不同曲霉感染大蜡螟幼虫模型的构建及曲霉毒力评价

The construction of different Aspergillus infection models of Galleria mellonella larvae and the evaluation of Aspergillus virulence

目的探讨不同曲霉感染大蜡螟幼虫模型的构建及曲霉毒力的鉴定。方法取烟曲霉ygy和黄曲霉LD-F菌株接种于马铃薯葡萄糖琼脂培养基(PDA),30℃培养5 d,分别收集曲霉孢子并配制1×10^(8) CFU/L(低剂量)、1×10^(9) CFU/L(中剂量)及1×10^(10) CFU/L(高剂量)孢子悬液;取大蜡螟幼虫60条随机均分为6组,分别注射上述2种曲霉孢子悬液高、中、低剂量各10μL,同时再取大蜡螟60条幼虫随机均分为6组,对应2种曲霉各设置3种形式对照,即空白对照(UTC)组(不做任何处理)、穿刺(PC)组[微量注射器穿刺但不注射磷酸盐缓冲液(PBS)]及PBS组(注射接种PBS 10μL),各组大蜡螟幼虫于37℃黑暗环境培养5 d,每日监测各组大蜡螟幼虫的体表颜色、应对刺激反馈及存活情况,采用生存曲线评价2种曲霉的毒力强弱;取2种曲霉孢子悬液高剂量组大蜡螟幼虫感染48 h后死亡幼虫和同时点的UTC组大蜡螟幼虫,采用苏木精和曙红(HE)染色检测幼虫虫体组织的病理学特征,采用六铵银染色检测幼虫体腔内菌丝形成情况,二者结合判断幼虫感染曲霉是否成功。结果烟曲霉和黄曲霉感染幼虫后,幼虫黑化,其黑化程度与感染孢子悬液质量浓度无相关性,但其毒力有质量浓度依赖性(P<0.0001);组织病理检查可见幼虫体腔结构破坏,真菌菌丝在幼虫体内定植,表明曲霉感染模型构建成功。结论成功构建曲霉感染的大蜡螟幼虫模型,烟曲霉毒力高于黄曲霉,相同曲霉菌株毒力有质量浓度依赖性。

Objective To construct different Aspergillus infection models of Galleria mellonella larvae and evaluate the Aspergillus virulence.Methods Aspergillus fumigatus strain ygy and Aspergillus flavus strain LD-F were cultured on potato dextrose agar medium(PDA)at 30℃for 5 days,then spores were harvested and prepared into concentrations of 1×10^(8) CFU/L(low-dose),1×10^(9) CFU/L(medium-dose),and 1×10^(10) CFU/L(high-dose).Sixty larvae of G.mellonella were randomly divided into 6 groups and injected with 10μL spore suspension of high-dose,medium-dose,and low-dose of the A.fumigatus and A.flavus respectively.At the same time,other 60 larvae of G.mellonella were randomly divided into 6 groups including UTC group(untouched larvae without any treatment as controls),PC group[microsyringe puncture without phosphate buffered saline(PBS)injection],and PBS group(with 10μL PBS injection).All the G.mellonella larvae were cultured in the dark at 37℃for 5 days.The body surface color,response to stimulus feedback,and survival of the G.mellonella larvae in each group were monitored daily.The survival curve was used to evaluate the virulence of A.fumigatus and A.flavus.In order to determine Aspergillus infection,the pathological characteristics of the larvae were checked with hematoxylin and eosin(HE),and the formation of mycelia in the lumen of larvae were detected with silver hexaammonium after 48 h of infection with high-dose compared with UTC group.Results The G.mellonella larvae were used as a model organism to assess the virulence of Aspergillus infection.Histopathological examination showed that the larva’s body cavity was destroyed,and the fungus colonized inside the larvae.Melanization was observed in all infected larvae,and there were significant differences in the survival rates of larvae injected with different concentrations of spore suspension(P<0.0001).Conclusion G.mellonella larvae could be used as Aspergillus infection model.The virulence of A.fumigatus is higher than that of A.flavus,and the virulence of each strain is dependent on concentration.