鸭疫里默氏杆菌recA基因突变对其生长及DNA损伤反应的影响

DNA损伤反应（SOS response）是细菌应对基因组DNA损伤的一种重要的保护机制。RecA蛋白在许多细菌中对SOS反应的诱导都起关键调控作用,其同源蛋白广泛存在于各种生物体中。本研究对鸭疫里默氏杆菌RecA蛋白是否调控其SOS反应进行探索。构建recA基因缺失株和回复株,检测亲本株、缺失株和回复株在紫外线辐照损伤下的生长能力、recA基因的转录水平以及SOS反应。结果表明,成功构建了recA基因缺失株ΔrecA和回复株cΔrecA;recA基因灭活对鸭疫里默氏杆菌RA-GD株的生长速率无明显影响;在紫外线辐照损伤下,亲本株recA基因的转录水平明显升高,缺失株的存活能力及其基因组的完整性都显著低于亲本株和回复株。本研究证实RecA蛋白参与鸭疫里默氏杆菌的SOS反应。     

鸭疫里默氏杆菌RA-LZ01株GE296_RS01450基因介导其对重金属离子产生耐受性的分析

本研究旨在阐明鸭疫里默氏杆菌RA-LZ01株GE296_RS01450基因介导其对重金属离子产生耐受性的作用。构建RA-LZ01株的GE296_RS01450基因缺失株Δ1450和回复株cΔ1450,测定菌株的生长曲线、抗菌素对菌株的最小抑菌浓度（MIC）、菌株对重金属离子的耐受程度以及GE296_RS01450基因在特异性重金属离子刺激下的转录水平。结果显示,与亲本株相比,缺失株的生长能力以及11类抗菌素对缺失株的MIC值均无明显变化;与亲本株和回复株相比,缺失株对重金属离子Ni、Mn和Co的敏感性显著上升,并且在Ni、Mn刺激下,GE296_RS01450基因的转录水平显著上调。上述结果表明,GE296_RS01450基因不参与亲本株的生长及菌株对抗菌素的耐药性,可介导菌株对Ni、Mn和Co产生耐受性。GE296_RS01450基因属于GE296_RS01445-GE296_RS01450-GE296_RS01455 RND外排泵,编码外膜蛋白,我们将其命名为RopM。     

鸭疫里默氏杆菌对鸭胚肝细胞粘附入侵能力的研究

为了分析鸭胚肝细胞用于鸭疫里默氏杆菌粘附入侵实验的可行性,本研究比较了鸭疫里默氏杆菌对鸭胚肝细胞和Vero细胞的粘附和入侵能力。首先将构建的互补质粒pRES1-ompA通过双亲本接合转导的方法导入ompA基因缺失株Th4ΔompA中,构建得到缺失株的回复菌株cTh4ΔompA;然后比较了野生株Th4、缺失株Th4ΔompA及回复株cTh4ΔompA菌株对鸭胚肝细胞和Vero细胞的粘附入侵能力。结果表明,与野生株Th4菌株相比,缺失株Th4ΔompA对鸭胚肝细胞和Vero细胞的粘附能力分别降低了3.32倍和3.12倍,入侵能力分别降低了2.25倍和7.58倍,而cTh4ΔompA对鸭胚肝细胞和Vero细胞的粘附入侵能力均得到部分回复。本研究结果表明鸭胚肝细胞可用于鸭疫里默氏杆菌粘附入侵能力的分析。     

鸭疫里默氏杆菌RIA_0940基因缺失株的构建及主要生物学特性测定

【目的】通过构建鸭疫里默氏杆菌RIA_0940基因缺失株并测定其生物学特性,探讨RIA_0940基因的潜在功能。【方法】以鸭疫里默氏杆菌RA-GD株为亲本株,扩增其左右同源臂及红霉素抗性基因（ermR）盒片段,构建左同源臂-ermR抗性基因盒-右同源臂融合片段,通过自然转化的方法缺失RA-GD株RIA_0940基因,并利用PCR筛选、鉴定RA-GDΔRIA_0940基因缺失株。分别对亲本株RA-GD和缺失株RA-GDΔRIA_0940的生长特性、对雏鸭的半数致死量、感染鸭血液和组织载菌量及对Vero细胞的黏附和入侵性能进行比较分析。【结果】试验成功构建了RA-GD株的RIA_0940基因缺失株（RA-GDΔRIA_0940）;生物学特性检测结果显示,RA-GDΔRIA_0940株体外生长能力较亲本株低,基因缺失株在生长后期生长受到显著或极显著抑制（P<0.05;P<0.01）;RA-GDΔRIA_0940株对雏鸭的半数致死量是亲本株的114倍;与亲本株相比,缺失株感染鸭血液和组织的载菌量显著或极显著下降（P<0.05;P<0.01）;缺失株对Vero细胞的黏附和入侵性能均极显著低于亲本株（P<0.01）。【结论】本试验成功构建RA-GD株RIA_0940基因缺失株,该缺失株在体外培养条件下生长能力较亲本株低,对雏鸭的致病力、感染鸭血液和组织载菌量及对Vero细胞的黏附和入侵性能均显著或极显著低于亲本株。本试验结果为深入研究鸭疫里默氏杆菌的分子致病机理和研制基因工程疫苗奠定了基础。     

Enolase在鸭疫里默氏杆菌侵袭鸭脑微血管内皮细胞中的作用

旨在明确鸭疫里默氏杆菌烯醇化酶(Enolase)在其侵袭鸭脑微血管内皮细胞(DBMEC)以及血脑屏障(BBB)中的作用。本研究以鸭疫里默氏杆菌RA-LZ01株为亲本株，利用同源重组和结合转移的方法构建enolase基因缺失株ΔEnolase和回复株cΔEnolase，并测定RA-LZ01、ΔEnolase和cΔEnolase对DBMEC黏附和侵袭能力的差异；用上述菌株感染雏鸭，测定雏鸭血液和脑组织中的载菌量。结果表明，与亲本株RA-LZ01相比，缺失株ΔEnolase对DBMEC的黏附率和入侵率均极显著降低；回复株cΔEnolase恢复了对DBMEC的黏附和入侵能力。感染RA-LZ01、ΔEnolase和cΔEnolase菌株的雏鸭的血液载菌量无显著差异；与感染RA-LZ01和cΔEnolase菌株的雏鸭相比，感染ΔEnolase菌株的雏鸭脑组织中的载菌量极显著降低。以上结果说明，Enolase与鸭疫里默氏杆菌黏附和入侵DBMEC以及入侵雏鸭脑组织显著相关，可能为介导鸭疫里默氏杆菌突破鸭血脑屏障的毒力因子。     

番鸭源鸭疫里默氏杆菌的分离鉴定及其耐药性分析

为了解番鸭源鸭疫里默氏杆菌的耐药性,本研究对疑似鸭疫里默氏杆菌感染的发病番鸭进行细菌分离培养、革兰氏染色镜检、病鸭病原检测、生化试验、16S rRNA序列分析、PCR鉴定、药敏试验和耐药基因检测。细菌分离结果显示,分离菌在鲜血琼脂培养基上长出表面光滑、边缘整齐、有光泽、半透明的奶油状针尖大小菌落;革兰氏染色镜检呈革兰氏阴性短小杆菌,命名为GZQN201907。GZQN201907生化试验中尿素反应阳性,葡萄糖、麦芽糖、乳糖等生化反应呈阴性。其16S rRNA系统进化树与鸭疫里默氏杆菌处于同一分支;并且分离菌鸭疫里默氏杆菌OmpA基因PCR鉴定结果为阳性。其对头孢呋辛、红霉素、头孢他啶等18种抗菌药耐药,对羧苄西林和环丙沙星中度敏感,对新霉素和复方新诺明敏感。而耐药基因能检测出β-内酰胺类耐药基因VIM、TEM,四环素类耐药基因tetB,大环内酯类耐药基因ermB和ermF。药敏试验与耐药基因检测结果说明,GZQN201907对β-内酰胺类、四环素类、大环内酯类3类药物的耐药表型和耐药基因检测结果一致。动物回归试验中接种GZQN201907的雏鸭在72 h内全部死亡,而对照组雏鸭未出现任何症状,说明GZQN201907对雏鸭有致病力。试验成功分离到1株番鸭源鸭疫里默氏杆菌,为鸭疫里默氏杆菌病的防治奠定基础。     

鸭疫里默氏杆菌KYF39＿09285基因缺失株的构建及生物学特性研究

为研究KYF39＿09285基因对鸭疫里默氏杆菌生物学特性及其对雏鸭致病力的影响，试验利用基因同源重组的原理构建KYF39＿09285的基因缺失株RA-YMΔKYF39＿09285，并使用穿梭质粒构建其基因回补菌株，评价其对鸭疫里默氏杆菌生物学特性以及雏鸭致病力的影响。结果显示：与亲本株相比，KYF39＿09285基因缺失株的生长速度、生化特性、对Vero细胞的黏附入侵能力均未发生改变，但缺失株中dnaK表达量降低，对高渗、青霉素、氨苄西林、庆大霉素和链霉素的敏感性增强，对雏鸭的致病力（LD50）降低1.84倍，45℃胁迫下的KYF39＿09285表达量显著降低。研究表明KYF39＿09285与鸭疫里默氏杆菌对高温、高渗和抗生素的耐受性以及致病力有关，结果为深入研究鸭疫里默氏杆菌的致病机制奠定基础。     

鸭疫里默菌aroA基因缺失突变株的构建及生物学特性研究

构建鸭疫里默菌aroA基因缺失株,并对其生物学特性及毒力进行研究。以本实验室分离的鸭疫里默菌YM株(RA-YM)为亲本株,建立pRE112-LSR重组自杀性质粒,通过结合转移的方法缺失RAYM株aroA基因,并利用PCR方法对突变株进行鉴定,成功构建了鸭疫里默菌aroA基因缺失株。生物学特性检测结果显示,鸭疫里默菌aroA基因缺失株不再水解精氨酸;体外生长曲线对数期细菌生长速度减缓,在稳定期趋于一致;半数致死量与野生株相比下降了3倍左右;aroA基因缺失株在血液、心、肝、脑等组织的载菌量与亲本株相比有所下降,但差异不显著。研究结果为深入研究鸭疫里默菌的分子致病机理和研制基因工程疫苗奠定了良好的基础。     

血清2型鸭疫里默氏杆菌铁载体受体蛋白突变体的构建及生物学特性研究

铁离子是致病菌重要的生长因子,研究表明铁离子摄取系统调控鸭疫里默氏杆菌(Riemerella anatipestifer,RA)铁离子的代谢,是鸭疫里默氏杆菌重要的致病因子之一。为了研究RA外膜ATP依赖的铁载体受体蛋白(siderophore receptor protein,SRP)在铁离子代谢中的作用,以及SRP与细菌毒力之间的关系,本研究构建了SRP基因的突变株,通过TSB培养基和限铁环境中的生长曲线比较,结果显示突变株在限铁环境中比TSB培养基生长慢约10 h,表明SRP基因是鸭疫里默氏杆菌在体内维持铁离子代谢至关重要的成份。动物试验表明突变菌株比野生型菌株晚24 h开始死亡,说明突变菌株在体内增殖并达到致死数量的速度明显要晚于野生型,从而提示SRP是鸭疫里默氏杆菌重要的毒力因子之一。     

鸭疫里默氏杆菌TonB依赖性受体TbdR2基因缺失株的构建

用PCR方法扩增tbdR2基因的左右臂和壮观霉素抗性基因表达盒,然后将以上3个片段组成的同源重组同源臂LSR连接到自杀性质粒pDS132上,构建得到重组自杀质粒pDS-TbdR2-LSR.再将此质粒通过接合转导的方法导入到鸭疫里默氏杆菌CH3株.用含卡那霉素和壮观霉素的TSA筛选可能的基因缺失株,并对其进行PCR鉴定,获得基因缺失株CH3△tbdR2.CH3△tbdR2稳定性检测结果表明该缺失株能够稳定存在.另外,CH3△tbdR2突变株在电镜下的细菌形态以及在TSA培养基上的菌落形态与野生株CH3相似.表明通过自杀质粒同源重组的方法获得了鸭疫里默氏杆菌CH3株TonB依赖受体TbdR2的基因缺失株CH3△tbdR2,为下一步研究TbdR2的功能奠定了基础.     

C5a-C5aR轴在鸭疫里氏杆菌感染致鸭肝损伤中的作用

本试验旨在阐明C5a-C5aR轴在鸭疫里氏杆菌（Riemerella anatipestifer）感染致雏鸭肝损伤中的作用。将保存的鸭疫里氏杆菌复苏培养，同时制备鸭抗凝血，将鸭疫里氏杆菌与鸭抗凝血混匀，分别与抗C5a抗体和抗C5aR抗体孵育后进行全血试验，利用ELISA检测C5a及各炎性因子表达水平；利用鸭疫里氏杆菌感染雏鸭，同时分别用抗C5a抗体和抗C5aR抗体处理进行动物试验，采集主要组织进行病理组织学检查；采集雏鸭外周血和肝组织进行细菌计数；采集外周血分离血清后，进行血清酶活性检测，应用ELISA检测血清C5a及炎性因子水平；采集肝组织，利用荧光定量PCR检测主要炎性因子mRNA转录水平。结果显示，在全血试验和动物试验中，与R.anatipestifer组相比，R.anatipestifer +anti-C5a组和R.anatipestifer +anti-C5aR组C5a、TNF-α、IL-6和IL-1β表达水平显著降低（P<0.05）；阻断C5a-C5aR轴可显著降低鸭疫里氏杆菌感染雏鸭的发病率和死亡率，同时减轻雏鸭心、肝和脾组织损伤；阻断C5a-C5aR轴能显著降低鸭疫里氏杆菌感染雏鸭外周血和肝细菌数及谷丙转氨酶（alanine aminotransferase，ALT）和谷草转氨酶（aspartate aminotransferase，AST）水平；荧光定量PCR检测发现，与R.anatipestifer组相比，R.anatipestifer +anti-C5a和R.anatipestifer +anti-C5aR组C5aR、Sphk1、FGL2、TNF-α和IL-1β mRNA转录水平显著降低（P<0.05）。本研究成功证实C5a-C5aR轴激活有助于鸭疫里氏杆菌感染雏鸭，阻断C5a-C5aR轴可显著减轻雏鸭组织损伤和炎症反应，为进一步研究抗鸭疫里氏杆菌感染药物提供新思路。     

Detection of acid production from carbohydrates by Riemerella anatipestifer and related organisms using the buffered single substrate test

One hundred and twenty-one Riemerella anatipestifer field strains from wild birds, domesticated poultry and pigs were examined for their ability to produce acid from carbohydrates by using conventional biochemical and buffered single substrate (BSS) test methods. The type strains of the species R. anatipestifer and taxometrically related genera Chryseobacterium and Bergeyella were included in the study. In contrast to 10 indole-positive R. anatipestifer variant strains, only a few of the 111 typical indole-negative R. anatipestifer strains produced acid from dextrin (32%), glucose (17%), maltose (14%) and trehalose (5%) when the conventional test procedure was used. Using the BSS test all the field isolates and the type strain of R. anatipestifer produced acid from one or more carbohydrates, most of them from dextrin (96%), maltose (91%), glucose (87%), mannose (83%), less frequently from fructose (38%) and only in some cases from trehalose (19%). One hundred and six (87%) of the R. anatipestifer strains could be assigned to 8 biovars, based on the diversity of the carbohydrate acidification patterns. The remaining 16 R. anatipestifer isolates gave delayed reactions and displayed 13 different carbohydrate acidification profiles. The Chryseobacterium and Bergeyella type strains also produced acid from more carbohydrates when the BSS test was used. The BSS-carbohydrate acidification pattern of the Chryseobacterium indologenes strain was similar to that of R. anatipestifer biovar 3.     

Isolation,Identification and Drug Resistance Analysis of Riemerella anatipestifer from Muscovy Duck

In order to understand the drug resistance of Riemerella anatipestifer from Muscovy duck,this study carried out bacterial isolation and culture,Gram staining microscopy,pathogen detection,biochemical test,16S rRNA sequence analysis,PCR identification,drug sensitivity test and drug resistance gene detection for Muscovy duck suspected of Riemerella anatipestifer infection.The results of bacterial isolation showed that on the blood agar medium,the isolated bacteria grew creamy needle tip size colonies with smooth surface,neat edge,luster and translucency.The Gram-negative bacillus brevis was detected by Gram-negative staining microscopy,and it was named GZQN201907.In the biochemical test of GZQN201907,urea reaction was positive,but glucose,maltose,lactose and other biochemical reactions were negative.The 16S rRNA phylogenetic tree was in the same branch as Riemerella anatipestifer.And the OmpA gene of PCR identification results of the isolated strain were positive.The drug sensitivity test was sensitive to 18 antibiotics including cefuroxime,erythromycin and ceftazidime,moderately sensitive to carboxypicillin and ciprofloxacin,and sensitive to neomycin and cotrimoxazole.And the resistance genes could detect the β-lactam resistance genes VIM and TEM,tetracycline resistance genes tetB,macrolide resistance genes ermB and ermF.The results of drug sensitivity test and drug resistance gene detection indicated that GZQN201907 showed the same resistance phenotype and gene detection results for β-lactam,tetracycline and macrolide.In the animal regression test,all the ducklings inoculated with GZQN201907 died within 72 h,while the control group showed no symptoms,indicating that GZQN201907 was virulent to the ducklings.One strain of Riemerella anatipestifer from Muscovy duck was successfully isolated,which laid a foundation for the prevention and treatment of Riemerella anatipestifer from muscovich.     

布鲁氏菌BPE159基因缺失株的构建及BPE159蛋白对细胞自噬因子表达的影响

【目的】构建布鲁氏菌BPE159基因缺失株,研究缺失株体外生长变化特征及其在宿主细胞中的存活能力,探究布鲁氏菌感染期间分泌蛋白BPE159对自噬因子表达的影响。【方法】同源重组方法构建布鲁氏菌BPE159基因重组质粒,电转化布鲁氏菌S2308感受态细胞构建BPE159基因缺失株S2308ΔBPE159。PCR扩增BPE159基因,连接转化构建pBBR1MCS-4-BPE159载体,提取质粒进行电转化,构建BPE159基因回补株S2308ΔBPE159-C。琼脂糖凝胶电泳检测缺失株和回补株遗传稳定性。构建布鲁氏菌感染小鼠巨噬细胞RAW264.7模型,实时荧光定量PCR检测布鲁氏菌侵染后自噬细胞因子ATG5、Beclin1、LC3a和LC3b基因表达水平。以S2308、S2308ΔBPE159和S2308ΔBPE159-C株侵染小鼠巨噬细胞,收集细胞总RNA,实时荧光定量PCR检测BPE159基因缺失对布鲁氏菌侵染后自噬细胞因子表达水平的影响。在相同起始浓度下培养S2308、S2308ΔBPE159及S2308ΔBPE159-C株,观察细菌生长变化趋势;评价S2308ΔBPE159株在不同...     

Screening and Identification of Outer Membrane Proteins of Riemerella anatipestifer Recruited Duck C4b-binding Protein

The purpose of this experiment was to screen and identify the outer membrane proteins of Riemerella anatipestifer (R. anatipestifer) interacting with duck C4b-binding protein (C4BP). The preserved R. anatipestifer was resuscitated, then the outer membrane proteins of R. anatipestifer were extracted and His pull-down and LC-MS/MS were conducted by using duck C4BPα as the bait protein, and the candidate outer membrane proteins that might interact with duck C4BP were screened out. The candidate proteins were cloned, prokaryotic expression was conducted and the polyclonal antibodies were prepared by immunizing the mice. Far-western blot was conducted to verify the outer membrane proteins of R. anatipestifer interacting with duck C4BP. The clone and prokaryotic expression of functional domains of candidate proteins and C4BPα were conducted, and Far-western blot was used to identify the interaction sites between candidate proteins and C4BP. The deposition of complement C3b, C4b and C4BP on the surface of R. anatipestifer were detected by ELISA to verify the function of the candidate proteins. The results showed that a total of 3 outer membrane proteins of R.anatipestifer interacting with duck C4BP were screened out by His pull-down and LC-MS/MS, namely ECE-1, SODs and Omp62. The polyclonal antibodies of 3 outer membrane proteins were successfully prepared, the titers of 3 polyclonal antibodies were more than 1∶6 400 by ELISA, and the result of Western blot showed that 3 polyclonal antibodies could specifically react with corresponding recombinant proteins. The results of Far-western blot showed that only ECE-1 could interact with C4BP, and only the full length of ECE-1 could interact with duck C4BP, and the interaction region between duck C4BP and ECE-1 was located in SCR 2 and SCR 3 of C4BPα. Anti-ECE-1 antibody could significantly increase the C3b and C4b deposition on the surface of R. anatipestifer using 3.125% normal duck serum (NDS, P<0.05), while anti-ECE-1 antibody could significantly decrease the deposition of C4BP on the surface of R. anatipestifer using 6.25% NDS (P<0.05). The study successfully screened out and identified one outer membrane protein (ECE-1) of R. anatipestifer interacting with duck C4BP, which provide a basis to further study the mechanism of R. anatipestifer immune escape.