本文選題：多殺性巴氏桿菌 + 毒力基因��； 參考：《華中農(nóng)業(yè)大學(xué)》2017年碩士論文

【摘要】：多殺性巴氏桿菌(Pasteurella multocida,Pm)是常見的共生菌或機(jī)會性病原菌,該菌宿主范圍廣,通常能在大多數(shù)畜禽以及野生動物的上呼吸道分離到。就畜牧養(yǎng)殖業(yè)而言,該菌可引發(fā)豬肺疫、禽霍亂、牛出血性敗血癥、兔出血性敗血癥等,給畜牧業(yè)生產(chǎn)造成巨大的經(jīng)濟(jì)損失。此外,Pm也是一種人獸共患病原菌。人發(fā)生本病后,主要表現(xiàn)為傷口感染。Pm依據(jù)莢膜抗原、菌體抗原、脂多糖抗原的差異,可分為多個血清型,各血清型之間多數(shù)無交叉免疫原性。另外,特定的疾病往往與Pm特定的血清型相關(guān)。按照莢膜分型法,禽霍亂、豬肺疫常與Pm A型菌株相關(guān);而按照赫德爾斯頓脂多糖分型法,引起禽霍亂的菌株其脂多糖血清型常為Pm 1型和3型。然而,這些特定血清型與宿主疾病相關(guān)性的分子和細(xì)胞基礎(chǔ),至今仍不清楚。本研究對我國14個省份與豬肺炎相關(guān)的Pm進(jìn)行了分離與鑒定。通過莢膜多重PCR、脂多糖多重PCR、毒力基因的檢測、多位點(diǎn)序列分型等方法,對所分離的菌株進(jìn)行研究,旨在了解此類菌株的流行情況以及分子特征,為防控巴氏桿菌病奠定基礎(chǔ)。主要研究結(jié)果如下:1.豬源Pm的分離鑒定2015年~2016年從我國14省份的1597份臨床豬肺炎肺臟中共分離115株P(guān)m,分離率為7.2%。對所分離菌株進(jìn)行莢膜多重PCR分型,其中A型57(50%)株,D型53(46%)株,未定型5(4%)株。2.豬源Pm的脂多糖分型對所分離的115株P(guān)m進(jìn)行脂多糖多重PCR分型,結(jié)果發(fā)現(xiàn)所有菌株均能定型。而脂多糖型僅有L3和L6兩種,其中L3型26株,占23%,L6型89株,占77%。我們把莢膜血清型與脂多糖基因型進(jìn)行組合后,發(fā)現(xiàn)莢膜D型菌株僅與脂多糖L6型相對應(yīng)。3.豬源Pm毒力基因分布應(yīng)用PCR對115株P(guān)m的23個毒力基因進(jìn)行檢測,并分析這些毒力基因的分布情況。結(jié)果發(fā)現(xiàn)其中一些毒力基因總的檢出率為100%,這些基因包括黏附因子相關(guān)基因ptfA、fimA、hsf-2;鐵攝取相關(guān)基因exbB、exbD、tonB;外膜蛋白基因opmA、ompH、omp87;超氧化物歧化酶相關(guān)基因sodA、sodC。也有一些毒力基因總的檢出率很低,如轉(zhuǎn)鐵結(jié)合蛋白基因tbpA,檢出率為0,又如PMT毒素基因toxA,檢出率為2.6%。在不同型(莢膜血清型、脂多糖基因型、莢膜:脂多糖組合型)的菌株間一些毒力基因的檢出率有較大的差別。以莢膜分型為例,pfhA、tadD、pmHAS、hgbA基因在A型菌株中的檢出率顯著高于D型菌株中的檢出率(P0.05),而hsf-1、hgbB基因在D型菌株中的檢出率顯著高于A型菌株中的檢出率(P0.05)。同樣的,以脂多糖分型為例,pfhA、tadD、pmHAS、hgbA、nanH基因在L3型菌株中的檢出率顯著高于L6型菌株中的檢出率(P0.05),而hgbB、nanB基因在L6型菌株中的檢出率顯著高于L3型菌株中的檢出率(P0.05)。通過進(jìn)一步統(tǒng)計(jì)分析莢膜:脂多糖組合型間毒力基因的差異,首次發(fā)現(xiàn)pfhA等基因的檢出率可能與莢膜型無關(guān),而和脂多糖型相關(guān)。4.豬源Pm的MLST研究本研究對我國不同省份的23株P(guān)m分離株進(jìn)行MLST分型,結(jié)果發(fā)現(xiàn)了ST3、ST10、ST11三個ST型。而在湖北、廣東等多個省份,豬源Pm不止一個ST型。ST11序列類型的菌株在我國七個省份均有分離到,表明了該類型菌株在我國分布的廣泛性。因此,需要加強(qiáng)對此類型菌株的流行病學(xué)監(jiān)測和防控。本研究中從山西、湖北、廣東分離的3株產(chǎn)毒素Pm都屬于ST11型,而本實(shí)驗(yàn)室先前保存的8株產(chǎn)毒素Pm也屬于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.

【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S852.61

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