本文選題：兔皮膚真菌病 + 分離鑒定��； 參考：《山東農業(yè)大學》2012年碩士論文

【摘要】：本研究旨在確定兔皮膚病原真菌的種類，掌握該地區(qū)兔皮膚真菌病的流行特點；建立一種快速、準確的檢測兔皮膚病原真菌的分子生物學方法；探明兔皮膚病原真菌的基本生物學特性，并篩選出敏感的抗真菌的藥物。從而為人畜健康以及食品安全體系提供理論支持。為此，本研究從以下三個方面進行： 首先，為深入了解該地區(qū)兔皮膚真菌病的流行病學特點，確定皮膚病原真菌的種類，我們歷時一年（2009.6～2010.5）采集了10個兔場的880份疑似病兔的結痂和皮屑，用沙堡弱培養(yǎng)基(SDA)進行培養(yǎng)，并根據菌絲特點確定其種類，同時收集兔群的相關流行病學資料，并用統(tǒng)計學方法對其發(fā)病特點進行分析。結果顯示：確診兔皮膚真菌病病例為162例(18.4㳠)，其中須毛癬菌73例(45.1㳠),犬小孢子菌56例(34.6㳠)，石膏樣小孢子菌31例(19.1㳠)；兔群在0～3月齡時最易感染該病，感染率高達14.3㳠，夏季為本病的多發(fā)季節(jié)（3.0㳠）。以上結果為該地區(qū)合理防控兔皮膚真菌病提供了第一手資料。 其次，為快速準確地診斷皮膚病原真菌，我們以微衛(wèi)星序列(GACA)4為引物，對須癬毛癬菌、犬小孢子菌和石膏樣小孢子菌的3種標準菌株的DNA進行擴增，并用NTSYS-pc2.10軟件分析其DNA指紋的特點，確定該方法鑒定皮膚病原真菌的可行性；然后以標準菌株為陽性對照，用該方法對已經形態(tài)學鑒定的須癬毛癬菌（10株）、犬小孢子菌（7株）和石膏樣小孢子菌（4株）分離株進行鑒定。結果表明：3種不同的病原真菌均呈現特異性的DNA指紋，條帶差異明顯、直觀，聚類分析圖中，同種菌株間的相似性均在90%-100%之間。根據此方法（相似性大于90%可視為由同種株菌），21株待檢菌株的鑒定結果與形態(tài)學鑒定結果相一致。由此可見，微衛(wèi)星結合NTSYS-pc2.10軟件分析鑒定皮膚病原真菌的方法是可行有效的。 最后，對這3種常見皮膚病原真菌的基本生物學特性（生長速率、分生孢子最佳生長環(huán)境、對常見抗真菌藥物敏感性和致病性）進行了研究，結果顯示：在SDA、酵母完全培養(yǎng)基（YEPD）、改良馬丁、麥芽抽提培養(yǎng)基（MEA）和燕麥瓊脂五種培養(yǎng)基中，，生長速度最快的均為石膏樣小孢子菌，其次為犬小孢子菌，須癬毛癬菌生長速度相對較慢；3種不同病原真菌在YEPD和MEA培養(yǎng)基中的菌絲生長速度均高于其余3種培養(yǎng)基（SDA、改良馬丁和燕麥瓊脂培養(yǎng)基）；確定了3種病原真菌分生孢子的最適培養(yǎng)基，須癬毛癬菌在YEPD上所產生的分生孢子數量最高，犬小孢子菌在MEA培養(yǎng)基中所產生的分生孢子數量最高，石膏樣小孢子菌在改良馬丁培養(yǎng)基中所產生的分生孢子數量最多；利用紙片擴散法檢測3種病原菌對8種常見抗真菌藥物的敏感性，結果表明這3種病原菌對氟康唑、5-氟胞嘧啶和兩性霉素B均產生了耐藥性；采用成熟的砂紙法制備不同病原真菌感染豚鼠和伊拉肉兔的動物模型，檢測其致病性，結果顯示，豚鼠和伊拉肉兔對這3種病原真菌的感染率均達到100%。該試驗為進一步研究皮膚真菌病提供了科學依據。 通過對青島地區(qū)10個兔場皮膚真菌病原的分離鑒定以及深入分析該病的發(fā)病特點和規(guī)律，使我們了解了兔皮膚真菌病在該地區(qū)的流行特點，為合理防控兔皮膚真菌病提供了依據；通過利用NTSYS-pc2.10軟件對微衛(wèi)星引物PCR電泳條帶進行分析，我們可以獲得同種真菌間，標準菌株和分離菌株間的相似性大小，從而更直觀準確的確定其遺傳性關系，從而建立了一種快速準確的分子生物學鑒定方法；通過對3種常見病原真菌基本生物學特性的研究，我們對皮膚病原真菌的特點有了進一步的了解，為合理有效的防治皮膚真菌病提供了科學依據。
[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.
【學位授予單位】：山東農業(yè)大學
【學位級別】：碩士
【學位授予年份】：2012
【分類號】：R756

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本文編號：1919902