羊腐蹄病微生物多样性及主要病原菌分离与耐药性分析
发布时间：2018-08-08 15:58
【摘要】：本研究在河北地区某羊场采集了19份患腐蹄病羊的蹄部拭子，随机分成3个混合样品，即sFR_1（n=6）、sFR_2（n=6）和sFR_3（n=7）；同时，采集3个健康羊蹄部拭子样品，混成1个样品sFR_C（n=3），用于对照试验。利用基于细菌16Sr RNA基因的V3-V4可变区的Illumina MiSeq二代测序技术，对sFR_1、sFR_2、sFR_3和sFR_C样品的细菌16S rRNA基因进行深度测序，通过生物信息学，分析患腐蹄病羊蹄部拭子的微生物多样性。根据微生物多样性分析的结果，本研究进一步对患腐蹄病羊蹄部拭子样品中高丰度的坏死杆菌进行了分离和PCR鉴定，并分析了坏死杆菌分离菌的耐药性。微生物多样性结果表明，在sFR_1、sFR_2、sFR_3和sFR_C样品中，获得了246478条细菌16S rRNA基因序列，产生308个独立操作分类单元（OTU）；组成成分分析显示，，患腐蹄病样品sFR_1、sFR_2和sFR_3明显的不同与健康样品sFR-C；在细菌门和属的水平上，sFR_1、sFR_2和sFR_3样品中普遍存在梭杆菌、拟杆菌、厚壁菌门和变形菌，其中坏死杆菌在所有患病样品中相对丰度最高；与健康样品sFR-C相比较，化脓杆菌在患病样品sFR_2、sFR_3中呈现出高丰度。利用厌氧培养法在患腐蹄病拭子样品中，成功分离出坏死杆菌，通过PCR确定了坏死杆菌主要毒力因子白细胞毒素的存在。药敏试验结果显示，分离的坏死杆菌对头孢吡肟、卡那霉素、庆大霉素、多粘菌素B、恩诺沙星、环丙沙星抗生素均敏感，对青霉素、红霉素、阿奇霉素、克林霉素、林可霉素、杆菌肽、磺胺异恶唑、复方新诺明均耐药。本研究明确了河北地区爆发的羊腐蹄病蹄部微生物多样性，明确了主要病原菌及其敏感药物，为羊腐蹄病的防治提供了基础。
[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.
【学位授予单位】：黑龙江八一农垦大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.26
本文编号：2172291
[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.
【学位授予单位】：黑龙江八一农垦大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：S858.26
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