中国农业科学 ›› 2015, Vol. 48 ›› Issue (14): 2876-2883.doi: 10.3864/j.issn.0578-1752.2015.14.019

• 研究简报 • 上一篇    下一篇

宁夏肉牛皮肤病原真菌的分离与鉴定

葛松1,蒋万2,何生虎1,余永涛1,张蕾蕾1,郭澍强1,王静1   

  1. 1宁夏大学农学院,银川750021
    2宁夏大学实验农场,银川750021
  • 收稿日期:2014-06-06 出版日期:2015-07-16 发布日期:2015-07-16
  • 通讯作者: 何生虎,E-mail:heshenghu308@163.com
  • 作者简介:葛松,E-mail:gesong2099@163.com
  • 基金资助:
    宁夏科技攻关重点专项(412-0164)

Isolation and Identification of Dermatophytes from Beef Cattle in Ningxia

GE Song1, JIANG Wan2, HE Sheng-hu1, YU Yong-tao1, ZHANG Lei-lei1, GUO Shu-qiang1, WANG Jing1   

  1. 1College of Agronomy, Ningxia University, Yinchuan 750021
    2Teaching Experimental Farm, Ningxia University, Yinchuan 750021
  • Received:2014-06-06 Online:2015-07-16 Published:2015-07-16

摘要: 【目的】从患真菌性皮肤病肉牛的毛发、皮屑、痂皮等病料样本中分离皮肤病原真菌并对其进行种属分类鉴定,为牛真菌性皮肤病的诊断及防治提供参考依据。【方法】无菌采集宁夏地区患皮肤病肉牛的毛发、皮屑、痂皮,将病料样本置于载玻片上,滴加10% KOH溶液1滴,静置5 min后进行镜检观察;将病料样本接种于沙氏葡萄糖琼脂培养基(SDA)中进行真菌的分离、纯化培养,对分离的真菌用乳酸酚棉兰染液进行染色观察,根据真菌形态学特征对其进行初步分类;用分离的真菌制备浓度为1×108 cfu·mL-1的孢子悬液涂抹健康ICR小鼠皮肤,根据其致病性筛选病原真菌;提取病原真菌DNA,对其内部转录间隔区(ITS)进行PCR扩增并测序,登录NCBI GenBank数据库,对病原真菌ITS序列进行同源性比较并构建系统发育树,根据其同源性比较及系统发育分析结果,结合真菌形态学特征对病原真菌作出种属分类鉴定。【结果】病料中含真菌菌丝及链状厚垣孢子,毛发中存在大量小分生孢子;从病料样本中共分离、纯化到23株真菌,分属于毛癣菌属(Trichophyton)、曲霉属(Aspergillus)、横梗霉属(Lichtheimia)、链格孢属(Alternaria)、镰刀菌属(Fusarium)、附球菌属(Epicoccum)、毛霉菌属(Mucor);小鼠皮肤感染试验表明,分离的毛癣菌属真菌NXGY1、NXGY2具有较强的致病性,试验组小鼠均出现瘙痒、皮屑增多、形成结痂、脱毛等临床症状;毛癣菌属NXGY1、NXGY2 的ITS序列长度大小分别为660、662 bp,其同源性比较及系统发育分析结果表明,NXGY1、NXGY2与疣状毛癣菌(Trichophyton verrucosum)同源性高达99% ,在系统发育树上属于同一分支,遗传距离最近。【结论】根据真菌形态特征及系统发育分析结果,鉴定分离的肉牛皮肤病原真菌NXGY1、NXGY2为疣状毛癣菌(T. verrucosum)。

关键词: 肉牛, 皮肤癣菌, ITS序列, 系统发育分析, 疣状毛癣菌

Abstract: 【Objective】 An experiment was carried out to isolate and identify the pathogenic fungi from the skin scrapings of beef cattle affected by dermatophytes in Ningxia, in order to provide reference for the diagnosis, prevention and control of dermatophytes in cattle population. 【Method】 The samples of scrapings and hairs were collected from beef cattle affected by dermatophytes in Ningxia. Each sample was placed on microslide containing a drop of 10% potassium hydroxide solution and covered with a cover slip, and observed under the light microscope. Each sample was inoculated onto the Sabouraud’s dextrose agar medium(SDA)to isolate and purify the fungi, and the fungal isolates were observed under the light microscope containing lactophenol cotton blue stain, the identification and classification of each isolate were performed preliminarily according to the morphological characteristics. The conidial suspension at a density of 1×108 cfu/mL from the isolates was used to infect the healthy ICR mice’s skin to confirm whether the isolates do has pathogenicity or not and to screen pathogenic fungi. The suspected pathogenic fungi’s genome DNA were extracted, the ITS sequences of which were amplified and sequenced, and finally classified according to the homology comparision and phylogenetic analysis combined with the morphological characteristics. 【Result】  According to the microscopic examination, many fungal hyphae and chlamydospore were observed in chains in the samples, and a lot microcondia inside the hairs. Twenty-three fungi were isolated from the samples, and the isolates were classified into the genera like Trichophyton, Aspergillus, Lichtheimia, Alternaria, Fusarium, Epicoccum, Mucor. Two of Trichophyton NXGY1 and NXGY2 were confirmed to be pathogenic fungi according to the skin infection experiments in mice, and all the infected mice with clinical symptoms like itching, dandruff increasing, scabbing, unhairing. The ITS sequence lengths of NXGY1 and NXGY2 were 660 bp and 662 bp, and the homology compared with Trichophyton verrucosum was up to 99% according to the ITS sequences homology comparison. Phylogenetic analyses showed that NXGY1 and NXGY2 were also highly similar to T. verrucosum. 【Conclusion】 The results indicated that NXGY1, NXGY2 isolated in this research were Trichophyton verrucosum.

Key words: beef cattle, dermatophytes, ITS sequences, phylogenetic analysis, Trichophyton verrucosum