Riemerella anatipestifer的分离鉴定及荚膜多糖研究
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摘要
鸭疫里默氏杆菌(Riemerella anatipestifer，简称RA)引起鸭的传染性浆膜炎(Infectious serositis)，是危害养鸭业最重要的病原之一。鸭疫里默氏杆菌的血清型复杂，现被公认的有21个血清型，各血清型之间缺乏交叉免疫保护。在生产实际中，主要利用抗菌药物进行鸭传染性浆膜炎的预防和治疗，但耐药性越来越严重。因此，进行鸭疫里默氏杆菌的分离鉴定，弄清流行菌株的优势血清型及耐药性等是当前及今后一段时间内防治此病必须优先考虑的工作。本试验自2004年9月至2005年5月从重庆和四川地区收集可疑患病鸭、鹅，并进行鸭疫里默氏杆菌分离鉴定，弄清了流行菌株的血清型及耐药性，为该地区鸭传染性浆膜炎的有效防治提供了保障；另一方面，我们提取了细菌的荚膜成分，并纯化获得荚膜多糖及对荚膜多糖的单糖组成、生物学特性等进行了研究，为进一步研究鸭疫里默氏杆菌的荚膜多糖奠定了基础。本研究的主要结果如下：
1．自2004年9月至2005年5月从重庆和四川地区收集的浆膜炎可疑病料中利用巧克力琼脂平板分离培养，通过形态观察、生化鉴定、致病性测定及PCR扩增协同溶血素基因(cam)等鉴定出64株鸭疫里默氏杆菌。
2．利用平板凝集试验和琼脂双扩散试验将64株分离细菌分为8个不同血清型，其中GNRA、HNDJ和MY为新血清型，占总分离数的15.6％。生长曲线测定表明细菌在12-14小时达到生长高峰；鸭源和鹅源株对10日龄鸭的半数致死量(LD_(50))分别为1.9×10~7和6.0×10~7CFU／ml；体外抗生素敏感性测定表明对头孢三嗪、利福平、新霉素、头孢唑啉、恩诺沙星、环丙沙星、壮观霉素的敏感率分别为66％、64％、61％、55％、52％、48％、45％。
3．在20℃条件下，利用十六烷基三甲基溴化铵(CRAB)从鸭疫里默氏杆菌改良马丁肉汤培养物中提取出乳白色泥状、真空干燥后呈白色疏松状的荚膜提取物；经sevag法脱蛋白、DEAE-Sepharose和Sephadex G-200层析法纯化，得到AF、BSY、HNDJ及MY株的荚膜多糖，其中糖含量分别为54.0、33.5、31.6和37.6ug／ml，蛋白含量分别为10.3、20.3、11.0和11.4 ug／ml，核酸含量分别为4.2、5.0、4.1和5.3 ug／ml。在分子筛层析中，分别在490nm和280nm处检测糖与蛋白质的峰形一致，表明鸭疫里默氏杆菌的荚膜多糖可能为糖蛋白。
4．AF、BSY、HNDJ和MY株荚膜粗提物与鸡抗鸭疫里默氏杆菌卵黄抗体的琼扩效价分别为1：8、1：16、1：8和1：8；经初提纯纯化的荚膜物质与鸡抗鸭疫里默氏杆菌卵黄抗体的琼扩效价分别为1：1、1：2、1：4和1：2；经离子交换和分子筛层析层析纯化的荚膜多糖与鸡抗鸭疫里默氏杆菌卵黄抗体在琼扩试验中没有出现沉淀线。用AF、BSY、HNDJ和MY株纯化的荚膜多糖制作的矿物油乳剂免疫抗原分别在5和15日龄150ug／只的剂量免疫鸭，10天后用同型或异型菌株以10LD_(50)剂量攻毒，AF株荚膜多糖免疫试验鸭经AF、BSY、HNDJ、MY株攻毒的保护率分别为50％、12.5％、25％、0；BSY株分别为12.5％、50％、50％、0；HNDJ株分别为25％、50％、50％、0；MY株0、0、0、25％。
5．采用薄层层析法测定AF、BSY、HNDJ、MY株的荚膜多糖的单糖组成，AF株和HNDJ株荚膜多糖含有葡萄糖和半乳糖，BSY株含有半乳糖和鼠李糖，MY株含有半乳糖。试验证明可以根据鸭疫里默氏杆菌荚膜物质中多糖的单糖组成进行荚膜分群。
The pathogen named Riemerella anatipestifer (which is abbreviated as RA) is one of the most important causative agent that can attack the ducklings giving rise them to infectious serositis. The serotypes of Riemerella anatipestifer are so complicate that tween-one had been demarcated in the world, which is acknowledged to the personnel who are engaging in the research work about it around the world. There have little or no significant cross-protection against challenge among different serotypes of RA. The drug-sensitiveness is increasingly aggravated because antibiotic are utilized heavily to prevent and remedy the disease in practice.So, it is the prior task of consideration to thrown vast stamina of research on the work such as probing the prevailing serotypes and drug-resistance of RA. In view of the above.This research ascertained the principal serotypes and drug-resistance of RA obtained from the infected ducklings or gooselings during the time from September 2004 to may 2005. The results above provide effective indemnification for the cure to infectious serositis. On the other hand, the capsular polysaccharide of bacterials are extracted and purified by chormatgraphy technicals, then, the component and biological characterization of the capsular polysaccharide are determined by chormatography simultaneously. The first fruits laid the foundation for the further research of capsular polysaccharide of Rimerella anatipestifer. The main outcome summarized as follows:1. The total 64 strains isolated from the dubious infected ducklings which are collected from chongqing and sichuan regions are identified as Riemerella anatipestifer by observation of morphology , assay of biochemical characterization , determination of pathogenicity and amplification of cam gene through growth on chocolate plate.2. The combined test of plate agglutination and agarose gel double diffusion divided the 64 strains of them into eight serotypes, Five serotypes are known already. Additional investations showed that remaining ten strains amounting for 15.6% of the total represented three new occurred serotypes which are named tentatively as GNRA、HNDJ and My according to origination. It showed that the time from 12 to 14 is the peak growth value for RA. The median lethal dose (LD_(50)) value of the RA isolated from ducklings or gooselings are 1.9×10~7 and 6.0×10~7 colony formation unity(CFU/ml), respectively. The percentage of antibioic-sensitiveness to Cefazolin、Rifampicin、Neomycinum、Cefazolin、Enrofloxacin、Ciprofloxacin and Spectinomycin of Riemerella anatipestifer are 66%、64%、61%、55%、52%、48% and 45%, respectively.
3. The materials of capsular polysaccharide looks like muddy clump extracted from the centrifuged supematant of cultured RA and colored white-ivory after lyophilized. The crude capsular polysaccharide turned into refinment after purified by the technique of sevag protein removal, DEAE-sepharose and sephadex G-200 chromatgraphy. The contents of saccharide from strain AF、F、BSY、HNDJ and MY are 54.0、33.5、31.6 and 37.6ug/ml, respectively. The contents of protein are 10.3、20.3、11.0 and 11.4 ug/ml; the contents of nucleis are 4.2、5.0、4.1 and 5.3 ug/ml. The polysaccharide are probably acid glycoprotein on the grounds that the absorption peak of saccharide and protein on elution pattern curve are unanimous when they are monitored by ultraviolet spectrophotometer at 490 and 280 nanometer.
4. The agarose gel precipitin valence are 1: 8, 1: 16, 1: 8 and 1: 8 when the crude materials of capsular polysaccharide extracted from strain AF、BSY、HNDJ and MY reacted to yolk antibody made of homologous strain antigen; the agarose gel precipitin valence are 1: 1、1: 2、1: 4 and 1: 2 when primary purified materials of capsular polysaccharide reacted with yolk antibody, respectively. There is no precipitin lines emerged when the capsular polysaccharide refined by DEAE-sepharose and sephadex G-200 chromatgraphy are reacted with yolk antibody. The percentage protection of mineral oil vaccination made of polysaccharide of strain AF are 50%, 12.5%, 25% and 0% when challenged with serotype AF, BSY, HND Jand MY; The percentage protection of mineral oil vaccination made of polysaccharide of strain BSY are 50%, 12.5%, 25%and 0% when challenged with serotype AF, BSY, HNDJ and MY; The percentage protection of mineral oil vaccination made of polysaccharide of strain HNDJ are 25%, 50%, 50% and 0% when challenged with homologous serotype AF, BSY, HNDJband MY; The percentage protection of mineral oil vaccination made of polysaccharide of strain M Y are 0%, 0%, 0% and 25% when challenged with serotype AF, BSY, HNDJ and MY.
5. The monosaccharide component of polysaccharide of strain AF、BSY、HNDJ、MY are determined by thin-layer chromatgrphy. Glucose and galactose are expiscated from strain AF and HNDJ, galactose and rhamnose expiscated from strain BSY, galactose expiscated from strain MY. It can divide the antigen of capsular into several representative groups according to the composition of monosaccharide of RA
1．自2004年9月至2005年5月从重庆和四川地区收集的浆膜炎可疑病料中利用巧克力琼脂平板分离培养，通过形态观察、生化鉴定、致病性测定及PCR扩增协同溶血素基因(cam)等鉴定出64株鸭疫里默氏杆菌。
2．利用平板凝集试验和琼脂双扩散试验将64株分离细菌分为8个不同血清型，其中GNRA、HNDJ和MY为新血清型，占总分离数的15.6％。生长曲线测定表明细菌在12-14小时达到生长高峰；鸭源和鹅源株对10日龄鸭的半数致死量(LD_(50))分别为1.9×10~7和6.0×10~7CFU／ml；体外抗生素敏感性测定表明对头孢三嗪、利福平、新霉素、头孢唑啉、恩诺沙星、环丙沙星、壮观霉素的敏感率分别为66％、64％、61％、55％、52％、48％、45％。
3．在20℃条件下，利用十六烷基三甲基溴化铵(CRAB)从鸭疫里默氏杆菌改良马丁肉汤培养物中提取出乳白色泥状、真空干燥后呈白色疏松状的荚膜提取物；经sevag法脱蛋白、DEAE-Sepharose和Sephadex G-200层析法纯化，得到AF、BSY、HNDJ及MY株的荚膜多糖，其中糖含量分别为54.0、33.5、31.6和37.6ug／ml，蛋白含量分别为10.3、20.3、11.0和11.4 ug／ml，核酸含量分别为4.2、5.0、4.1和5.3 ug／ml。在分子筛层析中，分别在490nm和280nm处检测糖与蛋白质的峰形一致，表明鸭疫里默氏杆菌的荚膜多糖可能为糖蛋白。
4．AF、BSY、HNDJ和MY株荚膜粗提物与鸡抗鸭疫里默氏杆菌卵黄抗体的琼扩效价分别为1：8、1：16、1：8和1：8；经初提纯纯化的荚膜物质与鸡抗鸭疫里默氏杆菌卵黄抗体的琼扩效价分别为1：1、1：2、1：4和1：2；经离子交换和分子筛层析层析纯化的荚膜多糖与鸡抗鸭疫里默氏杆菌卵黄抗体在琼扩试验中没有出现沉淀线。用AF、BSY、HNDJ和MY株纯化的荚膜多糖制作的矿物油乳剂免疫抗原分别在5和15日龄150ug／只的剂量免疫鸭，10天后用同型或异型菌株以10LD_(50)剂量攻毒，AF株荚膜多糖免疫试验鸭经AF、BSY、HNDJ、MY株攻毒的保护率分别为50％、12.5％、25％、0；BSY株分别为12.5％、50％、50％、0；HNDJ株分别为25％、50％、50％、0；MY株0、0、0、25％。
5．采用薄层层析法测定AF、BSY、HNDJ、MY株的荚膜多糖的单糖组成，AF株和HNDJ株荚膜多糖含有葡萄糖和半乳糖，BSY株含有半乳糖和鼠李糖，MY株含有半乳糖。试验证明可以根据鸭疫里默氏杆菌荚膜物质中多糖的单糖组成进行荚膜分群。
The pathogen named Riemerella anatipestifer (which is abbreviated as RA) is one of the most important causative agent that can attack the ducklings giving rise them to infectious serositis. The serotypes of Riemerella anatipestifer are so complicate that tween-one had been demarcated in the world, which is acknowledged to the personnel who are engaging in the research work about it around the world. There have little or no significant cross-protection against challenge among different serotypes of RA. The drug-sensitiveness is increasingly aggravated because antibiotic are utilized heavily to prevent and remedy the disease in practice.So, it is the prior task of consideration to thrown vast stamina of research on the work such as probing the prevailing serotypes and drug-resistance of RA. In view of the above.This research ascertained the principal serotypes and drug-resistance of RA obtained from the infected ducklings or gooselings during the time from September 2004 to may 2005. The results above provide effective indemnification for the cure to infectious serositis. On the other hand, the capsular polysaccharide of bacterials are extracted and purified by chormatgraphy technicals, then, the component and biological characterization of the capsular polysaccharide are determined by chormatography simultaneously. The first fruits laid the foundation for the further research of capsular polysaccharide of Rimerella anatipestifer. The main outcome summarized as follows:1. The total 64 strains isolated from the dubious infected ducklings which are collected from chongqing and sichuan regions are identified as Riemerella anatipestifer by observation of morphology , assay of biochemical characterization , determination of pathogenicity and amplification of cam gene through growth on chocolate plate.2. The combined test of plate agglutination and agarose gel double diffusion divided the 64 strains of them into eight serotypes, Five serotypes are known already. Additional investations showed that remaining ten strains amounting for 15.6% of the total represented three new occurred serotypes which are named tentatively as GNRA、HNDJ and My according to origination. It showed that the time from 12 to 14 is the peak growth value for RA. The median lethal dose (LD_(50)) value of the RA isolated from ducklings or gooselings are 1.9×10~7 and 6.0×10~7 colony formation unity(CFU/ml), respectively. The percentage of antibioic-sensitiveness to Cefazolin、Rifampicin、Neomycinum、Cefazolin、Enrofloxacin、Ciprofloxacin and Spectinomycin of Riemerella anatipestifer are 66%、64%、61%、55%、52%、48% and 45%, respectively.
3. The materials of capsular polysaccharide looks like muddy clump extracted from the centrifuged supematant of cultured RA and colored white-ivory after lyophilized. The crude capsular polysaccharide turned into refinment after purified by the technique of sevag protein removal, DEAE-sepharose and sephadex G-200 chromatgraphy. The contents of saccharide from strain AF、F、BSY、HNDJ and MY are 54.0、33.5、31.6 and 37.6ug/ml, respectively. The contents of protein are 10.3、20.3、11.0 and 11.4 ug/ml; the contents of nucleis are 4.2、5.0、4.1 and 5.3 ug/ml. The polysaccharide are probably acid glycoprotein on the grounds that the absorption peak of saccharide and protein on elution pattern curve are unanimous when they are monitored by ultraviolet spectrophotometer at 490 and 280 nanometer.
4. The agarose gel precipitin valence are 1: 8, 1: 16, 1: 8 and 1: 8 when the crude materials of capsular polysaccharide extracted from strain AF、BSY、HNDJ and MY reacted to yolk antibody made of homologous strain antigen; the agarose gel precipitin valence are 1: 1、1: 2、1: 4 and 1: 2 when primary purified materials of capsular polysaccharide reacted with yolk antibody, respectively. There is no precipitin lines emerged when the capsular polysaccharide refined by DEAE-sepharose and sephadex G-200 chromatgraphy are reacted with yolk antibody. The percentage protection of mineral oil vaccination made of polysaccharide of strain AF are 50%, 12.5%, 25% and 0% when challenged with serotype AF, BSY, HND Jand MY; The percentage protection of mineral oil vaccination made of polysaccharide of strain BSY are 50%, 12.5%, 25%and 0% when challenged with serotype AF, BSY, HNDJ and MY; The percentage protection of mineral oil vaccination made of polysaccharide of strain HNDJ are 25%, 50%, 50% and 0% when challenged with homologous serotype AF, BSY, HNDJband MY; The percentage protection of mineral oil vaccination made of polysaccharide of strain M Y are 0%, 0%, 0% and 25% when challenged with serotype AF, BSY, HNDJ and MY.
5. The monosaccharide component of polysaccharide of strain AF、BSY、HNDJ、MY are determined by thin-layer chromatgrphy. Glucose and galactose are expiscated from strain AF and HNDJ, galactose and rhamnose expiscated from strain BSY, galactose expiscated from strain MY. It can divide the antigen of capsular into several representative groups according to the composition of monosaccharide of RA
引文
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