藏羚羊链球菌新种的发现及我国动物源性产志贺毒素大肠埃希菌的分析

发布时间：2018-01-06 13:27

  本文关键词：藏羚羊链球菌新种的发现及我国动物源性产志贺毒素大肠埃希菌的分析　出处：《中国疾病预防控制中心》2016年博士论文　论文类型：学位论文

  更多相关文章： 高原动物 藏羚羊 链球菌新种 产志贺毒素大肠埃希菌 流行

【摘要】：近年来在不断出现的新发传染病中,动物源性病原体占绝大多数。分布在高原、热带等特殊气候地理环境的某些重要野生动物,可携带和传播某些已知和未知的病原体。本课题集中在对我国青藏高原藏羚羊携带可能未知病原体的发现及其相关致病性研究,并系统地开展以青藏高原特殊地理环境中野生动物为主的我国动物源性产志贺毒素大肠埃希菌的流行情况调查及菌株特征分析。通过尝试不同的细菌分离培养方法,我们成功从两份藏羚羊粪便标本中分离出两株新细菌,菌株编号TA26、TA421。通过生化鉴定、形态学观察、培养特性、16S rRNA序列分析及基因组杂交,最终确定这两株新细菌属于链球菌属新种,建议命名为藏羚羊链球菌(Streptococcus pantholopis sp. nov.)。新种链球菌在血平板上24 h后生长,形成白色、圆形隆起、边缘不规则、直径0.75-1.00 mm的细小菌落,培养48 h后出现α溶血现象。菌体呈成双或数个短链状排列,革兰染色阳性。链球菌新种在22、30、37、42℃培养均时生长,4、15℃下培养不生长。菌株在含3.5% NaCl的BHI培养基上生长,在含4.5、6.5% NaCl的BHI上不生长。生化实验结果显示链球菌新种氧化酶阴性,发酵乳糖、山梨糖、麦芽糖,生化特征与同属内相近的链球菌种存在明显差异。16S rRNA基因进行进化分析显示,新种链球菌处在一个独立的进化分支上,属于变异链球菌群。对藏羚羊链球菌新种模式菌株TA 26T进行基因组测序及序列分析,S. pantholopis新种基因组全长2,241,302 bp,G+C含量为42 mo1%,预测含有2,251个编码基因CDSs,66个tRNAs,15个rRNAs,7个不完整的前噬菌体。通过与GenBank中其他已知链球菌种进行基因组杂交,发现藏羚羊链球菌与已知链球菌种相似性较低(17.1%-46.1%)。与已知链球菌种基因组进化分析显示,新种链球菌处于一个独立的进化分支。同时在基因组分析中我们发现了相关毒力因子,如肺炎球菌粘附因子A(pavA)、其他粘附相关因子(hasC、cna)、内毒素及毒力表达调控因子等,提示藏羚羊链球菌可能具有致病性。产志贺毒素大肠埃希菌(Shiga toxin-producing Escherichia coli, STEC)是一类重要的人兽共患病原体,许多家禽家畜、野生动物为STEC的储存宿主。然而STEC在我国不同动物中的流行情况缺乏系统的调查研究,尤其是野生动物中STEC的流行情况尚处于空白。本研究系统地开展以青藏高原动物为主,包括高原鼠兔、藏羚羊、早獭、牦牛,及其他动物源性宿主包括家养动物、不同类生肉中产志贺毒素大肠埃希菌的流行情况调查。研究从1116份高原鼠兔中分离出22株STEC菌株,从187份藏羚羊粪便中分离出2株菌,162份旱獭粪便中分离出4株菌,100份肉牛粪便分离出5株菌,从853份生肉标本中分离出63株菌,分离率分别为1.97%、1.07%、2.47%、5.0%及6.8%,表明非0157 STEC在我国多种动物源性宿主中存在,同时证实了高原动物也是STEC的重要宿主。研究首次重点对青藏高原特殊地理环境下高原鼠兔中STEC的流行及菌株特征进行分析,通过对菌株进行血清分型,毒力因子分析,耐药性检测,MLST及PFGE分型分析,显示高原鼠兔STEC分离株呈现出表型和基因型多态性。鼠兔STEC分离株与我们之前分离自同一地区牦牛STEC分离株有9个相同的ST型别(;ST40、 ST297、ST737、ST906、ST1001、ST3686、ST3692、ST3883、ST3884),相同的流行血清型02：H45及相似的毒力基因型,提示STEC菌株在同一地区不同动物之间广泛传播。将我国不同动物源性STEC分离株与国际上报道的STEC致病株及主要流行血清型菌株进行MLST分析发现,我国高原地区动物包括鼠兔、藏羚羊、早獭、牦牛的STEC分离株、山羊STEC分离株、不同生肉分离株均存在与HUSEC、流行血清型菌株相同或相近的ST型别,结合菌株血清型、毒力基因型及进化分析推测我国动物源性宿主中存在致病潜力的菌株。
[Abstract]:In recent years, the emergence of new infectious diseases, animal pathogens accounted for the vast majority. Located in the plateau, the special geographical environment of the tropical climate of some important wild animal, can carry and spread some known and unknown pathogens. This study focused on China's Qinghai Tibet Plateau and its related pathogenic research may carry the Tibetan antelope the discovery of unknown pathogens, analysis the prevalence and characteristics of strains and the system to carry out the wild animal special geographical environment on the Qinghai Tibet Plateau in the main animal source in China of Shiga toxin producing Escherichia coli. Try through the methods of isolation and culture of different bacteria, we successfully from two samples isolated from stool specimens of two Tibetan antelope new strains of bacteria, strains TA26, TA421. through biochemical identification, morphological observation, culture characteristics, 16S rRNA sequence analysis and genomic hybridization, and ultimately determine the two new strains of bacteria In Streptococcus species, suggested named Streptococcus (Streptococcus pantholopis sp. Nov. Tibetan antelope). New species of Streptococcus in blood agar after 24 h growth, the formation of white, rounded protuberance, irregular edge, small colony diameter 0.75-1.00 mm, alpha hemolytic culture phenomenon after 48 h. A number of bacteria in pairs or short chains. Gram positive species were cultured. Streptococcus growth at 22,30,37,42 DEG 4,15 DEG C, cultivation of growth. Strains grow on medium containing 3.5% NaCl BHI, with 4.5,6.5% NaCl do not grow in BHI. The experimental results show that the new biochemical Streptococcus oxidase negative, fermentation of lactose, sorbitol, maltose, Streptococcus a significant difference of.16S rRNA gene phylogenetic analysis revealed that the biochemical characteristics and are similar, species of Streptococcus in a independent evolutionary branch, which belongs to the group of Streptococcus mutans on Tibetan antelope chain. The new model of 26T aureus strain TA genome sequencing and sequence analysis, S. new species of pantholopis genome is 2241302 BP, the content of G+C is 42 mo1%, containing 2251 predicted genes encoding CDSs, 66 tRNAs, 15 rRNAs, 7 incomplete prophage and GenBank. Through it he known for Streptococcus genomic hybridization. The Tibetan antelope found streptococcus and Streptococcus strains known low similarity (17.1%-46.1%). With the known streptococcal species genome evolution analysis showed that the species of Streptococcus in an independent evolutionary branch. At the same time in the analysis of genomes we find the virulence factors, such as pneumococcal adhesion factor A (pavA), other adhesion related factors (hasC, CNA), endotoxin and virulence factor expression, suggesting that the Tibetan antelope may have Pathogenic Streptococcus. Shiga toxin producing Escherichia coli (Shiga toxin-producing Escherichia coli, S TEC) is a kind of important zoonotic pathogens, many poultry and livestock, wild animal STEC reservoirs. But the lack of systematic investigation and study the epidemic situation of STEC in different animal in our country, especially the prevalence of STEC in wild animal is still in the blank. This study systematically carried out in Tibetan Plateau animal including, plateau pika, Tibetan antelope, marmots, yak, and other animal hosts including domestic animal epidemic investigation, different kinds of meat of Shiga toxin producing Escherichia coli. The 22 strains of STEC were isolated from 1116 strains of plateau pika, 2 bacterial strains were isolated from 187 samples of Tibetan antelope, separation 4 strains of 162 samples of feces in 100 marmots, beef cattle manure isolated 5 strains of bacteria, 63 bacterial strains were isolated from 853 raw meat samples, the isolation rate was 1.97%, 1.07%, 2.47%, 5% and 6.8%, showed that 0157 non STEC in a variety of animal in China There is the source host, also confirmed the important host animal plateau is STEC. The first study focused on the STEC special geographical environment of the Tibetan Plateau Pika in epidemic strains and characteristics were analyzed, the strains were serotype, virulence factor analysis, drug resistance detection, analysis of MLST and PFGE, showed that plateau pika STEC isolates showed phenotypic and genotypic polymorphism. STEC isolates and pikas before we isolated from the same region of yak STEC isolates have 9 of the same type of ST (ST40, ST297; ST737, ST906, ST1001, ST3686, ST3692, ST3883, ST3884, 02:H45) serotypes of the same or similar the virulence genes, suggesting that STEC strains are widely spread in the same area in different animal. STEC animal pathogenic strains isolated in China STEC and the main serotypes of strains with the reported MLST analysis Found that the plateau animal in China include the pika, Tibetan antelope, marmots, yak STEC isolates, goat STEC isolates, different raw strains were present with HUSEC, the popular serotypes of the same or similar type of ST, combined with the strains of serotype, virulence genotype and phylogenetic analysis of putative pathogenic potential animal source I in the host strain.

【学位授予单位】：中国疾病预防控制中心
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S852.61
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本文编号：1388065