我国部分省区猪源多杀性巴氏杆菌的分子流行病学调查

发布时间：2018-04-18 09:38

本文选题：多杀性巴氏杆菌 + 毒力基因　；参考：《华中农业大学》2017年硕士论文

【摘要】：多杀性巴氏杆菌(Pasteurella multocida,Pm)是常见的共生菌或机会性病原菌,该菌宿主范围广,通常能在大多数畜禽以及野生动物的上呼吸道分离到。就畜牧养殖业而言,该菌可引发猪肺疫、禽霍乱、牛出血性败血症、兔出血性败血症等,给畜牧业生产造成巨大的经济损失。此外,Pm也是一种人兽共患病原菌。人发生本病后,主要表现为伤口感染。Pm依据荚膜抗原、菌体抗原、脂多糖抗原的差异,可分为多个血清型,各血清型之间多数无交叉免疫原性。另外,特定的疾病往往与Pm特定的血清型相关。按照荚膜分型法,禽霍乱、猪肺疫常与Pm A型菌株相关;而按照赫德尔斯顿脂多糖分型法,引起禽霍乱的菌株其脂多糖血清型常为Pm 1型和3型。然而,这些特定血清型与宿主疾病相关性的分子和细胞基础,至今仍不清楚。本研究对我国14个省份与猪肺炎相关的Pm进行了分离与鉴定。通过荚膜多重PCR、脂多糖多重PCR、毒力基因的检测、多位点序列分型等方法,对所分离的菌株进行研究,旨在了解此类菌株的流行情况以及分子特征,为防控巴氏杆菌病奠定基础。主要研究结果如下:1.猪源Pm的分离鉴定2015年~2016年从我国14省份的1597份临床猪肺炎肺脏中共分离115株Pm,分离率为7.2%。对所分离菌株进行荚膜多重PCR分型,其中A型57(50%)株,D型53(46%)株,未定型5(4%)株。2.猪源Pm的脂多糖分型对所分离的115株Pm进行脂多糖多重PCR分型,结果发现所有菌株均能定型。而脂多糖型仅有L3和L6两种,其中L3型26株,占23%,L6型89株,占77%。我们把荚膜血清型与脂多糖基因型进行组合后,发现荚膜D型菌株仅与脂多糖L6型相对应。3.猪源Pm毒力基因分布应用PCR对115株Pm的23个毒力基因进行检测,并分析这些毒力基因的分布情况。结果发现其中一些毒力基因总的检出率为100%,这些基因包括黏附因子相关基因ptfA、fimA、hsf-2;铁摄取相关基因exbB、exbD、tonB;外膜蛋白基因opmA、ompH、omp87;超氧化物歧化酶相关基因sodA、sodC。也有一些毒力基因总的检出率很低,如转铁结合蛋白基因tbpA,检出率为0,又如PMT毒素基因toxA,检出率为2.6%。在不同型(荚膜血清型、脂多糖基因型、荚膜:脂多糖组合型)的菌株间一些毒力基因的检出率有较大的差别。以荚膜分型为例,pfhA、tadD、pmHAS、hgbA基因在A型菌株中的检出率显著高于D型菌株中的检出率(P0.05),而hsf-1、hgbB基因在D型菌株中的检出率显著高于A型菌株中的检出率(P0.05)。同样的,以脂多糖分型为例,pfhA、tadD、pmHAS、hgbA、nanH基因在L3型菌株中的检出率显著高于L6型菌株中的检出率(P0.05),而hgbB、nanB基因在L6型菌株中的检出率显著高于L3型菌株中的检出率(P0.05)。通过进一步统计分析荚膜:脂多糖组合型间毒力基因的差异,首次发现pfhA等基因的检出率可能与荚膜型无关,而和脂多糖型相关。4.猪源Pm的MLST研究本研究对我国不同省份的23株Pm分离株进行MLST分型,结果发现了ST3、ST10、ST11三个ST型。而在湖北、广东等多个省份,猪源Pm不止一个ST型。ST11序列类型的菌株在我国七个省份均有分离到,表明了该类型菌株在我国分布的广泛性。因此,需要加强对此类型菌株的流行病学监测和防控。本研究中从山西、湖北、广东分离的3株产毒素Pm都属于ST11型,而本实验室先前保存的8株产毒素Pm也属于ST11型。
[Abstract]:Pasteurella multocida (Pasteurella multocida Pm) is a commensal bacterium or common opportunistic pathogen, the pathogen host range, usually isolated in the upper respiratory tract of livestock and poultry and wild animal. Most on animal husbandry, the bacteria can cause lung disease, fowl cholera, cattle hemorrhagic septicemia, hemorrhage in rabbits septicemia, causing huge economic losses to livestock production. In addition, Pm is a zoonosis pathogen. The occurrence of the disease, mainly for the.Pm on the basis of capsular antigen, infected wound cell antigen, LPS antigen differences, can be divided into multiple serotypes among serotypes most no cross immunogenicity. In addition, certain diseases are often associated with Pm serotype specific. In accordance with the capsule type method, fowl cholera, lung disease is often associated with Pm type A strain; according to Huddleston lipopolysaccharide classification, caused by avian cholera strains The lipopolysaccharide serotype often Pm type 1 and type 3. However, the molecular and cellular basis of these specific serotypes and host disease, is still unclear. In this study, the isolation and identification of 14 provinces in China associated with swine pneumonia Pm. Through multiple capsular PCR, lipopolysaccharide multiple PCR. Detection of virulence genes, multilocus sequence typing method to study the isolated strains, in order to understand the epidemic situation of the strain and molecular characteristics, lay the foundation for the prevention and control of pasteurellosis. The main results are as follows: 1. isolation and identification of porcine Pm in 2015 ~2016 from 1597 clinical isolated pig lung pneumonia 115 strains of Pm 14 provinces of our country, the isolation rate of 7.2%. PCR on isolated from multiple capsular types, including A type 57 (50% strains), D 53 (46%) strains of undetermined type 5 (4%) strains of porcine.2. lipopolysaccharide Pm type of fat from 115 line Pm Polysaccharide multiple PCR types, the results showed that all strains could shape. Only L3 and L6 and lipopolysaccharide type two species, including 26 strains of type L3, accounting for 23%, 89 strains of L6, accounting for 77%. we put a combination of capsular serotypes and genotypes of lipopolysaccharide, found capsular type D strains only with lipopolysaccharide L6 the corresponding.3. distribution of porcine Pm virulence genes using PCR 23 virulence genes of 115 strains of Pm were detected, and the analysis of the distribution of these virulence genes. The results showed that some virulence genes of the total detection rate was 100%. These genes include adhesion factor related genes ptfA, fimA, hsf-2; iron uptake related gene exbB. ExbD, tonB; outer membrane protein gene opmA, ompH, omp87; superoxide dismutase gene related to sodA, sodC. also has some virulence genes in the overall detection rate is very low, such as the transferrin binding protein gene tbpA, the detection rate was 0, and PMT toxA gene, the detection rate of 2.6%. in different Type (capsular serotype, genotype: lipopolysaccharide, lipopolysaccharide combined capsular type) between strains of some virulence genes detection rate is different. The capsule type as an example, pfhA, tadD, pmHAS, the detection rate of hgbA gene in A strains was significantly higher than the detection rate of D strain in P0.05 (), while hsf-1, the detection rate of hgbB gene in D strains was significantly higher than the detection rate of A strain in (P0.05). Similarly, the type of LPS for example, pfhA, tadD, pmHAS, hgbA, the detection rate of nanH gene in L3 strains was significantly higher than that of L6 strain in the examination rate (P0.05), and hgbB, the detection rate of nanB gene in L6 strains was significantly higher than the detection rate of L3 strain in (P0.05). Through further statistical analysis: the difference between lipopolysaccharide combined capsular virulence gene, first found pfhA gene detection rate may have nothing to do with the capsule type, and lipopolysaccharide type.4. swine Pm The MLST of this study in different provinces of our country 23 Pm isolates were typed by MLST, found that ST3, ST10, ST11 three ST. In Hubei, Guangdong and other provinces, more than one ST of porcine Pm.ST11 sequence type strains isolated in China seven provinces have, show that the type of strain in the wide distribution in China. Therefore, the need to strengthen this type strains of epidemiological surveillance and prevention. This study from Shanxi, Hubei, Guangdong, 3 strains of toxigenic Pm belong to ST11 type, while the laboratory previously saved 8 strains belong to Pm toxin type ST11.

【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S852.61

【参考文献】