【摘要】：本研究在河北地區(qū)某羊場采集了19份患腐蹄病羊的蹄部拭子，隨機分成3個混合樣品，即sFR_1（n=6）、sFR_2（n=6）和sFR_3（n=7）；同時，采集3個健康羊蹄部拭子樣品，混成1個樣品sFR_C（n=3），用于對照試驗。利用基于細菌16Sr RNA基因的V3-V4可變區(qū)的Illumina MiSeq二代測序技術，對sFR_1、sFR_2、sFR_3和sFR_C樣品的細菌16S rRNA基因進行深度測序，通過生物信息學，分析患腐蹄病羊蹄部拭子的微生物多樣性。根據微生物多樣性分析的結果，本研究進一步對患腐蹄病羊蹄部拭子樣品中高豐度的壞死桿菌進行了分離和PCR鑒定，并分析了壞死桿菌分離菌的耐藥性。微生物多樣性結果表明，在sFR_1、sFR_2、sFR_3和sFR_C樣品中，獲得了246478條細菌16S rRNA基因序列，產生308個獨立操作分類單元（OTU）；組成成分分析顯示，，患腐蹄病樣品sFR_1、sFR_2和sFR_3明顯的不同與健康樣品sFR-C；在細菌門和屬的水平上，sFR_1、sFR_2和sFR_3樣品中普遍存在梭桿菌、擬桿菌、厚壁菌門和變形菌，其中壞死桿菌在所有患病樣品中相對豐度最高；與健康樣品sFR-C相比較，化膿桿菌在患病樣品sFR_2、sFR_3中呈現(xiàn)出高豐度。利用厭氧培養(yǎng)法在患腐蹄病拭子樣品中，成功分離出壞死桿菌，通過PCR確定了壞死桿菌主要毒力因子白細胞毒素的存在。藥敏試驗結果顯示，分離的壞死桿菌對頭孢吡肟、卡那霉素、慶大霉素、多粘菌素B、恩諾沙星、環(huán)丙沙星抗生素均敏感，對青霉素、紅霉素、阿奇霉素、克林霉素、林可霉素、桿菌肽、磺胺異惡唑、復方新諾明均耐藥。本研究明確了河北地區(qū)爆發(fā)的羊腐蹄病蹄部微生物多樣性，明確了主要病原菌及其敏感藥物，為羊腐蹄病的防治提供了基礎。
[Abstract]:In this study, 19 hoof swabs were collected from a sheep farm in Hebei province, and were randomly divided into 3 mixed samples, sFR_1 (nt6) s FR2 (nFR2) and sFR_3 (N7), and three healthy swabs were collected and mixed into a sample of sFR_C (n3), which was used in a control trial. Using the second generation Illumina MiSeq sequencing technique of V3-V4 variable region based on bacterial 16Sr RNA gene, the 16s rRNA gene of sFR1 and sFR_C samples were deeply sequenced. The microbial diversity of hoof swabs was analyzed by bioinformatics. According to the results of microbial diversity analysis, the high abundance necrotic bacilli in the swabs of hoof rot were isolated and identified by PCR, and the drug resistance of necrotic bacteria was analyzed. The results of microbial diversity showed that 246478 16s rRNA gene sequences of bacteria were obtained from sFRStack2sFR3 and sFR_C samples, and 308 independent operation units (OTU);) were produced. The results showed that sFR1 / sFR2 and sFR_3 were significantly different from healthy samples (sFR-C). At the level of phylum and genus of bacteria, Clostridium, Bacteroides, phylum and Proteus are prevalent in the samples of sFR1, sFR2 and sFR_3, of which necrotic bacilli are the most abundant among all the diseased samples; compared with healthy samples, sFR-C. Pseudomonas pyogenes showed high abundance in sFRs 2 / sFR _ 3. Necrosis bacilli were isolated successfully from swabs of hoof rot by anaerobic culture method. The existence of leukocyte toxin of main virulence factor of Necrosis bacillus was determined by PCR. The drug sensitivity test showed that the isolated necrotic bacteria were sensitive to cefepime, kanamycin, gentamicin, polymyxin B, enrofloxacin, ciprofloxacin, penicillin, erythromycin, azithromycin, clindamycin, lincomycin. Bacitracin, sulfamethoxazole and compound sulfamethoxazole were all resistant. In this study, the microbial diversity of sheep hoof disease outbreak in Hebei province was confirmed, and the main pathogenic bacteria and its sensitive drugs were identified, which provided the basis for the prevention and control of sheep hoof rot disease.
【學位授予單位】：黑龍江八一農墾大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：S858.26

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