利用免疫抑制小鼠模型检测真菌病毒对黄曲霉菌致病力影响

Mycoviruses affect the virulence of Aspergillus flavus to immunosuppressed mouse model

摘要:

【目的】构建用于比较黄曲霉(Aspergillus flavus，A. flavus)菌株之间致病力差异的小鼠感染模型，并利用该模型评价真菌病毒AfPV1对宿主A. flavus致病力的影响。【方法】用不同浓度环磷酰胺腹腔注射Institute of Cancer Research (ICR)小鼠，根据白细胞的数量判断小鼠免疫抑制程度；通过滴鼻和尾静脉两种感染方法接种不同浓度的A. flavus孢子量，统计14 d以内小鼠的死亡率，确定A. flavus最佳的孢子接种量；通过小鼠组织的菌落负荷量以及肺部组织的病理观察，确定A. flavus感染是否成功；最后利用该小鼠模型评价真菌病毒AfPV1对寄主A. flavus致病力的影响。【结果】腹腔注射环磷酰胺的浓度为250 mg/kg时，能够达到免疫抑制水平；小鼠组织真菌负荷和病理组织切片观察显示A. flavus成功感染接种的ICR小鼠组织；在滴鼻接种模型中，A. flavus的孢子接种量为40 μL (1×10⁶ CFU/mL)时比较合适评价A. flavus菌株之间的差异；在尾静脉接种的模型中，A. flavus的孢子接种量在50 μL (1×10⁶ CFU/mL)比较合适评价A. flavus菌株之间的差异；病毒AfPV1能够引起A. flavus的致病力下降，但是不影响A. flavus在小鼠肺部的定殖量。【结论】本研究构建的小鼠感染模型能够评价A. flavus菌株之间致病力的差异；真菌病毒AfPV1的感染降低了宿主A. flavus的致病力。

关键词:Aspergillus flavus;真菌病毒;免疫抑制小鼠;感染模型;尾静脉注射;滴鼻接种

Abstract:

[Objective] To compare the virulence of different Aspergillus flavus strains and the effect of the mycovirus AfPV1 on A.flavus virulence to the established immunosuppressed mouse model. [Methods]Institute of Cancer Research (ICR) mice were intraperitoneally injected with different concentrations of cyclophosphamide, and the degree of immunosuppression was determined based on the number of white blood corpuscles. Different concentrations of A.flavus spores were inoculated through nasal drip and caudal vein, and the optimal inoculation amount ofA.flavus spores was determined based on the mortality rate of mice within 14 days. Fungal load and pathological changes of lung tissue were employed to determine whether the mice were infected by A.flavus. The effect of AfPV1 on the virulence of A.flavus was finally evaluated with the mouse model.[Results]The cyclophosphamide at a dose of 250 mg/kg caused the immunosuppression of ICR mice. Fungal load and histological changes demonstrated that ICR mice were successfully infected byA.flavus. In the nasal inoculation model, the inoculation with 40 μL (1×10⁶ CFU/mL spores) A.flavus was suitable for the evaluation of A.flavus virulence. In the model of caudal vein inoculation, the suitable inoculation dose ofA.flavus was 50 μL (1×10⁶ CFU/mL spores). AfPV1 weakened the virulence of A.flavus while did not affect the load of A.flavus in mice. [Conclusion] The constructed mouse infection model could evaluate the pathogenicity of A.flavus strains, and the mycovirus AfPV1 infection reduced the pathogenicity ofA.flavus.

Key words:Aspergillus flavus;mycovirus;immunosuppressed mice;infection model;caudal vein inoculation;nasal inoculation