肠球菌性羔羊脑炎的发现及其病原特性和诊断方法研究
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摘要
肠球菌是动物和人类正常菌群的重要组成部分，多作为有益菌进行研究。然而有研究表明，在需氧革兰阳性球菌中，它是仅次于葡萄球菌的重要院内感染致病菌。美国医院感染监视系统(NISS)已将其列为引起医院感染的第二大病原菌。最近几年新疆生产建设兵团部分规模化羊场连续在同一季节发生一种以20-40日龄羔羊神经症状和败血症为主要症状和病变特征的传染性疾病，羔羊的病死率高达20％左右，经病原分离鉴定和回归试验确诊为肠球菌所致羔羊脑炎，这是羔羊的一种新的疾病，本文围绕该病的病原特性和诊断方法开展了系列研究，以期为阐明肠球菌对羔羊的致病机理和有效防治该病提供科学数据，结果如下：
1．肠球菌性羔羊脑炎的发现及其病原分离鉴定：自2002年新疆生产建设兵团部分规模化羊场连续几年在冬季产羔季节发生了一种病程很短，无品种差异，主要感染羔羊、可引起死亡的、以败血症和神经症状为主要特征的传染病。先后从不同羊场发生脑炎症状的羔羊体内分离到11株细菌，经形态学、培养特性、生化反应、血清学以及动物试验确定是由肠球菌属的某些种引起。
2．检测羔羊肠球菌性脑炎间接ELISA方法建立和初步应用：利用超声波裂解制备致羔羊脑炎粪肠球菌裂解物作为抗原包被酶标板，应用棋盘滴定法，通过条件筛选成功建立了致羔羊肠球菌抗体测定的间接ELISA方法。应用该方法检测羔羊肠球菌阳性血清，其灵敏度是微量凝集反应的25-100倍。利用建立的方法检测来自未免疫羊场的50份成年羊血清和30份羔羊血清，结果显示有2份成年羊血清呈阳性。
3．检测羔羊肠球菌性脑炎PCR方法建立和初步应用：以致羔羊脑炎粪肠球菌基因组为模板，以相对保守的肠球菌的tuf基因为基础设计一对引物，通过条件筛选成功的建立了用于致羔羊脑炎肠球菌快速诊断的PCR方法，其灵敏度在10fg。应用该方法检测自然感染羔羊和人工感染发病死亡羔羊以及人工感染发病死亡小鼠主要器官组织，即在心、肝、脾、肾、脑、脑脊液等组织中均检测到了肠球菌DNA，其中2只人工感染死亡羔羊还在肺门淋巴结和肠系膜淋巴结中检测到肠球菌DNA。
4．免疫组化检测羔羊粪肠球菌性脑炎及其抗原定位的研究：以导致羔羊脑炎的粪肠球菌为研究对象，人工感染小鼠，通过条件优化，建立了肠球菌性羔羊脑炎检测的间接免疫组化方法。用该方法定期检测粪肠球菌在试验小鼠体内的分布，结果显示：4h时可在小鼠的肝脏中检测到粪肠球菌的阳性颗粒；14h时在肾脏和心脏中检测到阳性颗粒；18h可在食道中检测到粪肠球菌阳性颗粒；20h在脑中检测到阳性颗粒；22h在肺脏和气管中检测到粪肠球菌阳性颗粒；24h可在除脾脏外所有受检组织即脑、肝、心脏、肾、气管、肺、食道及小肠中观察到粪肠球菌的阳性颗粒。
5．致羔羊脑炎肠球菌毒力因子的分子流行病学调查：对11株致羔羊脑炎肠球菌ace、efa、cylA、gelE、asal和asa373、esp、EF0591和EF3314等9种毒力因子基因的PCR检测结果表明，5／11株肠球菌检测到esp、cylA、asal、ace、efa、EF0591和EF3314等7种毒力因子基因。1／11株检测到esp、gelE、asal、ace、efa和EF3314等6种毒力因子基因，1／11株检测到cylA、asal、ace、efa、EF0591和EF3314等6种毒力因子基因，1／11株检测到esp、cylA、asal、ace、EF0591和EF3314。1／11株菌仅携带esp基因。2／11株菌未检测上述9种毒力因子基因任何一种。与人类医学其它来源肠球菌毒力因子携带情况不同。
6．致羔羊脑炎粪肠球菌8种毒力因子基因片段克隆和序列特征研究：将致羔羊脑炎粪肠球菌检测到的的8个毒力因子8ce、efa、cylA、gelE、asal、esp、EF3314和EF0591基因部分片段克隆到pMD19-T载体上，应用PCR及酶切方法检测出阳性克隆并进行序列测定。通过BLAST软件对致羔羊脑炎粪肠球菌各毒力基因扩增片段的测定序列与GeneBank公布的医学临床分离株相应序列进行同源性分析比较，结果显示两种来源8种毒力因子基因目的片段序列的同源性分别为99.3％、99.56％、99.3％、97.85％、96.64％、99.9％、99.29％和98.11％。
7．致羔羊脑炎肠球菌与临床健康羔羊分离的肠球菌生物学特性的比较研究：通过研究发现羔羊两种来源肠球菌的培养特性和生化特性基本一致；对某些抗生素有不同程度的耐药性；致病株有更多的毒力因子组合；对两者共有的毒力因子基因片段进行克隆与序列分析，并同时与GeneBank来自医学临床分离株相应毒力因子基因片段比对分析，其同源性很高，均在95％以上。正常菌群株不能引起小鼠的死亡，而致病株可导致小鼠的死亡。
8．致羔羊脑炎粪肠球菌在人工感染小鼠疾病模型体内分布规律及病理学研究：经不同途径用致羔羊脑炎粪肠球菌分离株感染小鼠，最佳途径是腹腔注射，皮下注射次之，鼻腔吸入不引起感染。对小鼠的LD_(50)为9.3×10~(10)CFU。定期剖杀腹腔接种致羔羊脑炎粪肠球菌的小白鼠，用2种不同的方法检测在细菌主要脏器中的分布。结果发现感染2h后用PCR方法即可从脑、肝和心血检测到粪肠球菌DNA。6h后不仅能检测到肠球菌DNA，而且从上述组织中分离到肠球菌。感染4-6h后各组织脏器开始出现病理组织学变化，超微结构变化出现于4h。因此致羔羊脑炎粪肠球菌在人工感染小鼠体内分布广泛，引起的病理变化也是全身性的。
Enterococcus are usually studied as useful microorganism bcause of commensals of the intestinal tract of humans and animals but have emerged in recent decades as a major cause of nosocomial infections Enterococcus infections represent the second most common cause of bacteraemia and endocarditis after Staphylococcus in US hospitals from NISS Survey. Recently, there is a infectious diseases of 20-40 day old lamb which main characteristics are neurological symptoms and the septicemia in large-scale farms for the sheep of Xinjiang Production and Construction Corps in the same season. Lamb mortality is about 20%. It is identified as lamb encephalitis induced by Enterococusi through the isolation of bacteria and regression test. It is a new infectious diseases of lamb. A series of research n pathogen characteristics and diagnostic method are done in order to provide scientific data for pathogenic mechanism and control of the disease effectively about Enterococcus.
1 The Finding of Lamb Encephalitis Infected by Enterococcus Isolated and Identified 11 Enterococcus isolated from lamb encephalitis in partial breeding farm of Xinjiang Production and Construction Corps are identified by a series of tests. They belong to serum type D,G and unidentifiable type by Latex. They can led to mouse and partial guinea pigs or rabbits dead. The 11 bacterum have resistance for streptomycin,gentamicin, erythromycin and partial resistance to norfloxacin, penicillin, tetracycline, but sensitivity to nitrofurantoin, rifampin, chloramphenicol and vancomycin.
2 Development of ELISA to Detecte the Antibody for Enterococcus Inducing Lamb Encephalitis and Preliminary Application A ELISA are developed by phalanx titration with coating antigen of Enterococcus faecalis cracked by ultrasonic. It is more 25-100 times sensitive than micro-agglutination test through some positive serum for Enterococcus, and It has the more specific characteristic, easy or quick to operate. 2 serum in 50 adult sheep serum are positive in adult sheep serum detected.
3 Development of PCR to Detect Antigen for Enterococcus Inducing Iamb Encephalitis and Preliminary Application A PCR are developed base of Conservative gene tuf gene to detect Enterococcus. The optimal reaction conditions is that nuclease refolding temperature is 55℃, Mg~(2+) Concentration is 2.5um and reaction mode is 95℃5min, 94℃30s, 55℃30s, 72℃30s, 35 cycle, 72℃10 min. The sensitivity of this mathod is 10fg. The other 10 Enterococcus, the tissue from lamb and mouse infected by Enterococcus faecalis can be amplified special electrophoresis bands about 112bp and no any bands can be amplified in S.equi s, bsp.equi, Streptococcus G,Streptococcus A,S.aureus, L.monocytogenes,M.ovipneumoniae, Br.ovis and Enterococcus faecalis inducing lamb encephalitis.
4 A Study on the Detection of Enterococcus faecalis Inducing Lamb Encephalitis and Antigen Location by Immunoenzymatic Histochemistry An indirect Immunoenzymatic Histochemistry is developed to detect the distribution of Enterococcus faecalis in mouse artificial infected. The result indicated that positive particles of Enterococcus faecaIis can be detected in liver in 4h, in kidney and heart at 14h, in esophagus at 18h, in encephalon and in all tissues except for spleen of mouse infeted at 24h.
5 The research of Molecular epidemiology for Enterococcus inducing lamb encephalitis 9 virulence factor genes(ace,efaA,cylA, gelE, asaland asa373,esp,EF0591 and EF3314)in 11 Enterococcus inducing lamb encephalitis are detected. The results indicated that there is 7viru-lence factor genes in 5 bacteria (5/11),6 virulence factor genes in 3 bacteria (3/11), 1viru-lence factor gene in 1 bacteria (1/11),no virulence factor genes 2 bacteria (2/11).
6 Gene Cloning and Sequence Analysis of Virulence Factor Genes in Enterococeus facealis Inducing Lamb Encephalitis 8 virulence factor genes (ace, efa,cylA,gelE, asa1, esp, EF0591, EF3314) segments amplyfied are cloned in pMD19-T and the positive clone are going to determin the nucleotide Sequence, the results indicated that the homology is 99.3%、99.56%、99.3%、97.85%、96.64%、99.9%、99.29% and 98.11% between the Enterococcus faecalis from lamb encephalitis and corresponding sequence from GeneBank.
7 A Comparative Study of Biological Characteristics of Enterococci from Different Sources of Lamb The study on cultural characteristics, biochemical characteristics, hemo- lytic properties, drug sensitivity and the types, distribution of virulence factor gene of Enterococcus inducing lamb encephalitis and normal flora indecate that basic biochemical chara- cteristics of enterococci from two different sources of lamb are consistent. Pathogens have resistance for Streptomycin, gentamicin and erythromycin, one bacterial from normal flora have resistance for tetracycline, erythromycin, streptomycin, esp、cylA、asal、ace、efa、EF0591 and EF3314 can be detected in 5/11 from Enterococcus inducing lamb encephalitis. gelE and EF3314 can be detected in 3/30 from normal flora and gelE、EF3314 and asal can be detected in 1/30 from normal flora. The homology of 3 gene sequence is more than 95% between GeneBank and bacteria from normal flora, the corresponding sequence is more than 96% with patho-genic strains, pathogens can cause the death of mice, whereas the Enterococci from normal flora can not cause the death of mice.
8 A study on distribution and pathological changes in artificial infectious mice disease model forEnterococcus faecalis inducing Lamb encephalitis A study on 135 mouse infec ted by Enterococcus faecalis inducing Lamb encephalitis with different ways indicates that intraperitoneal injection is the best way, subcutaneous implant is second way and nasal inhal-ation can not cause infectious. LD_(50) of the bacteria is9.3×10~(10)CFU. Enterococcus faecalis DNA can be detected in encephalon, liver and blood spleen in artificial infectious mouse at 2h. Enterococcus faecalis and its DNA can be detected in the tissues at 6h. Signifiant histopatho-logical changes are observed during 4-6h and the changes of ultrastructural organization are observed at 4h.
1．肠球菌性羔羊脑炎的发现及其病原分离鉴定：自2002年新疆生产建设兵团部分规模化羊场连续几年在冬季产羔季节发生了一种病程很短，无品种差异，主要感染羔羊、可引起死亡的、以败血症和神经症状为主要特征的传染病。先后从不同羊场发生脑炎症状的羔羊体内分离到11株细菌，经形态学、培养特性、生化反应、血清学以及动物试验确定是由肠球菌属的某些种引起。
2．检测羔羊肠球菌性脑炎间接ELISA方法建立和初步应用：利用超声波裂解制备致羔羊脑炎粪肠球菌裂解物作为抗原包被酶标板，应用棋盘滴定法，通过条件筛选成功建立了致羔羊肠球菌抗体测定的间接ELISA方法。应用该方法检测羔羊肠球菌阳性血清，其灵敏度是微量凝集反应的25-100倍。利用建立的方法检测来自未免疫羊场的50份成年羊血清和30份羔羊血清，结果显示有2份成年羊血清呈阳性。
3．检测羔羊肠球菌性脑炎PCR方法建立和初步应用：以致羔羊脑炎粪肠球菌基因组为模板，以相对保守的肠球菌的tuf基因为基础设计一对引物，通过条件筛选成功的建立了用于致羔羊脑炎肠球菌快速诊断的PCR方法，其灵敏度在10fg。应用该方法检测自然感染羔羊和人工感染发病死亡羔羊以及人工感染发病死亡小鼠主要器官组织，即在心、肝、脾、肾、脑、脑脊液等组织中均检测到了肠球菌DNA，其中2只人工感染死亡羔羊还在肺门淋巴结和肠系膜淋巴结中检测到肠球菌DNA。
4．免疫组化检测羔羊粪肠球菌性脑炎及其抗原定位的研究：以导致羔羊脑炎的粪肠球菌为研究对象，人工感染小鼠，通过条件优化，建立了肠球菌性羔羊脑炎检测的间接免疫组化方法。用该方法定期检测粪肠球菌在试验小鼠体内的分布，结果显示：4h时可在小鼠的肝脏中检测到粪肠球菌的阳性颗粒；14h时在肾脏和心脏中检测到阳性颗粒；18h可在食道中检测到粪肠球菌阳性颗粒；20h在脑中检测到阳性颗粒；22h在肺脏和气管中检测到粪肠球菌阳性颗粒；24h可在除脾脏外所有受检组织即脑、肝、心脏、肾、气管、肺、食道及小肠中观察到粪肠球菌的阳性颗粒。
5．致羔羊脑炎肠球菌毒力因子的分子流行病学调查：对11株致羔羊脑炎肠球菌ace、efa、cylA、gelE、asal和asa373、esp、EF0591和EF3314等9种毒力因子基因的PCR检测结果表明，5／11株肠球菌检测到esp、cylA、asal、ace、efa、EF0591和EF3314等7种毒力因子基因。1／11株检测到esp、gelE、asal、ace、efa和EF3314等6种毒力因子基因，1／11株检测到cylA、asal、ace、efa、EF0591和EF3314等6种毒力因子基因，1／11株检测到esp、cylA、asal、ace、EF0591和EF3314。1／11株菌仅携带esp基因。2／11株菌未检测上述9种毒力因子基因任何一种。与人类医学其它来源肠球菌毒力因子携带情况不同。
6．致羔羊脑炎粪肠球菌8种毒力因子基因片段克隆和序列特征研究：将致羔羊脑炎粪肠球菌检测到的的8个毒力因子8ce、efa、cylA、gelE、asal、esp、EF3314和EF0591基因部分片段克隆到pMD19-T载体上，应用PCR及酶切方法检测出阳性克隆并进行序列测定。通过BLAST软件对致羔羊脑炎粪肠球菌各毒力基因扩增片段的测定序列与GeneBank公布的医学临床分离株相应序列进行同源性分析比较，结果显示两种来源8种毒力因子基因目的片段序列的同源性分别为99.3％、99.56％、99.3％、97.85％、96.64％、99.9％、99.29％和98.11％。
7．致羔羊脑炎肠球菌与临床健康羔羊分离的肠球菌生物学特性的比较研究：通过研究发现羔羊两种来源肠球菌的培养特性和生化特性基本一致；对某些抗生素有不同程度的耐药性；致病株有更多的毒力因子组合；对两者共有的毒力因子基因片段进行克隆与序列分析，并同时与GeneBank来自医学临床分离株相应毒力因子基因片段比对分析，其同源性很高，均在95％以上。正常菌群株不能引起小鼠的死亡，而致病株可导致小鼠的死亡。
8．致羔羊脑炎粪肠球菌在人工感染小鼠疾病模型体内分布规律及病理学研究：经不同途径用致羔羊脑炎粪肠球菌分离株感染小鼠，最佳途径是腹腔注射，皮下注射次之，鼻腔吸入不引起感染。对小鼠的LD_(50)为9.3×10~(10)CFU。定期剖杀腹腔接种致羔羊脑炎粪肠球菌的小白鼠，用2种不同的方法检测在细菌主要脏器中的分布。结果发现感染2h后用PCR方法即可从脑、肝和心血检测到粪肠球菌DNA。6h后不仅能检测到肠球菌DNA，而且从上述组织中分离到肠球菌。感染4-6h后各组织脏器开始出现病理组织学变化，超微结构变化出现于4h。因此致羔羊脑炎粪肠球菌在人工感染小鼠体内分布广泛，引起的病理变化也是全身性的。
Enterococcus are usually studied as useful microorganism bcause of commensals of the intestinal tract of humans and animals but have emerged in recent decades as a major cause of nosocomial infections Enterococcus infections represent the second most common cause of bacteraemia and endocarditis after Staphylococcus in US hospitals from NISS Survey. Recently, there is a infectious diseases of 20-40 day old lamb which main characteristics are neurological symptoms and the septicemia in large-scale farms for the sheep of Xinjiang Production and Construction Corps in the same season. Lamb mortality is about 20%. It is identified as lamb encephalitis induced by Enterococusi through the isolation of bacteria and regression test. It is a new infectious diseases of lamb. A series of research n pathogen characteristics and diagnostic method are done in order to provide scientific data for pathogenic mechanism and control of the disease effectively about Enterococcus.
1 The Finding of Lamb Encephalitis Infected by Enterococcus Isolated and Identified 11 Enterococcus isolated from lamb encephalitis in partial breeding farm of Xinjiang Production and Construction Corps are identified by a series of tests. They belong to serum type D,G and unidentifiable type by Latex. They can led to mouse and partial guinea pigs or rabbits dead. The 11 bacterum have resistance for streptomycin,gentamicin, erythromycin and partial resistance to norfloxacin, penicillin, tetracycline, but sensitivity to nitrofurantoin, rifampin, chloramphenicol and vancomycin.
2 Development of ELISA to Detecte the Antibody for Enterococcus Inducing Lamb Encephalitis and Preliminary Application A ELISA are developed by phalanx titration with coating antigen of Enterococcus faecalis cracked by ultrasonic. It is more 25-100 times sensitive than micro-agglutination test through some positive serum for Enterococcus, and It has the more specific characteristic, easy or quick to operate. 2 serum in 50 adult sheep serum are positive in adult sheep serum detected.
3 Development of PCR to Detect Antigen for Enterococcus Inducing Iamb Encephalitis and Preliminary Application A PCR are developed base of Conservative gene tuf gene to detect Enterococcus. The optimal reaction conditions is that nuclease refolding temperature is 55℃, Mg~(2+) Concentration is 2.5um and reaction mode is 95℃5min, 94℃30s, 55℃30s, 72℃30s, 35 cycle, 72℃10 min. The sensitivity of this mathod is 10fg. The other 10 Enterococcus, the tissue from lamb and mouse infected by Enterococcus faecalis can be amplified special electrophoresis bands about 112bp and no any bands can be amplified in S.equi s, bsp.equi, Streptococcus G,Streptococcus A,S.aureus, L.monocytogenes,M.ovipneumoniae, Br.ovis and Enterococcus faecalis inducing lamb encephalitis.
4 A Study on the Detection of Enterococcus faecalis Inducing Lamb Encephalitis and Antigen Location by Immunoenzymatic Histochemistry An indirect Immunoenzymatic Histochemistry is developed to detect the distribution of Enterococcus faecalis in mouse artificial infected. The result indicated that positive particles of Enterococcus faecaIis can be detected in liver in 4h, in kidney and heart at 14h, in esophagus at 18h, in encephalon and in all tissues except for spleen of mouse infeted at 24h.
5 The research of Molecular epidemiology for Enterococcus inducing lamb encephalitis 9 virulence factor genes(ace,efaA,cylA, gelE, asaland asa373,esp,EF0591 and EF3314)in 11 Enterococcus inducing lamb encephalitis are detected. The results indicated that there is 7viru-lence factor genes in 5 bacteria (5/11),6 virulence factor genes in 3 bacteria (3/11), 1viru-lence factor gene in 1 bacteria (1/11),no virulence factor genes 2 bacteria (2/11).
6 Gene Cloning and Sequence Analysis of Virulence Factor Genes in Enterococeus facealis Inducing Lamb Encephalitis 8 virulence factor genes (ace, efa,cylA,gelE, asa1, esp, EF0591, EF3314) segments amplyfied are cloned in pMD19-T and the positive clone are going to determin the nucleotide Sequence, the results indicated that the homology is 99.3%、99.56%、99.3%、97.85%、96.64%、99.9%、99.29% and 98.11% between the Enterococcus faecalis from lamb encephalitis and corresponding sequence from GeneBank.
7 A Comparative Study of Biological Characteristics of Enterococci from Different Sources of Lamb The study on cultural characteristics, biochemical characteristics, hemo- lytic properties, drug sensitivity and the types, distribution of virulence factor gene of Enterococcus inducing lamb encephalitis and normal flora indecate that basic biochemical chara- cteristics of enterococci from two different sources of lamb are consistent. Pathogens have resistance for Streptomycin, gentamicin and erythromycin, one bacterial from normal flora have resistance for tetracycline, erythromycin, streptomycin, esp、cylA、asal、ace、efa、EF0591 and EF3314 can be detected in 5/11 from Enterococcus inducing lamb encephalitis. gelE and EF3314 can be detected in 3/30 from normal flora and gelE、EF3314 and asal can be detected in 1/30 from normal flora. The homology of 3 gene sequence is more than 95% between GeneBank and bacteria from normal flora, the corresponding sequence is more than 96% with patho-genic strains, pathogens can cause the death of mice, whereas the Enterococci from normal flora can not cause the death of mice.
8 A study on distribution and pathological changes in artificial infectious mice disease model forEnterococcus faecalis inducing Lamb encephalitis A study on 135 mouse infec ted by Enterococcus faecalis inducing Lamb encephalitis with different ways indicates that intraperitoneal injection is the best way, subcutaneous implant is second way and nasal inhal-ation can not cause infectious. LD_(50) of the bacteria is9.3×10~(10)CFU. Enterococcus faecalis DNA can be detected in encephalon, liver and blood spleen in artificial infectious mouse at 2h. Enterococcus faecalis and its DNA can be detected in the tissues at 6h. Signifiant histopatho-logical changes are observed during 4-6h and the changes of ultrastructural organization are observed at 4h.
引文
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