禽源烟曲霉菌和黄曲霉菌的分离鉴定及几种中药抑菌作用研究

目的] 对保定市临床真菌感染病鸭进行菌株分离鉴定，并筛选分离菌株的体外抑菌药物。方法] 通过形态学观察、PCR扩增、测序及动物回归试验对菌株进行鉴定，并采用微量稀释法测定最小抑菌浓度（MIC）及体外药敏试验统计菌落数量和直径来探究石菖蒲、黄柏、决明子、苦参、辣蓼、白头翁和蒲公英对烟曲霉菌（Aspergillus fumigatus）和黄曲霉菌（Aspergillus flavus）的体外抑菌活性。结果] 成功分离到烟曲霉菌和黄曲霉菌。动物回归试验结果显示，雏鸡出现与病鸭相似的症状，剖检可见肝脏长出灰白色结节，且病变组织出现肉芽肿病变，说明2种病原菌均可使雏鸡感染。烟曲霉菌MIC₈₀结果显示，石菖蒲、黄柏、决明子和苦参MIC₈₀分别为8、16、32和64 μg/μL，辣蓼、白头翁和蒲公英均为128 μg/μL；黄曲霉菌的MIC₈₀结果显示，石菖蒲、决明子和黄柏MIC₈₀分别为8、16和32 μg/μL，苦参和辣蓼均为64 μg/μL，白头翁和蒲公英均为128 μg/μL。体外药敏试验结果显示，在石菖蒲浓度为16 μg/μL时培养基长出烟曲霉菌和黄曲霉菌菌落，在决明子浓度为32 μg/μL时培养基长出黄曲霉菌菌落，黄柏浓度为32 μg/μL时培养基中长出烟曲霉菌菌落，而在苦参、辣蓼、白头翁和蒲公英浓度128 μg/μL培养基中就已长出直径较大的烟曲霉菌和黄曲霉菌菌落。结论] 石菖蒲、黄柏和决明子对烟曲霉菌和黄曲霉菌的生长具有显著的抑制作用，其中石菖蒲的抑制作用最为显著。本试验可为抗家禽曲霉病的中药研发提供新的思路。

Isolation and Identification of Aspergillus fumigatus and Aspergillus flavus from Poultry and Bacteriostasis of Several Traditional Chinese Medicines

Objective] The purpose of this study was to isolate and identify the isolated strains of ducks with clinical fungal infections in Baoding city, and screening the antibacterial drugs for the isolated strains in vitro. Method] The strains were identified by morphological observation, PCR amplification, sequencing and animal regression test. Microdilution method was used to determine the minimum inhibitory concentration (MIC) and in vitro drug sensitivity test was used to calculate the colony count and diameters to investigate the in vitro antibacterial activities of Acorus gramineus, Phellodendron chinensis, Cassia obtusifolia, Sophora flavescens, Polygonum hydropiper, Pulsatilla adans and Herba taraxaci against Aspergillus fumigatus and Aspergillus flavus. Result] Aspergillus fumigatus and Aspergillus flavus were successfully isolated. The results of the animal regression test showed that the chicks had the symptoms similar with diseased ducks, the liver had gray-white nodules on autopsy, and granulomatous lesions appeared in the diseased tissues, indicating that both pathogens could infect the chicks. The MIC₈₀ results of Aspergillus fumigatus showed that the MIC₈₀ of Acorus gramineus Phellodendron chinensis, Cassia obtusifolia and Sophora flavescens were 8, 16, 32 and 64 μg/μL, respectively, and Polygonum hydropiper, Pulsatilla adans and Herba taraxaci were all 128 μg/μL. The MIC₈₀ results of Aspergillus flavus showed that the MIC₈₀ of Acorus gramineus, Cassia obtusifolia and Phellodendron chinensis were 8, 16 and 32 μg/μL, respectively, and Sophora flavescens and Polygonum hydropiper were both 64 μg/μL, and Pulsatilla adans and Herba taraxaci were both 128 μg/μL. The results of drug sensitivity test in vitro showed that when the concentration of Acorus gramineus was 16 μg/μL, Aspergillus fumigatus and Aspergillus flavus colonies were grown on the medium, and when the concentration of Cassia obtusifolia was 32 μg/μL, colonies of Aspergillus flavus were grown on the medium, and the concentration of Phellodendron chinensis was 32 μg/μL, Aspergillus fumigatus colonies grew in the medium, while the larger diameter Aspergillus fumigatus and Aspergillus flavus colonies were grown in the medium with a concentration of 128 μg/μL of Sophora flavescens, Polygonum hydropiper, Pulsatilla adans and Herba taraxaci. Conclusion] Acorus gramineus, Phellodendron chinensis, Cassia obtusifolia had significant inhibitory effects on the growth of Aspergillus fumigatus and Aspergillus flavus, among which Acorus gramineus had the most significant inhibitory effect. This experiment could provide new ideas for the research and development of traditional Chinese medicines against poultry aspergillosis.