仔猪水肿病灭活疫苗的研制
摘要
猪水肿病(Edema disease, ED)是由产志贺毒素大肠杆菌(STEC)引起的以头部、肠系膜和胃壁浆液性水肿为特征的一种肠毒血症,常伴有共济失调、麻痹或惊厥等神经症状。本病多发生于断奶前后的仔猪,呈地方性流行和散发性,猪水肿病发病率虽然只有10%～30%,但致死率可高达80%～100%,是影响养猪业发展的重要传染病之
随着猪场养殖规模的扩大和仔猪饲养密度的增加,猪水肿病日益严重,成为导致断奶仔猪死亡的重要传染病。本病的防治目前主要采取加强饲养管理及对病程稍长的仔猪对症治疗的综合性防治措施,但治愈率不高。由于本病的致死率高,所以疫苗免疫已成为防制本病的最经济的、有效的途径之一
能引起猪水肿病的大肠杆菌称为产志贺毒素大肠杆菌(STEC), STEC有两类毒力因子：黏附素(adhesin)和志贺毒素(Stx)。黏附素包括菌毛、外膜蛋白(OMP)、紧密素(intimin)等。F18菌毛是STEC菌株的一个重要的毒力因子,它有助于细菌在猪肠黏膜上皮细胞定居和繁殖。而志贺毒素系STEC所产生的一种蛋白质性细胞毒素。STEC有多种血清型,常见的有O139：K82、O138：K81、O141：K85、O45、O8等。近年来有关STEC分离及鉴定的报道比较多,但疫苗研制的效果并不理想,目前还没有安全稳定、免疫效果确实的商品化疫苗被国家批准和面市。
本课题以从中国兽医药品监察所引进的临床常见血清型的标准株为研究对象,旨在研发出免疫效果确实、应用范围较广的高效安全的商品化疫苗。我们引进的菌种为O138、O139、O141三个血清型的大肠杆菌C839O5、C83684、C83527的冻干菌种,首先,按照疫苗生产工艺和新兽药申报的有关要求,进行了菌种的纯粹性检验、菌体形态及培养特性观察、生化特性鉴定、血清型鉴定、毒力测定、免疫原性测定及稳定性测定等试验。
结果表明此三株菌种均为革兰氏阴性菌,生化特性符合大肠杆菌的特性,菌种C83905株的菌体抗原血清型为O138,C83527株为O141、C83684株为O139；菌株对小鼠的最小致死量(MLD)分别为：C83905株3.6×108CFU,C83527株3.0×108CFU,C83684株2.0×108CFU；C83905、C83684、C83527三株菌的毒素对小鼠的最小致死量均为0.1mL/只,混合毒素为0.2mL/只；C83905、C83684、C83527三株菌毒素对14～18日龄仔猪的最小致死量分别为30mL、40mL、40mL,混合毒素的最小致死量为30mL/头；用三个菌株制备的灭活疫苗(灭活前含活菌数为3.0×108CFU/mL),分别以0.05mL/只免疫小鼠和以2mU头免疫14～18日龄仔猪,分别进行致死量的强毒菌和强毒菌毒素的攻击,均能达到80%～100%的保护率,充分表明了三株菌具有非常好的免疫原性。综上所述,引进的菌种具备了作为兽用生物制品菌种的基本条件。
在此基础上,又对菌种的培养条件、灭活条件及疫苗佐剂选择进行了优化,如使用改良的LB培养基、以0.4%浓度甲醛37℃灭活48h和使用氢氧化铝胶作为佐剂等等,最终成功研制了仔猪水肿病三价(0138、0139、0141)灭活疫苗。根据实验结果,初步制定了仔猪水肿病灭活疫苗制造与检验试行规程和质量标准。
通过对实验室试制的6批疫苗进行安全性试验、效力试验、最小免疫剂量测定及疫苗的免疫期试验和保存期试验等,表明了制备的疫苗具有良好的安全性和良好的保护力。安全性试验结果表明,用16-18g小鼠每只皮下注射0.3mL疫苗和用14日龄仔猪每头肌肉注射4mL疫苗均安全；最小免疫剂量试验表明,免疫小鼠能达50%以上保护的免疫剂量是0.01mL/只,免疫仔猪能达50%以上保护的免疫剂量1.0mL/头；此疫苗免疫小鼠能达100%以上保护的最小免疫剂量是0.05mL/只,免疫仔猪能达100%以上保护的最小免疫剂量1.5mL/头,为保证免疫效果确实,将此疫苗的使用剂量确定为2mL/头。经检验,该疫苗的甲醛、硫柳汞含量均符合制品的有关规定,免疫期至少可达3个月。此疫苗在2-8℃条件下保存到24个月,物理性状仍达到规程要求的标准,保存到18个月,效力仍能达到80%保护以上。另外,关于菌株毒力、疫苗安全性和保护性试验证明,对小鼠的使用剂量与对仔猪的使用剂量具有相关性,在实际生产中可用小鼠代替仔猪进行检验。
根据实验室的试制情况,我们完善了仔猪水肿病灭活疫苗制造与检验试行规程和质量标准,并按照试行规程进行了中试生产,经检验,试制的6批疫苗经检验符合制定的质量标准。
田间试验和扩大田间试验结果表明,实验室试制的6批疫苗,均具有良好的安全性和确实的效力,保护率达95%以上。中试疫苗在沈阳市、大连市、鞍山市、锦州市、铁岭市、辽阳市等地区免疫仔猪10万余头,均安全有效,总保护率达95%以上,对控制仔猪水肿病收到了满意的结果。
以溶血性大肠杆菌C83905、C83684、C83527研制的仔猪水肿病灭活疫苗,按照新兽药申报的要求进行了注册申报,经过多次完善和补充试验,制备的三批样品,经中国兽医药品监察所进行质量标准复核检验,结果均达到制定的质量标准要求。该疫苗于2006年8月份率先获得农业部颁发的国家二类新兽药证书[证号：(2006)新兽药证字30号]
Edema disease of swine (ED) is an enterotoxemia caused mainly by Shiga toxin E.coli (STEC) which can prod cutaneous dropsy at face, conjunctive tunic, dental caries, eyelid, submaxilla, gasyric wall of piglets and nerval symptom. This disease which display the feature of endemic epidemic usually attack weaned piglets. Although the incidence rate is less than10%-30%, the mortality rate reaches to80%-100%. It's one of the most important infectious disease which impacts the development of the swine industry. With the enlargment of the cultivation field and the increase of piglets breeding density, Ederma disease gradually becomes a significant communicable disease causing death of post-weaning pigs. The preventive and therapeutic measures to the disease depend on strengthening breeding administration and relieving the symptoms at present, but the recovery rate is low. Consequently, vaccine immunifaction has become one of the most economic and effective pathway to control this disease.
The Bacterium coli which causing Edema disease is called Shiga toxin E.coli (STEC). It produces two virulence facts:adhesin and Shiga toxin (Stx). Adhesin includes fimbria, OMP, intimin and so on. Fimbria F18is an important virulence fact which helps to ecesis and reproduce in cellula epithelialis of mucous membrane. Stx is a protein cytotoxin produced by Shiga toxin E.coli. It has many kinds of serotype. The frequent serotype is0139, K82,0138, K81,0141, K85,045, and08and so on. Recently, there were many papers that reported on isolation and identification of piglet enteropathogenic E.coli, but the developments of the vaccine were not satisfactory simultaneously. There is not safe and effective commercialization vaccine which obtained the nation license nowadays.
In this study, three different serotype bacterial strains (C83905:O138、C83684:O139、 C83527:O141) of E.coli were introduced from China Institute of Veterinary Drugs Control to develop the high performance inactivated vaccines against Porcine Edema disease. According to the applying requirement of the new veterinary drugs, the purity, cultural character, biochemistry character, serotype, virulence, immunity of the strains have been detected separately and yet the stability tests have been executed.
The results showed that the three strains were Gram-negative bacteria, and the serotypes of C83905, C83527, and C83684were0138,0141, and0139respectively. The strains'minimum lethal dose for mice were3.6×108CFU(C83905),3.0×108CFU (C83527),2.0×108CFU (C83684). The MLD of the toxinum of the three bacteria were all0.1mL per mouse, while the toxinum mixture of the three bacteria for mice was0.2mL per mouse, and for14d-18d old piglets, the MLD was30mL,40mL,40mL respectively. And the MLD of toxinum mixture was30mL per pig. When receiving the inactivated vaccine prepared respectively with the three strains, the mice and the piglets which were injected0.05mL and2mL respectively could counteract the challenge with the fatal dose of the strains and the toxinum, and showed80%-100%protection rates. All the results showed that the three strains corresponded completely with the requirement of vaccine preparation as we expected.
On the basis of the laboratory process, the culture medium, inactivated condition and adjuvant were optimized under industry producing conditions. In consideration of (the stability of bacterial stain and the cost of production, the improved LB medium was selected as the optimal one. The optimal concentration of formaldehyde was0.4%and the inactivating condition was48hours at37℃. Finally, Trivalent (O138、O139、0141) inactivated vaccine against piglet edema disease was developed successfully. According to the experimental results, the initial manufacture and test regulations and the quality standards of this vaccine were carried out.
Then, six batches of the laboratory processing vaccine had been prepared, and the tests of safety, efficacy, min-immunizing-dose, immunity and keeping time were tested. All the results showed that the products which could induce pronounced immune response were safe and effective. The min-immunizing-dose which could obtain100%protection is0.05mL for mouse and2mL for piglet. The immune period could last for three months at least, and the storage period could be twenty-four months at2-8℃.
In addition, the correlation about the immunizing dose of strains and vaccines for mice and piglets were detected during the virulence, safety and protection test. It showed that piglets could be replaced by mice for tests in practical production.
Six batches semi-works products were tested according the regulation and the standard severely. The protection rate of six batches semi-works products was higher than95%in two field experiments. The production were used for more than100,000pigs in Shenyang, Dalian, Anshan, Jinzhou, Tieling, Liaoyang of China and so on, and showed better safety and excellent effect.
The trivalent(O138、O139、0141) inactivated vaccine against piglet edema disease had been registered and it had been awarded the second-class certificate of national new veterinary drugs in Aug,2006.
随着猪场养殖规模的扩大和仔猪饲养密度的增加,猪水肿病日益严重,成为导致断奶仔猪死亡的重要传染病。本病的防治目前主要采取加强饲养管理及对病程稍长的仔猪对症治疗的综合性防治措施,但治愈率不高。由于本病的致死率高,所以疫苗免疫已成为防制本病的最经济的、有效的途径之一
能引起猪水肿病的大肠杆菌称为产志贺毒素大肠杆菌(STEC), STEC有两类毒力因子：黏附素(adhesin)和志贺毒素(Stx)。黏附素包括菌毛、外膜蛋白(OMP)、紧密素(intimin)等。F18菌毛是STEC菌株的一个重要的毒力因子,它有助于细菌在猪肠黏膜上皮细胞定居和繁殖。而志贺毒素系STEC所产生的一种蛋白质性细胞毒素。STEC有多种血清型,常见的有O139：K82、O138：K81、O141：K85、O45、O8等。近年来有关STEC分离及鉴定的报道比较多,但疫苗研制的效果并不理想,目前还没有安全稳定、免疫效果确实的商品化疫苗被国家批准和面市。
本课题以从中国兽医药品监察所引进的临床常见血清型的标准株为研究对象,旨在研发出免疫效果确实、应用范围较广的高效安全的商品化疫苗。我们引进的菌种为O138、O139、O141三个血清型的大肠杆菌C839O5、C83684、C83527的冻干菌种,首先,按照疫苗生产工艺和新兽药申报的有关要求,进行了菌种的纯粹性检验、菌体形态及培养特性观察、生化特性鉴定、血清型鉴定、毒力测定、免疫原性测定及稳定性测定等试验。
结果表明此三株菌种均为革兰氏阴性菌,生化特性符合大肠杆菌的特性,菌种C83905株的菌体抗原血清型为O138,C83527株为O141、C83684株为O139；菌株对小鼠的最小致死量(MLD)分别为：C83905株3.6×108CFU,C83527株3.0×108CFU,C83684株2.0×108CFU；C83905、C83684、C83527三株菌的毒素对小鼠的最小致死量均为0.1mL/只,混合毒素为0.2mL/只；C83905、C83684、C83527三株菌毒素对14～18日龄仔猪的最小致死量分别为30mL、40mL、40mL,混合毒素的最小致死量为30mL/头；用三个菌株制备的灭活疫苗(灭活前含活菌数为3.0×108CFU/mL),分别以0.05mL/只免疫小鼠和以2mU头免疫14～18日龄仔猪,分别进行致死量的强毒菌和强毒菌毒素的攻击,均能达到80%～100%的保护率,充分表明了三株菌具有非常好的免疫原性。综上所述,引进的菌种具备了作为兽用生物制品菌种的基本条件。
在此基础上,又对菌种的培养条件、灭活条件及疫苗佐剂选择进行了优化,如使用改良的LB培养基、以0.4%浓度甲醛37℃灭活48h和使用氢氧化铝胶作为佐剂等等,最终成功研制了仔猪水肿病三价(0138、0139、0141)灭活疫苗。根据实验结果,初步制定了仔猪水肿病灭活疫苗制造与检验试行规程和质量标准。
通过对实验室试制的6批疫苗进行安全性试验、效力试验、最小免疫剂量测定及疫苗的免疫期试验和保存期试验等,表明了制备的疫苗具有良好的安全性和良好的保护力。安全性试验结果表明,用16-18g小鼠每只皮下注射0.3mL疫苗和用14日龄仔猪每头肌肉注射4mL疫苗均安全；最小免疫剂量试验表明,免疫小鼠能达50%以上保护的免疫剂量是0.01mL/只,免疫仔猪能达50%以上保护的免疫剂量1.0mL/头；此疫苗免疫小鼠能达100%以上保护的最小免疫剂量是0.05mL/只,免疫仔猪能达100%以上保护的最小免疫剂量1.5mL/头,为保证免疫效果确实,将此疫苗的使用剂量确定为2mL/头。经检验,该疫苗的甲醛、硫柳汞含量均符合制品的有关规定,免疫期至少可达3个月。此疫苗在2-8℃条件下保存到24个月,物理性状仍达到规程要求的标准,保存到18个月,效力仍能达到80%保护以上。另外,关于菌株毒力、疫苗安全性和保护性试验证明,对小鼠的使用剂量与对仔猪的使用剂量具有相关性,在实际生产中可用小鼠代替仔猪进行检验。
根据实验室的试制情况,我们完善了仔猪水肿病灭活疫苗制造与检验试行规程和质量标准,并按照试行规程进行了中试生产,经检验,试制的6批疫苗经检验符合制定的质量标准。
田间试验和扩大田间试验结果表明,实验室试制的6批疫苗,均具有良好的安全性和确实的效力,保护率达95%以上。中试疫苗在沈阳市、大连市、鞍山市、锦州市、铁岭市、辽阳市等地区免疫仔猪10万余头,均安全有效,总保护率达95%以上,对控制仔猪水肿病收到了满意的结果。
以溶血性大肠杆菌C83905、C83684、C83527研制的仔猪水肿病灭活疫苗,按照新兽药申报的要求进行了注册申报,经过多次完善和补充试验,制备的三批样品,经中国兽医药品监察所进行质量标准复核检验,结果均达到制定的质量标准要求。该疫苗于2006年8月份率先获得农业部颁发的国家二类新兽药证书[证号：(2006)新兽药证字30号]
Edema disease of swine (ED) is an enterotoxemia caused mainly by Shiga toxin E.coli (STEC) which can prod cutaneous dropsy at face, conjunctive tunic, dental caries, eyelid, submaxilla, gasyric wall of piglets and nerval symptom. This disease which display the feature of endemic epidemic usually attack weaned piglets. Although the incidence rate is less than10%-30%, the mortality rate reaches to80%-100%. It's one of the most important infectious disease which impacts the development of the swine industry. With the enlargment of the cultivation field and the increase of piglets breeding density, Ederma disease gradually becomes a significant communicable disease causing death of post-weaning pigs. The preventive and therapeutic measures to the disease depend on strengthening breeding administration and relieving the symptoms at present, but the recovery rate is low. Consequently, vaccine immunifaction has become one of the most economic and effective pathway to control this disease.
The Bacterium coli which causing Edema disease is called Shiga toxin E.coli (STEC). It produces two virulence facts:adhesin and Shiga toxin (Stx). Adhesin includes fimbria, OMP, intimin and so on. Fimbria F18is an important virulence fact which helps to ecesis and reproduce in cellula epithelialis of mucous membrane. Stx is a protein cytotoxin produced by Shiga toxin E.coli. It has many kinds of serotype. The frequent serotype is0139, K82,0138, K81,0141, K85,045, and08and so on. Recently, there were many papers that reported on isolation and identification of piglet enteropathogenic E.coli, but the developments of the vaccine were not satisfactory simultaneously. There is not safe and effective commercialization vaccine which obtained the nation license nowadays.
In this study, three different serotype bacterial strains (C83905:O138、C83684:O139、 C83527:O141) of E.coli were introduced from China Institute of Veterinary Drugs Control to develop the high performance inactivated vaccines against Porcine Edema disease. According to the applying requirement of the new veterinary drugs, the purity, cultural character, biochemistry character, serotype, virulence, immunity of the strains have been detected separately and yet the stability tests have been executed.
The results showed that the three strains were Gram-negative bacteria, and the serotypes of C83905, C83527, and C83684were0138,0141, and0139respectively. The strains'minimum lethal dose for mice were3.6×108CFU(C83905),3.0×108CFU (C83527),2.0×108CFU (C83684). The MLD of the toxinum of the three bacteria were all0.1mL per mouse, while the toxinum mixture of the three bacteria for mice was0.2mL per mouse, and for14d-18d old piglets, the MLD was30mL,40mL,40mL respectively. And the MLD of toxinum mixture was30mL per pig. When receiving the inactivated vaccine prepared respectively with the three strains, the mice and the piglets which were injected0.05mL and2mL respectively could counteract the challenge with the fatal dose of the strains and the toxinum, and showed80%-100%protection rates. All the results showed that the three strains corresponded completely with the requirement of vaccine preparation as we expected.
On the basis of the laboratory process, the culture medium, inactivated condition and adjuvant were optimized under industry producing conditions. In consideration of (the stability of bacterial stain and the cost of production, the improved LB medium was selected as the optimal one. The optimal concentration of formaldehyde was0.4%and the inactivating condition was48hours at37℃. Finally, Trivalent (O138、O139、0141) inactivated vaccine against piglet edema disease was developed successfully. According to the experimental results, the initial manufacture and test regulations and the quality standards of this vaccine were carried out.
Then, six batches of the laboratory processing vaccine had been prepared, and the tests of safety, efficacy, min-immunizing-dose, immunity and keeping time were tested. All the results showed that the products which could induce pronounced immune response were safe and effective. The min-immunizing-dose which could obtain100%protection is0.05mL for mouse and2mL for piglet. The immune period could last for three months at least, and the storage period could be twenty-four months at2-8℃.
In addition, the correlation about the immunizing dose of strains and vaccines for mice and piglets were detected during the virulence, safety and protection test. It showed that piglets could be replaced by mice for tests in practical production.
Six batches semi-works products were tested according the regulation and the standard severely. The protection rate of six batches semi-works products was higher than95%in two field experiments. The production were used for more than100,000pigs in Shenyang, Dalian, Anshan, Jinzhou, Tieling, Liaoyang of China and so on, and showed better safety and excellent effect.
The trivalent(O138、O139、0141) inactivated vaccine against piglet edema disease had been registered and it had been awarded the second-class certificate of national new veterinary drugs in Aug,2006.
引文
[1]蔡宝祥.家畜传染病[M].北京：中国农业出版社,1998,54～55.
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[3]曹军平,周绍凤,陈德元等.仔猪水肿病灭活菌苗的制备及对小鼠的保护试验[J].中国畜禽传染病,1997,(4)：1-2.
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[5]成大荣,徐建生,孙怀昌等.大肠杆菌F18菌毛及其亚型的PCR鉴定[J].微生物学报.2004.24(5)：683-685.
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[3]曹军平,周绍凤,陈德元等.仔猪水肿病灭活菌苗的制备及对小鼠的保护试验[J].中国畜禽传染病,1997,(4)：1-2.
[4]成大荣,孙怀昌,徐建生等.断奶仔猪源大肠杆菌F18菌毛的分子流行病学研究[J].中国预防兽医学报,2006,28(2)：232-234
[5]成大荣,徐建生,孙怀昌等.大肠杆菌F18菌毛及其亚型的PCR鉴定[J].微生物学报.2004.24(5)：683-685.
[6]成大荣,徐建生,刘秀梵.黏附索菌毛F18的研究近况[J].中国预防兽医学报,2004,26,(5)：388-390.
[7]丁永龙,孙银华,倪俊兵,等.海安县猪水肿病的流行病学调查[J].中国兽医科技,2005,35(1)：71-73.
[8]高落,张如宽.志贺氏样毒素与动物大肠杆菌病[J].中国兽医科技.199525(3)：14-17.
[9]黄志琴,黄国伟,叶春芳等.猪水肿病多价灭活苗应用效果试验[J].中国兽医杂志,1999,25(2)：58
[10]黄纪金,张理谨.猪水肿病诊断和防制[J].中国兽医杂志,1996,(1)：29.
[11]陆承平.兽医微生物学(第四版)[M].北京：中国农业出版社,2007,101～106.
[12]廖延雄.兽医微生物实验诊断手册[M].北京：中国农业大学出版社,1995,81-114.
[13]刘纯杰,苏维萍.产肠毒素性大肠杆菌毒力相关质粒研究进展[J].中国畜禽传染病,1994,76(3)：63～65.
[14]刘国平,吴斌,刘梦元,何启盖,陈焕春.猪大肠杆菌水肿毒素SLT IIeA基因的克隆和序列分析[J].动物医学进展,2004,25(1)：122-125
[15]刘国平,吴斌,刘梦元,何启盖唐先春,郭黎明,黄晓东,陈焕春.致仔猪水肿病大肠杆菌的分离、鉴定及生物学特性.[J].中国兽医学报.2005,25(1)：31-33
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[17]林艺远,刘国平,杨明柳,吴斌.猪水肿病发病机理研究进展.2006,27(7)：10-15
[18]李鹏,王家乡,殷裕斌等.仔猪水肿病多价蜂胶灭活疫苗免疫原性的研究[J].中国兽医科技[J].2003,5：54-56.
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[11]李高明,何仁川,李家齐.仔猪水肿病的诊断[J].中国兽医杂志.2000,01：29.
[20]穆永才,罗天瑶.齐齐哈尔市猪水肿病病原的分离鉴定及药敏试验[J].动物医学进展,2007,28(4)：47-49
[21]马云燕,李金璋,陈章水.仔猪水肿病病理形态学研究[J].动物医学进展.1999,20(3)：108-110.
[22]倪振亚,焦新安,高秘,张如宽,刘秀梵SLT II vB基因的克隆与表达.微生物学报[J]2000.40(6)：591-597
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[29]许崇波,于凤芹,卫广森等.大肠杆菌肠毒素ST LTB融合基因的构建[J].中国兽医学报,1998,21(5)：231-236.
[30]杨建泉,等.猪水肿病高免血清的制备及其疗效观察[J].畜牧与兽医,1999,(1)：25-27.
[31]杨健美,施慧,高裕,陈祥,刘秀梵.表达致PWD和ED大肠杆菌保护性抗原基因的重组菌的构建与免疫保护试验.第一届中国养猪生产和疾病控制技术大会——2005中国畜牧兽医学会学术年会.中国济南.2005：20-25.
[32]严振龙,徐建生,董国雄,李俊宝.致仔猪水肿病大肠杆菌快速检测方法的建立[J].中国动物检疫.2003,3：21-23
[33]严振龙,陈雷,董国雄等F18ab(F107)菌毛单抗的制备.鉴定与初步应用[J].中国预防兽医学报,2002.4.
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[42]Agbodaze D.Verocytotoxins (Shiga-like toxins) produced by Escherichia coli:a miniview of their classification, clinical presentations and management of a heterogeneous family of cytotoxins. Comp Immun Microbiol Infect Dis,1999,22:221-230
[43]A. Smeds M. Pertovaara, T. Timonen, T. Pohjanvirta S. Pelkonen, A. Palva.Mapping the Binding Domain of the F18 Fimbrial Adhesin. Infect Immun.2003 April;71(4) 2163-2172
[44]Ball HJ, Finlay D, ZafarA, Wilson T. The detection ofverocytotoxins in bacterial cultures from human diarrhoea) samples with monoclonal antibody-based ELISAs. JMed Microbiol,1996, 44:273-276
[45]Cheng D, Sun H, Xu J,et al. Prevalence of fimbial colonization factors F18ab and F18ac in Escherichia col i isolates from weaned piglets with edema and/or diarrhea in China [J]. Vet Microbiol,2005,110 (122):35-39.
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[47]Hein Imberechts, Henri de Greve, Schlicker Ch, et al. Characterization of F107 fimbriae of Escherich ia coli 107/86,which causes edema disease in pigs, and nucleo tide sequence of the F107 major fimbrial subunit gene, fedA [J]. Infect Immun,1992, (5):1963-1971.
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[51]Moxley R A. Edema disease[J].VetClin North Am Food Anim Pract,2000,16 (1):175-185.
[52]Main R G, Dritz S S, Tokach M D, et al. Increasing weaningage improves pig performance in a multisite production system[J] J Anim Sci,2004,82 (5):1499-1507.
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