青岛地区兔皮肤病原真菌的分离鉴定及其基本生物学特性的研究

发布时间：2018-05-21 16:16

本文选题：兔皮肤真菌病 + 分离鉴定　；参考：《山东农业大学》2012年硕士论文

【摘要】：本研究旨在确定兔皮肤病原真菌的种类，掌握该地区兔皮肤真菌病的流行特点；建立一种快速、准确的检测兔皮肤病原真菌的分子生物学方法；探明兔皮肤病原真菌的基本生物学特性，并筛选出敏感的抗真菌的药物。从而为人畜健康以及食品安全体系提供理论支持。为此，本研究从以下三个方面进行：首先，为深入了解该地区兔皮肤真菌病的流行病学特点，确定皮肤病原真菌的种类，我们历时一年（2009.6～2010.5）采集了10个兔场的880份疑似病兔的结痂和皮屑，用沙堡弱培养基(SDA)进行培养，并根据菌丝特点确定其种类，同时收集兔群的相关流行病学资料，并用统计学方法对其发病特点进行分析。结果显示：确诊兔皮肤真菌病病例为162例(18.4㳠)，其中须毛癣菌73例(45.1㳠),犬小孢子菌56例(34.6㳠)，石膏样小孢子菌31例(19.1㳠)；兔群在0～3月龄时最易感染该病，感染率高达14.3㳠，夏季为本病的多发季节（3.0㳠）。以上结果为该地区合理防控兔皮肤真菌病提供了第一手资料。其次，为快速准确地诊断皮肤病原真菌，我们以微卫星序列(GACA)4为引物，对须癣毛癣菌、犬小孢子菌和石膏样小孢子菌的3种标准菌株的DNA进行扩增，并用NTSYS-pc2.10软件分析其DNA指纹的特点，确定该方法鉴定皮肤病原真菌的可行性；然后以标准菌株为阳性对照，用该方法对已经形态学鉴定的须癣毛癣菌（10株）、犬小孢子菌（7株）和石膏样小孢子菌（4株）分离株进行鉴定。结果表明：3种不同的病原真菌均呈现特异性的DNA指纹，条带差异明显、直观，聚类分析图中，同种菌株间的相似性均在90%-100%之间。根据此方法（相似性大于90%可视为由同种株菌），21株待检菌株的鉴定结果与形态学鉴定结果相一致。由此可见，微卫星结合NTSYS-pc2.10软件分析鉴定皮肤病原真菌的方法是可行有效的。最后，对这3种常见皮肤病原真菌的基本生物学特性（生长速率、分生孢子最佳生长环境、对常见抗真菌药物敏感性和致病性）进行了研究，结果显示：在SDA、酵母完全培养基（YEPD）、改良马丁、麦芽抽提培养基（MEA）和燕麦琼脂五种培养基中，，生长速度最快的均为石膏样小孢子菌，其次为犬小孢子菌，须癣毛癣菌生长速度相对较慢；3种不同病原真菌在YEPD和MEA培养基中的菌丝生长速度均高于其余3种培养基（SDA、改良马丁和燕麦琼脂培养基）；确定了3种病原真菌分生孢子的最适培养基，须癣毛癣菌在YEPD上所产生的分生孢子数量最高，犬小孢子菌在MEA培养基中所产生的分生孢子数量最高，石膏样小孢子菌在改良马丁培养基中所产生的分生孢子数量最多；利用纸片扩散法检测3种病原菌对8种常见抗真菌药物的敏感性，结果表明这3种病原菌对氟康唑、5-氟胞嘧啶和两性霉素B均产生了耐药性；采用成熟的砂纸法制备不同病原真菌感染豚鼠和伊拉肉兔的动物模型，检测其致病性，结果显示，豚鼠和伊拉肉兔对这3种病原真菌的感染率均达到100%。该试验为进一步研究皮肤真菌病提供了科学依据。通过对青岛地区10个兔场皮肤真菌病原的分离鉴定以及深入分析该病的发病特点和规律，使我们了解了兔皮肤真菌病在该地区的流行特点，为合理防控兔皮肤真菌病提供了依据；通过利用NTSYS-pc2.10软件对微卫星引物PCR电泳条带进行分析，我们可以获得同种真菌间，标准菌株和分离菌株间的相似性大小，从而更直观准确的确定其遗传性关系，从而建立了一种快速准确的分子生物学鉴定方法；通过对3种常见病原真菌基本生物学特性的研究，我们对皮肤病原真菌的特点有了进一步的了解，为合理有效的防治皮肤真菌病提供了科学依据。
[Abstract]:The purpose of this study is to determine the species of the rabbit skin pathogenic fungi, to grasp the epidemic characteristics of the rabbit skin fungal disease in this area, to establish a rapid and accurate molecular biological method for detecting the pathogenic fungi of the rabbit skin, to explore the basic biological characteristics of the rabbit skin pathogenic fungi and to screen out the sensitive antifungal drugs. And provide theoretical support for the food safety system. For this purpose, the research is carried out in the following three aspects:
First, in order to understand the epidemiological characteristics of the rabbit skin mycosis in this area and determine the species of the skin pathogenic fungi, we took one year (2009.6 ~ 2010.5) to collect the scab and dandruff of 880 suspected sick rabbits in 10 rabbit farms, culture it with the sand castle weak culture medium (SDA), and determine the species according to the hypha characteristics, and collect the rabbit group at the same time. The epidemiological data and statistical methods were used to analyze the characteristics of the disease. The results showed that 162 cases (18.4?) were diagnosed as derma, including 73 cases of Trichophyton trichophys (45.1), 56 cases of Microsporum canis (34.6), 31 cases of Microsporum plaster (19.1), and the most susceptible to the disease at the age of 0~3 months, and the infection rate up to 14.3? The summer is the most frequent season for the disease (3?). The above results provide first-hand information for reasonable prevention and control of rabbit skin dercosis.
Secondly, in order to diagnose the skin pathogenic fungi quickly and accurately, we amplified the DNA of Trichophyton Trichophyton, Microsporum canine and gesso Microsporum by microsatellite sequence (GACA) 4, and analyzed the characteristics of DNA fingerprints by NTSYS-pc2.10 software, and then determined the feasibility of identifying the skin pathogenic fungi by this method. Using the standard strain as positive control, this method was used to identify Trichophyton Trichophyton (10 strains), 10 strains of Microsporum canis (7 strains) and 4 strains of Microsporum plaster (4 strains). The results showed that the 3 different pathogenic fungi showed specific fingerprints, and the differences of strip and bands were obvious and intuitionistic. The similarity is between 90%-100%. According to this method (the similarity is greater than 90% can be seen as the same strain of the same strain), the identification results of 21 strains to be tested are in accordance with the morphological identification results. Thus, it is feasible and effective to analyze and identify the skin pathogenic fungi by the microsatellite combined with NTSYS-pc2.10 software.
Finally, the basic biological characteristics of these 3 common skin pathogenic fungi (growth rate, the best growth environment of conidia, the sensitivity and pathogenicity of common antifungal agents) were studied. The results showed that in the SDA, the yeast complete medium (YEPD), the improved Martin, malt extract medium (MEA) and oat agar, five kinds of medium were produced. The fastest growth rate was the Microsporum gypsum like Microsporum, followed by Microsporum canis and tinea Trichophyton, the growth rate of Trichophyton tinea pedis was relatively slow. The growth rate of mycelium in the 3 different pathogenic fungi in YEPD and MEA medium was higher than that of the other 3 cultures (SDA, improved Martin and oat agar medium), and the most conidium of the 3 pathogenic fungi was determined. On the suitable medium, the number of spores produced by Trichophyton Trichophyton on YEPD is the highest. The number of conidia produced by Microsporum canis in the MEA medium is the highest. The number of conidium produced by the microspore Microsporum in the improved Martin culture medium is the most, and 3 kinds of common antifungal agents are detected by the paper spread method. The results showed that the 3 pathogenic bacteria were resistant to fluconazole, 5- fluorcytosine and amphotericin B, and a mature sand paper method was used to prepare the animal models of guinea pig and IRA rabbit with different pathogenic fungi. The results showed that the infection rate of these 3 pathogenic fungi reached 1 by the guinea pig and the IRA rabbit. 00%. this experiment provides a scientific basis for further study of skin mycosis.
Through the isolation and identification of skin fungal pathogens from 10 rabbit farms in Qingdao and the analysis of the characteristics and laws of the disease, we understand the epidemic characteristics of rabbit skin fungal disease in this area and provide the basis for the rational prevention and control of rabbit skin fungal disease. By using the NTSYS-pc2.10 software, the microsatellite primer PCR electrophoresis strip is carried out. Analysis, we can obtain the similarity between the same species of fungi, the standard strain and the isolated strain, so as to determine the genetic relationship more intuitively and accurately, thus establishing a fast and accurate molecular biological identification method. By studying the basic biological characteristics of the 3 common pathogenic fungi, we are on the skin pathogenic fungi. Further understanding of the characteristics provides a scientific basis for rational and effective prevention and treatment of skin mycosis.
【学位授予单位】：山东农业大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R756

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