【作者】 李继祥；

【导师】 周泽扬；

【作者基本信息】 西南大学 ， 特种经济动物饲养， 2009， 博士

【摘要】 鸭疫里默氏杆菌是一个多宿主的病原菌,除主要引起家鸭的渗出性炎症以外,还感染鹅、鸡、火鸡、鸽子、野鸭、鹦鹉等其它鸟类,甚至已从猪、青鱼体内分离鉴定出该细菌。目前鸭疫里默氏杆菌已成为危害养鸭业的主要病原之一,呈世界性流行趋势,发病率和死亡率一般为10-30%,但部分鸭场达95%以上。该细菌的危害除引起动物发病、死亡外,还因感染耐过动物饲料转化率低、生长发育迟缓造成严重的经济损失,甚至由于抗菌药物的大量使用而引起食品安全问题。自1982年以来,尤其是在东亚和东南亚地区,鸭疫里默氏杆菌感染严重制约了肉鸭的集约化养殖。鸭疫里默氏杆菌血清型复杂,各血清型菌株间缺乏交叉免疫保护;极易产生耐药性,流行菌株多表现为多重耐药。关于其致病相关因子尚不明确,而对于致病机制的研究处于起步阶段。作者在研究鸭疫里默氏杆菌液化明胶菌株的流行病学特征的过程中发现,鸭疫里默氏杆菌液化明胶特性与致病性有关。基于此,本文以液化明胶活性成分（明胶酶）为对象,研究鸭疫里默氏杆菌的致病相关因子,为进一步研究细菌的致病机制奠定基础。1.鸭疫里默氏杆菌流行菌株的分离鉴定及液化明胶特性测定利用巧克力营养琼脂平板从2004-2008年收集于重庆、四川等25个地区的661份疑似鸭疫里默氏杆菌感染的自然病例中分离、鉴定出548株鸭疫里默氏杆菌,其中512株对10日龄鸭具有毒力,25株细菌经鸭体内传1-3代后获得毒力,11株在鸭体内传7代仍不具有毒力。利用明胶-营养琼脂平板和明胶-琼脂平板分别测定细菌及培养滤液的液化明胶特性,有毒力的537株细菌及培养滤液都能液化明胶,占分离菌株的98%;不具有毒力的11株细菌及培养滤液不能液化明胶,占分离菌株的2%。鸭疫里默氏杆菌液化明胶特性稳定,液化明胶和不液化明胶菌株在巧克力培养基和鸭体内分别传30和10代后未发现明显的变化。液化明胶的鸭疫里默氏杆菌培养滤液的明胶酶谱呈现6条活性带,不同菌株各谱带的活性存在差异,且各谱带活性能被EDTA抑制。2.鸭疫里默氏杆菌产明胶酶的条件优化及酶的分离纯化本研究在建立定量测定鸭疫里默氏杆菌明胶酶活性的基础上,对鸭疫里默氏杆菌产酶条件进行了优化。研究结果表明,产酶培养基的基本组成为2%蛋白胨、0.5%酵母粉和0.5%氯化钠,初始pH值为7.4;培养基含终浓度5mMCa²⁺有利于酶的产生;最适培养温度为37℃,在振荡条件下的最适培养时间为24h。在最佳条件下培养AF株细菌,培养滤液经分子截流量为10kDa超滤膜浓缩、最终饱和度为70%硫酸铵盐析、DEAE-Sepharose Fast Flow阴离子交换层析、phenyl-sepharose CL-4B疏水柱层析及Superdex-75凝胶柱层析分离纯化,获得具有液化明胶活性的蛋白质组分（明胶酶）,活性回收率为28.4%。分离纯化的明胶酶的比活力为17076.0U/mg;经SDS-PAGE检测为分子量59kDa单带;明胶酶谱与培养滤液的第5活性条带一致。3.鸭疫里默氏杆菌明胶酶的生物学活性研究本研究利用鸭胚成纤维细胞和10日龄雏鸭模型对分离纯化的鸭疫里默氏杆菌明胶酶的生物学活性进行了研究。明胶酶对鸭胚成纤维细胞具有细胞毒性,半数致死浓度(LC₅₀)和半数生长抑制浓度(GI₅₀)分别为1.45U/ml和0.16U/ml;导致细胞形态发生异常,主要表现为细胞膜降解、细胞质丧失与细胞核浓缩。对30株鸭疫里默氏杆菌临床分离株的培养滤液检测发现,具有毒力及能液化明胶的27株细菌均能引起与明胶酶一致的细胞形态变化,而无毒力及不液化明胶的3株细菌的培养滤液不表现细胞毒性。明胶酶对雏鸭具有毒性,能增强鸭疫里默氏杆菌对动物的侵袭力,增加菌血症的严重程度,缩短感染潜伏期和致死动物的时间,显著提高动物的死亡率。颈静脉注射5000U/ml能致死10日龄雏鸭,2000U/ml能引起发病,而500U/ml的作用不明显。发病动物主要表现嗜睡懒动、腿软无力、甩头、张口呼吸及头颈歪斜等症状;死亡动物有肝脏轻微肿大、硬脑膜有出血点、大脑水肿等大体病理变化。在组织学上,2000U/ml作用8h鸭的心肌纤维肿胀变粗、横纹不明显,纤维间质水肿、可见微细颗粒和空泡;肝细胞索不规则,肝血窦变小,肝细胞肿胀、胞浆中出现大小不等的空泡;脾中央动脉内皮细胞肿胀、脱落,脾小体局灶性出血及小淋巴管扩张,淋巴鞘数量增多、管径扩大,淋巴细胞不同程度变性、坏死。在超微结构上,心肌细胞线粒体肿大,毛细血管的内皮细胞中多空泡、核膜不均一;肝细胞核浓缩、边缘有皱褶,毛细血管内皮细胞间连接不紧密、红细胞形态不规则;脾淋巴细胞核膜不规则、染色质核周边浓集。明胶酶经静脉注射后对雏鸭外周血血液指标的影响明显。雏鸭外周血中红细胞（RBC）的数量与C3b花环率显著下降,在9h最低,分别为（1.58±0.05）×10¹²·L^-1和（18.5±1.32）%。白细胞（WBC）的数量减少;对绵羊红细胞的吞噬率（BP）与吞噬指数（IP）降低,在6h时最低,分别为（27.7±2.0）%和1.053±0.015,与对照差异极显著（P＜0.01）;对金黄色葡萄球菌的杀菌指数下降,在9h时最低为0.4562±0.0205,与对照差异极显著（P＜0.01）。外周血中淋巴细胞（LT）数量减少;a-醋酸萘酯酶阳性T淋巴细胞百分率降低。血清谷草/谷丙转氨酶活性、碱性磷酸酶活性及总胆固醇和血清葡萄糖水平显著升高。血清总补体活性(CH₅₀)显著下降。明胶酶的作用对雏鸭脾脏损伤严重,导致脾淋巴细胞的增殖活性和生成IgG的能力下降,9h最低,分别为0.185±0.022和0.237±0.038,与对照差异极显著（p＜0.01）;可引起雏鸭血-脑屏障机能障碍,表现为脑脊液中的白蛋白含量升高,血-脑屏障的通透性增大,基底膜粗糙、部分节段的连续性中断,脑血管周围水肿、管腔面不光滑、内皮细胞肿胀、连接处开放。4.鸭疫里默氏杆菌明胶酶基因克隆及分析从AF株培养滤液中分离纯化的明胶酶N-氨基酸残基序列为:天冬酰胺-苏氨酸-甘氨酸-丙氨酸-丙氨酸-谷氨酸-甘氨酸-苏氨酸-组氨酸-甘氨酸（NTGAAEGTHG）,在NCBI中未能找到同源序列,仅第3-10位氨基酸残基与类鼻疽伯克霍尔德菌406e株的一个假设保守蛋白的第555-562一致。在此基础上,以N端氨基酸序列设计引物,用单特性引物PCR（SSP-PCR）从细菌基因组DNA文库中克隆出197bp的DNA片段,然后利用热不对称交错PCR（Tail-PCR）进行基因组步移,获得2150bp核苷酸序列。该序列中有一个1872bp的ORF,编码624aa,其中包含NTGAAEGTHG序列;1-24aa为信号肽;成熟蛋白存在3个跨膜区,分别在25-45aa、211-232aa和416-435aa;在蛋白质数据库中未能找到同源性序列。5.鸭疫里默氏杆菌明胶酶的理化特性测定鸭疫里默氏杆菌明胶酶不降解Ⅰ型胶原蛋白维、干酪素、牛血清白蛋白V及Azocoll试剂;降解明胶的最适作用温度和pH值分别为37℃和7.8,在4℃24h及37℃2h内稳定、65℃0.1h内完全失活;Ca²⁺、Mg²⁺、Na⁺和Zn²⁺对酶有一定激活作用,Cu²⁺、Cd²⁺、Mn²⁺、Hg²⁺及金属离子络合物EDTA和EGTA对酶活性有强烈的抑制作用,但络合物的抑制作用可以被Ca²⁺逆转;蛋白质化学修饰剂2-ME和DEPC不影响酶活,而PMSF、ADD和NBS导致酶活显著下降。本文在研究明胶酶生物学活性过程中,与鸭疫里默氏杆菌感染模型进行比较,结果表明二者在引起雏鸭血液、肝、脾及血-脑屏障等的生物化学与组织学变化及机能障碍方面相似。这进一步表明,明胶酶是鸭疫里默氏杆菌的致病相关因子之一,在其致病过程中起着重要的作用。更多还原

【Abstract】 Riemerella anatipestifer infection characterized by exudative septicaemia is primarily a disease of domestic ducks.This bacterium is also isolated from other domestic poultry,such as geese,chickens,turkeys,pigeons,wild ducks and parrots and was also reported from snail craps and domesticated pigs.Now,this bacterium is a major bacterial pathogen of ducks in all over the world.Mortality varies from 10 to 30%,but it can be as high as 95%and is influenced by predisposing viral and bacterial infections.R.anatipestifer not only accounts for major economic losses in industrialized duck production due to high mortality,reduced growth rate,poor feed conversion,increased condemnations,and high treatment costs,but also causes food safety for antibacterial.In east and south-east Asia,R.anatipestifer infection has been a problem in intensive production of meat ducks since 1982.The occurrence of different serotypes of R.anatipestifer in field cases has been reported.Unfortunately,no cross-protection has been observed among different serotypes of R.anatipestifer.Drug resistant strains of R.anatipestifer are very popular and some strains are multidrug resistant.In contrast,there has been little work on the virulence factor and pathogenesis of this organism.We have studied the epidemiological characteristic of R.anatipestifer strains which liquefacted gelatin,and it has been found that the pathogenicity of R.anatipestifer related to gelatin liquefaction.To facilitate study of the pathogenesis,gelatinase was identified and verified as a potential virulence factor of R.anatipestifer.1.Isolation of R.anatipestifer from ducks and determination of gelatin liquefactionIn the study,chocolate nutrient agar was used and 548 R.anatipestifer field strains were isolated from representing 661outbreaks of Riemerellosis in China between 2004 and 2008.537 strains had pathogenicity to 10-day-old ducks and 11 strains were avirulence.The nutrient gelatin agar plate and gelatin agar plate were used to evaluate gelatin liquefaction of R.anatipestifer and it’s culture filtrate,respectively.The results showed that the virulence R.anatipestifer and its culture filtrate liquefied gelatin and avirulence strains did not.When this bacterium was cultured 10 passages in vivo or 30 passages in vitro,its activity of gelatin liquefaction did not changed obviously. Zymography showed gelatinase activity in culture filtrate of gelatin liquefaction strains and there were 6 gelatinolytic bands on a darkly stained gel.The activity of gelatinase was estimated by using densitometry and there were different among R.anatipestifer strains.2.Optimal enzyme production condition for R.anatipestifer AF strain and purification of gelatinaseA quantitative assay method of gelatinase has been established and the fermentation was studied.The results showed that the suitable condition of R. anatipestifer AF strain producing gelatinase were 2%Peptone,0.5%yeast powder, 0.5%sodium chloride,initial pH 7.4,37℃and shaking culture 24 hours.At the same time,5mmol/L Ca²⁺ promoted the producing level of gelatinase.Purification protocol of active compound was designed according to typical protein purification strategy and R.anatipestifer AF strain active compound was purified with the following program.The purification of active compound was started by concentrating the supernatantfluid in an Amicon（M_t cut-off 10,000）.Second, concentrate was precipitated with ammonium sulfate at 15%saturation,and then at 70%saturation.The third step was DEAE-Sepharose anion-exchange chromatography. Fourth,phenyl-sepharose CL-4B was used in hydrophobic interaction chromatography. Finally,Superdex-75 column was used in gel permeation chromatography.The active compound obtained after steps 5 was SDS-PAGE-pure.The molecular weight was about 59kDa which was determined with SDS-PAGE.From now on,the active compound was named gelatinase.The specific activity of gelatinase was 17076.0 unit per milligram protein and it was accorded to the fifth bands of culture filtrate in zymography.3.Study on biological activity of R.anatipestifer gelatinaseIn this study,duck embryo fibroblast（DEF） and 10-day-old ducklings were used to research the biological activity of R.anatipestifer gelatinase.Cytotoxicity of gelatinase was observed using the DEF.The median lethal concentration(LC₅₀) and the median growth inhibiting(GI₅₀) were 1.45U/ml and 0.16U/ml,respectively.DEF treated with gelatinase resulted in striking morphologic changes including losses of cell membrane and cytoplasm,and nucleus being condensed. Most of the clinical strains 90%（27/30） had the capability of hydrolyzing gelatin, causing disease and altering the morphology of DEF,While the rest 10%（3/30） are on the contrary.Moreover,the damage after ducklings intrajugular injection of gelatinase was studied.The results showed gelatinase not only enhanced the invasion of R. anatipestifer,increased bacteremia,shortened stage of latency and course of disease, but also raised mortality obviously.10-days old duckling was died after intrajugular injection of 5000U/ml.Injection 2000U/ml,the duckling was fall ill,but clinical symptom did not been discovered injected 500U/ml each ducklings.Ducklings treated with gelatinase resulted in drowsiness,debility,throwing head,buccal respiration, neurological symptom,liver intumesce,bleeding point on cerebral dura mater,Cerebral edema,and so on.Though light microscope and electron microscope,the pathological changes and ultrastructural changes of main organs in the duckling which was treated with 2000U/ml by intrajugular injection were observed.After 8h,there were cardiac muscle fibers coarsened,transverse striation in nubibus,interstitial substance oedema and many vacuo-luses.Liver cell cords were irregularity and liver sinusoid shrinked. Hepatocyte was swollen and there were many vacuoluses in endochylema.Spleen resulted in endotheliocyte of central arterys swelling and ablate,acinus lienalis focal bleeding and angioleukectasia,leukomonocyte apomorphosis and necro-sis.Under electron microscope,parenchymal cells such as cadiocyte,hepatocyte,splenic lymphocyte and endothelial cell of micrangium were damaged.The structure of organelles were changed and the main manifestations were that mitochondria of cadiocyte was Swollen,cell nucleus of hepatocyte was condensed and crenulated, caryotheca of splenic lymphocyte was irregularity and caryotin was enriched at ambitus.Ducklings after intrajugular injection of gelatinase at 10 days of age were studied to determine the dynamic changes of some blood indices.The results showed that the akaryocyte and its chaplet rate of C3b decreased,the 9^th hour was the ravine and they were（1.58±0.05）×10¹²·L^-1 and（18.5±1.32）%,respectively.The numbers of white blood cell of peripheral blood decreased,the rate of phagocytosis and index of phagocytosis to sheep red blood cell were lowest at 9h,and they were（27.7±2.0）%and 1.053±0.01, respectively.Gelatinase decreased bactericidal index of WBC significantly when compared with controls,maximal damage was seen at 9h.Leukomonocyte and the percentage of ANAE⁺-T of peripheral blood were decreased,too.The results of serum biochemical indices showed that GOT,GPT,ALP,CHOI and GLU were all obviously enhanced,but serum total complement activity(CH₅₀) was declined.Gelatinase damaged spleen of ducklings and the worst damage was discovered at 9h.At that time,the generation activity and production IgG of splenic lymphocyte were 0.185±0.022 and 0.237±0.038,respectively.Blood brain barrier dysfunction occur in ducklings injected with gelatinase,and the damages showed at higher concentration of albumin in CSF and higher permeability in blood brain barrier,Cerebral vasogenic edema,lumens rough,junction break,basement membrane rough and break.4.Gene cloning and analysisN-terminal first ten amino acids of gelatinase protein were sequenced as NTGAAEGTHG.In NCBI,Sequence alignments indicated that the same sequence was not founded.According to the analysis of gelatinase N-terminal first ten amino acid sequence,the primer encoding sequence was designed and 197bp DNA fragment was cloned by single specific primer polymerase chain reaction（SSP-PCR） from genome DNA library of R.anatipestifer AF strain.And then,Genome walking by thermal asymmetric interlaced PCR（Tail-PCR） was used and 2150bp DNA fragment was cloned.One ORF was found out and it encoded 624 amino acids.According to bioinformation,N-terminal first twenty-four amino acids were signal peptide,and mature protein has three transmembrane domain.Unfortunately,homophylic sequence was not found in NCBI.The results showed that the gelatinase gene was a new gene probably.5.Study on physico-chemical property of R.anatipestifer gelatinaseThe activities of gelatinase were tested byⅠcollagen fibers,casein,bovine serum albumin V and Azocoll.As the results showed they were not substrate of gelatinase.The optimum pH and temperature of the gelatinase were found to be 7.8 and 37℃.The activity of gelatinase was retained after storing at 4℃for 24h and at 37℃for 2h,but activity was not detected after storing at 65℃for 0.1h.Enzyme testing for inhibition and activation of the gelatinase indicated that EDTA,EGTA,Cu²⁺,Cd²⁺,Mn²⁺,Hg²⁺, PMSF,ADD and NBS inhibited enzymatic activity,2-ME and DEPC did not affected activity,but Ca²⁺,Mg²⁺,Na⁺ and Zn²⁺ activated the activity.Moreover,Ca²⁺ counteracted the inhibition of EDTA and EGTA.Animal model of R.anatipestifer infection was studied in order to showing the relationship of R.anatipestifer and gelatinase.The results showed the effects of gelatinase to ducklings were similar to R.anatipestifer infection at dysfunction, pathohistology,biochemistry,and so on.All results indicated that the gelatinase was a virulence factor and it was important to the infection of R.anatipestifer.更多还原