;(2) Oral and vaginal tissue were resected and the following homogenates were examined for the dynamic changes of IL-17and IL-23using the method of Elisa. Results (1) Both strains of mice infected by SC5314had a prominently declined fungal burden at day3through fluconazole therapy and candida albicans could be totally cleared out at day5. On the contrary, mice infected by ATCC62342failed to reduce the fungal burden throughout one week;(2) IL-17in oral tissue began to rise after infection and reached maximum between day3and5, while IL-17in vaginal tissue have a elevated value only at day1and then descend to normal level. IL-23from both sites had a prominent elevation throughout the total observation time. Conclusion The mouse model with concurrent oral and vaginal mucosal infection was suitable for research of in vivo pharmacodynamics and mucosal immunology.Chapter II Virulence comparison of candida albicans isolated from different human candidiasis Section I Evaluation of extracellular enzymatic activities of candida albicans isolated from different human candidiasisObjective To investigate the enzymatic activities of64candida albicans isolated from different human candidiasis. Methods The egg yolk agar, bovine serum albumin agar, tween-80agar and blood agar were used to detect phopholipase, proteinase, esterase and haemolytic activities, respectively. Results (1) Phospholipase activities:strains from blood and oral mucosa showed most powerful, while vaginal mucosa the weakest;(2) Proteinase activities:strains from oral and vaginal mucosa revealed stronger proteinase activities than blood strains;(3) Esterase activities:strains isolated blood showed strongest activity than strains from other sources;(4) Haemolytic activities:isolations from oral and vaginal mucosa revealed stronger proteinase activities than blood isolations Conclusion Candida albicans from different origins had great variations in the activities of extracellular enzymes.Section II Antifungal susceptibility profiling of candida albicans isolated from different human candidiasisObjective To profile the susceptibility of candida albicans isolated from different human candidiasis to seven commonly used antifungal agents. Methods The antifungal susceptibility tests were performed according to the CLSI-A2protocol. The tested antifungal agents included amphotericin B, fluconazole, itraconazole, voriconazole, terbinafine, caspofungin and micafungin. Results All isolates of candida albicans were susceptible to amphotericin B, caspofungin and micafungin. Five strains resisted to fluconazole (1isolated from blood,1isolated from oral and3isolated from vagina); Six strains resisted to itraconazole (2isolated from blood,2isolated from oral and2isolated from vagina); Two strains resisted to voriconazole (1isolated from blood and1isolated from oral); Nine strains resisted to terbinafine (2isolated from blood,5isolated from oral and2isolated from vagina). Conclusion Candida albicans had variable susceptibility to different antifungal agents. There are also differences of in vitro antifungal susceptibility among candida albicans isolated from different origin to fluconazole, itraconazole, voriconazole and terbinafine. Strains of candida albicans from superficial candidiasis seemed more likely resisting to antifungal agents than strains from invasive candidiasis.