新生仔猪大肠杆菌性腹泻K_（88）ac-ST_1-LT_B三价基因工程灭活疫苗的研究与应用
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摘要
新生仔猪大肠杆菌性腹泻(Colibacillus diarrhea of newborn piglet)是由产肠毒素性大肠埃希氏菌(Enterotoxigenic E. coli, ETEC)引起的以1～7日龄新生仔猪下痢为特征的一种急性致死性多发性传染病。本病在世界范围内广泛流行，是影响仔猪成活率的主要因素之一。随着疫苗的研发与应用，我国新生仔猪大肠杆菌性腹泻得到了一定程度的控制，但疫苗在免疫原性、安全性等方面仍不理想。因此，研制高效、安全的新型多价基因工程疫苗迫在眉睫。
本研究利用分子生物学技术将ST_1基因与K_(88)ac和LT_B基因融合在一起，研制大肠杆菌三价基因工程灭活疫苗，以期达到增加疫苗的安全性，提高疫苗免疫效果的目的，解决新生仔猪大肠杆菌性腹泻免疫预防这一难题。
(1) 根据已发表的K_(88)ac、ST_1和LT_B基因序列，分别设计并合成一对K_(88)ac引物、三对ST_1引物和一对LT_B引物，利用PCR技术，从大肠杆菌C83902质粒中扩增出K_(88)ac基因、ST_1突变基因和LT_B基因。将质粒DNA和扩增的DNA片段进行分离、纯化、限制性内切酶酶切、T4 DNA连接酶连接，获得重组质粒pXK88acS73LT5并转化BL21(DE3)宿主菌，构建了含K_(88)ac-ST_1-LT_B融合基因表达载体的重组菌株BL21(DE3)(pXK88acST3LT5)。经酶切鉴定和DNA序列分析证实，构建的重组质粒pXK88acST3LT5中含有K_(88)ac-ST_1-LT_B融合基因，且基因序列和阅读框架均正确。将重组菌株BL21(DE3)(pXK88acST3LT5)按1％的量接种到含有Kan(50μg·mL~(-1))的LB液体培养基中，37℃振荡培养2h，然后用1mmol·L~(-1)IPTG诱导4h，融合蛋白K_(88)ac-ST_1-LT_B获得高效表达。经ELISA检测，重组菌株表达的K_(88)ac-ST_1-LT_B融合蛋白能够被ST_1单抗、LT_B和K_(88)ac抗体识别。将重组菌株BL21(DE3)(pXK88acST3LT5)培养物上清及菌体裂解物，分别进行了乳鼠灌胃试验，结果均为阴性(C／C≤0.083)，这表明该融合蛋白K_(88)ac-ST_1-LT_B已丧失天然ST_1肠毒素的活性。用该工程菌株制备的包涵体粗提物和工程菌灭活疫苗免疫小鼠，再用C83902大肠杆菌强毒菌株攻毒均获得了较好的保护，试
新生仔猪大肠杆菌性腹泻际ac一STI一LTI，二价基因「程灭活疫苗的研究与应用
里理里旦巴月里巴里里里里里里里里
验结果表明，肠ac一sT一LT。融合蛋白能够诱发小鼠产生抗体，该抗体具有中和天然ST:
肠毒素毒性的作用，证实了构建的重组菌株可以作为预防新生仔猪大肠杆菌性腹泻基
因工程疫苗的候选菌株。
(2)对构建的表达K::aC一ST!一LT。融合蛋白的重组菌株BL21(DE3)(pXK88aeST3LTS)
进行了染色特性、培养特性、生化特性、质粒稳定性、免疫原性、保存条件等实验室
试验。结果1一10代菌种染色特性、培养特性、生化特性均符合大肠杆菌特性的要求;
在无选择压力(Kan一)T BL21(DE3)(pXK88aeST3LTS)菌株连续培养20代，统计质粒
丢失率，其重组质粒保留率为1 00%，表明重组质粒pXK88aeST3LTS在受体菌BL21(DE3)
中能较稳定传代:重组菌株BL21(DE3)(pXK88aeST3LTS)经诱导后的培养物，用SDS-
聚丙烯酞胺凝胶电泳和薄层凝胶扫描分析，结果表明融合蛋白占菌体总蛋白的相对含
量能稳定在73%以上;分另，J用BL21(DE3)(pXK88aeST3LTS)菌株第1代和第10代基础
种子的培养物制备的疫苗免疫小鼠后，用1 MLD(5/10吕CFU)强毒菌液攻击，其保护
率为90.6，表明1一10代基础种子均可作为疫苗生产用菌株，其免疫原性稳定。因此，
基础种子代次暂定为1一10代;菌种保存试验确定，甘油保存菌种在一20℃保存期为
2年，冻干保存菌种在一20℃保存期暂定为3年;最小免疫剂量测定结果表明小鼠的
最小免疫剂量每只为0.lmL，妊振母猪的最小免疫剂量每头为2.smL;制备的5批
实验室产品均安全有效;试验中本疫苗保存18个月，仍具有良好的效力，保护率达
8既以上，为了保证疫苗质量，并考虑疫苗在保存和应用中的各种不确定因素，将本
疫苗的保存期暂定为12个月。为了进一步验证疫苗的安全性和有效性，在不同地区
进行T田间试验，免疫妊娠母猪6 50头，产仔7 1 56头，平均保护率为98.2406，试验
证明本疚苗安全性良好，新生仔猪通过吮吸母乳获得被动免疫可有效地预防新生仔猪
大肠性腹泻的发生。
(3)以实验室工艺为基础，进行了工业化生产工艺的研究。用发酵罐培养，改
良LB培养基培养的菌数与普通肉汤培养基的菌数基本一致，明显高于LB培养基的菌
数，根据BL21(DE3)(pXK88aeST3LTS)菌株培养的稳定性和降低生产成本的要求，故改
良LB培养基为该菌株的最佳培养基;经诱导剂筛选试验分析，100 mmol·L一，乳糖组
诱导效果略低于1 mmol·L一，IpTG组，但优于10 mmol·L一，乳糖组，1 mmol·L一，乳糖
组诱导效果最差，在工业化生产上为了降低生产成本，故选择乳糖作为诱导剂;
摘要
aL21(nE3)(pxK88acsT3LTS)菌株添加终浓度为100 mm。l·L一，乳糖进行诱导后，目的
蛋白于Zh呈现表达，随着诱导时间的延长，总菌数和目的蛋白表达总数均相应增加，
5一6h趋于稳定，确定了乳糖诱导的浓度为10Omm。l·L一，，诱导时间不低于6h;通
气培养条件试验表明，BLzl(。Es)(pxKssacST3LTs)菌株以2、10，mL发酵罐通气培养，
在500L·min一?
Colibacillus diarrhea of newborn piglet is a kind of the most common infectious disease.which was caused by enterotoxigenic E.coli (ETEC) and characterized by '"yellow scour" of the 1 to 7-day-old newborn piglet and high morbidity and mortality. With the development and application of vaccine. colibacillus diarrhea of newborn piglet has been controlled to a certain extent. but the innocuity and the immunogenicity of the vaccine are still unsatisfied so that it is exigent to develop a new multivalence gene engineering inactivated vaccine which has high innocuity and immunogenicity.In this study. the genes of ST1 K88ac and LTB were linked and the trivalent gene engineering vaccine K88ac-ST1-LTB was constructed successfully with molecular bio-technology. The purpose of this study was to improve the innocuity and the immunity efficacy of the vaccine and promote prevention level against the colibacilius diarrhea of newborn piglet.(1)According to the published DNA sequence of K88ac.ST and LTb, one pair of K88ac primer, three pairs of ST1 primers and one pair of LTb primer had been designed and synthesized .With the technology of PCR. K88ac gene. ST1 mutant gene and LTb gene were amplified respectively. After isolating . purifying, restriction endonuclease-digesting and linking with T4 DNA Ligase .the recombinant plasmid pXK88acST3LT5 was obtained and transformed into BL21 ( DE3) thus the recombinant strain BL21 C DE3 )(pXK88acST3LT5) expressing fusion protein K88ac-ST1-LTB was constructed . It was proved that the recombinant plasmid pXK88acST3LT5 contained the fusion gene K88ac-ST1-LTB which had correct sequence and ORF by identification of endonuclease-digesting and sequence analysis. The recombinant strain BL21 (DE3 )( pXK88acST3LT5) was inoculated into LB medium at the ratio of 1%, and was cultured for 2 hours at 37 . then was induced bv 1mmol L-1 1PTG for 4 h, finally the fusion
protein K88ac-ST1-LTB was expressed at high level. The expressed protein can be recognized by MAb of ST1, antibodies of LTB and K88ac with ELISA method. With the supernatant and lysate of the recombinant strain BL21 (DE3) (pXK88acST3LT5) respectively, the test of pouring stomach of sucking mice was done and the result was negative (G/C 0.083) ,which indicated that the fusion protein lost the toxicity of ST1.The mice had been protected which were immunized with inclusion body or the inactivated vaccine and then challenged with C83902 virulent strain. The result showed that the fusion protein K88ac-ST1-LTB can stimulate the mice to produce antibody which has the function of neutralizing the natural STi toxin. Therefore, the recombinant strain could be used as a candidate strain of gene engineering inactivated vaccine against colibacillus diarrhea of newborn piglet.(2)The recombinant strain BL21 (DE3) (pXK88acST3LT5) was tested for the properties of bacterial staining, culturing, plasmid stability, preservation condition and so on in laboratory. The properties of bacterial staining, culturing, biochemistry of the bacterium seeds from the first to the tenth generation were all in accord with E.coli; Under the unselected pressure (Kan") , the recombinant strain BL21 (DE3) (pXK88acST3LT5) can be cultured for twenty generations continually and the reserved rate of recombinant plasmid was 100%.The result indicated that the recombinant plasmid pXK88acST3LT5 can be transmitted stably in BL21 (DE3) .The induced culture of the recombinant strain BL21 (DE3) (pXK88acST3LT5) was analyzed with SDS-PAGE and thin-layer gel scan, the result showed that the relative content of fusion protein was above 73% of total protein; The mice immunized respectively with the culture of the first and the tenth basic seeds were challenged with 1 MLD (5 108 CFU) virulent bacterium liquid, and the protected rate was 90%, which indicated all of the seeds from the first to the tenth generation can be used to produce vaccine. So they can be chosen as basic seeds .By use of kept term detecting test, the term of seed kept in glycerin at -20 癈 was two years,while it
本研究利用分子生物学技术将ST_1基因与K_(88)ac和LT_B基因融合在一起，研制大肠杆菌三价基因工程灭活疫苗，以期达到增加疫苗的安全性，提高疫苗免疫效果的目的，解决新生仔猪大肠杆菌性腹泻免疫预防这一难题。
(1) 根据已发表的K_(88)ac、ST_1和LT_B基因序列，分别设计并合成一对K_(88)ac引物、三对ST_1引物和一对LT_B引物，利用PCR技术，从大肠杆菌C83902质粒中扩增出K_(88)ac基因、ST_1突变基因和LT_B基因。将质粒DNA和扩增的DNA片段进行分离、纯化、限制性内切酶酶切、T4 DNA连接酶连接，获得重组质粒pXK88acS73LT5并转化BL21(DE3)宿主菌，构建了含K_(88)ac-ST_1-LT_B融合基因表达载体的重组菌株BL21(DE3)(pXK88acST3LT5)。经酶切鉴定和DNA序列分析证实，构建的重组质粒pXK88acST3LT5中含有K_(88)ac-ST_1-LT_B融合基因，且基因序列和阅读框架均正确。将重组菌株BL21(DE3)(pXK88acST3LT5)按1％的量接种到含有Kan(50μg·mL~(-1))的LB液体培养基中，37℃振荡培养2h，然后用1mmol·L~(-1)IPTG诱导4h，融合蛋白K_(88)ac-ST_1-LT_B获得高效表达。经ELISA检测，重组菌株表达的K_(88)ac-ST_1-LT_B融合蛋白能够被ST_1单抗、LT_B和K_(88)ac抗体识别。将重组菌株BL21(DE3)(pXK88acST3LT5)培养物上清及菌体裂解物，分别进行了乳鼠灌胃试验，结果均为阴性(C／C≤0.083)，这表明该融合蛋白K_(88)ac-ST_1-LT_B已丧失天然ST_1肠毒素的活性。用该工程菌株制备的包涵体粗提物和工程菌灭活疫苗免疫小鼠，再用C83902大肠杆菌强毒菌株攻毒均获得了较好的保护，试
新生仔猪大肠杆菌性腹泻际ac一STI一LTI，二价基因「程灭活疫苗的研究与应用
里理里旦巴月里巴里里里里里里里里
验结果表明，肠ac一sT一LT。融合蛋白能够诱发小鼠产生抗体，该抗体具有中和天然ST:
肠毒素毒性的作用，证实了构建的重组菌株可以作为预防新生仔猪大肠杆菌性腹泻基
因工程疫苗的候选菌株。
(2)对构建的表达K::aC一ST!一LT。融合蛋白的重组菌株BL21(DE3)(pXK88aeST3LTS)
进行了染色特性、培养特性、生化特性、质粒稳定性、免疫原性、保存条件等实验室
试验。结果1一10代菌种染色特性、培养特性、生化特性均符合大肠杆菌特性的要求;
在无选择压力(Kan一)T BL21(DE3)(pXK88aeST3LTS)菌株连续培养20代，统计质粒
丢失率，其重组质粒保留率为1 00%，表明重组质粒pXK88aeST3LTS在受体菌BL21(DE3)
中能较稳定传代:重组菌株BL21(DE3)(pXK88aeST3LTS)经诱导后的培养物，用SDS-
聚丙烯酞胺凝胶电泳和薄层凝胶扫描分析，结果表明融合蛋白占菌体总蛋白的相对含
量能稳定在73%以上;分另，J用BL21(DE3)(pXK88aeST3LTS)菌株第1代和第10代基础
种子的培养物制备的疫苗免疫小鼠后，用1 MLD(5/10吕CFU)强毒菌液攻击，其保护
率为90.6，表明1一10代基础种子均可作为疫苗生产用菌株，其免疫原性稳定。因此，
基础种子代次暂定为1一10代;菌种保存试验确定，甘油保存菌种在一20℃保存期为
2年，冻干保存菌种在一20℃保存期暂定为3年;最小免疫剂量测定结果表明小鼠的
最小免疫剂量每只为0.lmL，妊振母猪的最小免疫剂量每头为2.smL;制备的5批
实验室产品均安全有效;试验中本疫苗保存18个月，仍具有良好的效力，保护率达
8既以上，为了保证疫苗质量，并考虑疫苗在保存和应用中的各种不确定因素，将本
疫苗的保存期暂定为12个月。为了进一步验证疫苗的安全性和有效性，在不同地区
进行T田间试验，免疫妊娠母猪6 50头，产仔7 1 56头，平均保护率为98.2406，试验
证明本疚苗安全性良好，新生仔猪通过吮吸母乳获得被动免疫可有效地预防新生仔猪
大肠性腹泻的发生。
(3)以实验室工艺为基础，进行了工业化生产工艺的研究。用发酵罐培养，改
良LB培养基培养的菌数与普通肉汤培养基的菌数基本一致，明显高于LB培养基的菌
数，根据BL21(DE3)(pXK88aeST3LTS)菌株培养的稳定性和降低生产成本的要求，故改
良LB培养基为该菌株的最佳培养基;经诱导剂筛选试验分析，100 mmol·L一，乳糖组
诱导效果略低于1 mmol·L一，IpTG组，但优于10 mmol·L一，乳糖组，1 mmol·L一，乳糖
组诱导效果最差，在工业化生产上为了降低生产成本，故选择乳糖作为诱导剂;
摘要
aL21(nE3)(pxK88acsT3LTS)菌株添加终浓度为100 mm。l·L一，乳糖进行诱导后，目的
蛋白于Zh呈现表达，随着诱导时间的延长，总菌数和目的蛋白表达总数均相应增加，
5一6h趋于稳定，确定了乳糖诱导的浓度为10Omm。l·L一，，诱导时间不低于6h;通
气培养条件试验表明，BLzl(。Es)(pxKssacST3LTs)菌株以2、10，mL发酵罐通气培养，
在500L·min一?
Colibacillus diarrhea of newborn piglet is a kind of the most common infectious disease.which was caused by enterotoxigenic E.coli (ETEC) and characterized by '"yellow scour" of the 1 to 7-day-old newborn piglet and high morbidity and mortality. With the development and application of vaccine. colibacillus diarrhea of newborn piglet has been controlled to a certain extent. but the innocuity and the immunogenicity of the vaccine are still unsatisfied so that it is exigent to develop a new multivalence gene engineering inactivated vaccine which has high innocuity and immunogenicity.In this study. the genes of ST1 K88ac and LTB were linked and the trivalent gene engineering vaccine K88ac-ST1-LTB was constructed successfully with molecular bio-technology. The purpose of this study was to improve the innocuity and the immunity efficacy of the vaccine and promote prevention level against the colibacilius diarrhea of newborn piglet.(1)According to the published DNA sequence of K88ac.ST and LTb, one pair of K88ac primer, three pairs of ST1 primers and one pair of LTb primer had been designed and synthesized .With the technology of PCR. K88ac gene. ST1 mutant gene and LTb gene were amplified respectively. After isolating . purifying, restriction endonuclease-digesting and linking with T4 DNA Ligase .the recombinant plasmid pXK88acST3LT5 was obtained and transformed into BL21 ( DE3) thus the recombinant strain BL21 C DE3 )(pXK88acST3LT5) expressing fusion protein K88ac-ST1-LTB was constructed . It was proved that the recombinant plasmid pXK88acST3LT5 contained the fusion gene K88ac-ST1-LTB which had correct sequence and ORF by identification of endonuclease-digesting and sequence analysis. The recombinant strain BL21 (DE3 )( pXK88acST3LT5) was inoculated into LB medium at the ratio of 1%, and was cultured for 2 hours at 37 . then was induced bv 1mmol L-1 1PTG for 4 h, finally the fusion
protein K88ac-ST1-LTB was expressed at high level. The expressed protein can be recognized by MAb of ST1, antibodies of LTB and K88ac with ELISA method. With the supernatant and lysate of the recombinant strain BL21 (DE3) (pXK88acST3LT5) respectively, the test of pouring stomach of sucking mice was done and the result was negative (G/C 0.083) ,which indicated that the fusion protein lost the toxicity of ST1.The mice had been protected which were immunized with inclusion body or the inactivated vaccine and then challenged with C83902 virulent strain. The result showed that the fusion protein K88ac-ST1-LTB can stimulate the mice to produce antibody which has the function of neutralizing the natural STi toxin. Therefore, the recombinant strain could be used as a candidate strain of gene engineering inactivated vaccine against colibacillus diarrhea of newborn piglet.(2)The recombinant strain BL21 (DE3) (pXK88acST3LT5) was tested for the properties of bacterial staining, culturing, plasmid stability, preservation condition and so on in laboratory. The properties of bacterial staining, culturing, biochemistry of the bacterium seeds from the first to the tenth generation were all in accord with E.coli; Under the unselected pressure (Kan") , the recombinant strain BL21 (DE3) (pXK88acST3LT5) can be cultured for twenty generations continually and the reserved rate of recombinant plasmid was 100%.The result indicated that the recombinant plasmid pXK88acST3LT5 can be transmitted stably in BL21 (DE3) .The induced culture of the recombinant strain BL21 (DE3) (pXK88acST3LT5) was analyzed with SDS-PAGE and thin-layer gel scan, the result showed that the relative content of fusion protein was above 73% of total protein; The mice immunized respectively with the culture of the first and the tenth basic seeds were challenged with 1 MLD (5 108 CFU) virulent bacterium liquid, and the protected rate was 90%, which indicated all of the seeds from the first to the tenth generation can be used to produce vaccine. So they can be chosen as basic seeds .By use of kept term detecting test, the term of seed kept in glycerin at -20 癈 was two years,while it
引文
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[7] Evans MG, Waxler GL, Newman JP. Prevalence of K_(88), K_(99), and 987P pili of Escherichia coli in neonatal pigs with enteric colibacillosis[J]. Am J Vet Res, 1986, 47(11):2431～4.
[8] Flores-Abuxapqui JJ, Suarez-Hoil GJ, Puc-Franco, et al. Frequency of adhesive factors and enterotoxins in strains of EscherJchia coli isolated from piglets with diarrhea[J]. Rev Latinoam Microbiol, 1997, 39(3-4):145～51.
[9] Fairbrother JM, Lariviere S, Johnson WM . Pathogenicity of Escherichia coli O_(115):K'V165" strains isolated from pigs with diarrhea[J].Am J Vet Res, 1988, 49(8):1325～8.
[10] J. Harel, H. Lapointe, A. Fallara, et al. Detection of Genes for Fimbrial Antigens and Enterotoxins Associated with Escherichia coli Serogroups Isolated from Pigs with Diarrhea[J]. Journal Of Clinical Microbiology, 1991,745～752.
[11] Martins MF, Martinze—Rossi Nm, Ferreira A,et al. Pathogenic characteristics of Escherichia coli strains isolated from newborn piglets with diarrhea in Brazil[J].Vet Microbiol, 2000, 76(1):51～9.
[12] Wittig W, Fabricius C. Escherichia coli types isolated from porcine E. coli infections in Saxony from 1963 to 1990[J]. Zentralbl Bakteriol, 1992, 277(3): 389～402.
[13] Osek J Truszczynski M. Occurrence of fimbriae and enterotoxins in Kscherichia coli strains isolated from piglets in Poland [J].Comp Immunol Microbiol Infect Dis, 1992, 15(4):285～292.
[14] Nakazawa M, Sugimoto C, Isayama Y, et al. Virulence factors in Escherichia coli isolated from piglets with neonatal and post-weaning diarrhea in Japan[J].Vet microbial, 1987, 13(4):291～300.
[1
[15] Choi C, Chae C. Genotypic prevalence of F4 variants (ab, ac, and ad) in Escherichia coli isolated from diarrheic piglets in Korea[J].Vet Microbiol, 1999, 67(4): 307～10.
[16] Ha SK, Choi C, Jung K, et al. Genotypic prevalence of the adhesin involved in diffuse adherence in Escherichia coli isolates in pre-weaned pigs with diarrhoea in Korea[J]. J. Vet Med Infect Dis Vet Public Health, 2004, 51(4):166～8.
[17] Supar, Hirst RG, Patten BE. The importance of enterotoxigenic Escherichia coli containing the 987P antigen in causing neonatal colibacillosis in piglets in Indonesia[J]. Vet Microbiol, 1991, 26(4):393～400.
[18] HTTP://WWW.CPST. NET. CN/KXJ/ZGKXJSZJ/CX/NXB/PE/YZ08033001. HTM.
[19] 何明清，廖德惠，谢镜怀，等．仔猪白痢与黄痢的相关性研究[J]．家畜传染病，1983，18(4)：5～8．
[20] 毛盛英，郭秀琦，王友田，等．仔猪腹泻致病性大肠埃希氏菌血清型及其日龄分布的调查研究[J]．中国兽医科技，1983，8：6～8．
[21] 辛英伊，丁洪明，李元芳，等．新生仔猪大肠杆菌病致病性大肠杆菌的分离和鉴定[J]．吉林畜牧兽医，1990，6：30～32．
[22] 蔡葵蒸，靳国琴，柴君秀，等．仔猪大肠杆菌性腹泻病的调查及病原学特性的研究[J]．动物医学进展，2002，23(5)：101～103．
[23] 陈祥，高崧，王雷，等．华东地区致初生仔猪腹泻大肠杆菌的O血清型和毒力因子[J]．微生物学报，2004，44(1)：96～100．
[24] 陆承平．兽医微生物学(第三版)[M]．北京：中国农业出版社，2001，215～223．
[25] 王开功，周碧君，潘仕文等．仔猪大肠杆菌流行病学调查研究[J]．贵州畜牧兽医，1997，21(2)：3～5．
[26] 李常纯．应加强仔猪黄痢的预防[J]．黑龙江畜牧兽医，1999，4：48．
[27] 陈螺眉，郭金森，黄瑜．福州地区哺乳仔猪腹泻研究 Ⅰ．福州地区哺乳仔猪腹泻流行病学调查[J]．福建畜牧兽医，2000，22(5)：2．
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[29] 王红宁，刘世贵，刘书亮，等．规模化猪场仔猪黄、白痢的防治研究[J]．中国预防兽医学报，1999，21(4)：264～267．
[30] 王琛，方英．仔猪黄痢的诊断和预防[J]．中国兽医杂志，2001，37(7)：22～23．
[31] 张永红，崔德凤，沈永舟，等．北京地区猪致病性大肠埃希氏菌的分离鉴定．中国兽医科技，1999，29(4)：29～30．
[32] 韩庆彦，杨小玲，高生智，等．甘肃省集约化养猪场猪大肠杆菌的调查及防治试验[J]．甘肃畜牧兽医，2001，5：10～12．
[33] 刘书亮，王红宁，陶勇，等．规模化猪场仔猪黄白痢大肠杆菌的药敏区系调查[J]．中国兽医杂志，2001，37(9)：18(20)．
[34] 李文刚，杨治田，兰尊海，等．河南省猪致病性大肠杆菌分离株的鉴定[J]．河南农业科学，2001，11：28～29．
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[4] 崔治中．K_(88)肠病原性大肠杆菌在我国仔猪黄痢发病中的作用[J]．畜牧兽医学报，1982，13(2)：131～136．
[5] 费恩阁．动物传染病学[M]．长春：吉林科学技术出版社，1995，163～167．
[6] Wilson RA Francis DH. Fimbriae and enterotoxins associated with Escherichia coli serogroups isolated from pigs with colibacillosis[J]. Am J Vet Res, 1986, 47(2):213～7.
[7] Evans MG, Waxler GL, Newman JP. Prevalence of K_(88), K_(99), and 987P pili of Escherichia coli in neonatal pigs with enteric colibacillosis[J]. Am J Vet Res, 1986, 47(11):2431～4.
[8] Flores-Abuxapqui JJ, Suarez-Hoil GJ, Puc-Franco, et al. Frequency of adhesive factors and enterotoxins in strains of EscherJchia coli isolated from piglets with diarrhea[J]. Rev Latinoam Microbiol, 1997, 39(3-4):145～51.
[9] Fairbrother JM, Lariviere S, Johnson WM . Pathogenicity of Escherichia coli O_(115):K'V165" strains isolated from pigs with diarrhea[J].Am J Vet Res, 1988, 49(8):1325～8.
[10] J. Harel, H. Lapointe, A. Fallara, et al. Detection of Genes for Fimbrial Antigens and Enterotoxins Associated with Escherichia coli Serogroups Isolated from Pigs with Diarrhea[J]. Journal Of Clinical Microbiology, 1991,745～752.
[11] Martins MF, Martinze—Rossi Nm, Ferreira A,et al. Pathogenic characteristics of Escherichia coli strains isolated from newborn piglets with diarrhea in Brazil[J].Vet Microbiol, 2000, 76(1):51～9.
[12] Wittig W, Fabricius C. Escherichia coli types isolated from porcine E. coli infections in Saxony from 1963 to 1990[J]. Zentralbl Bakteriol, 1992, 277(3): 389～402.
[13] Osek J Truszczynski M. Occurrence of fimbriae and enterotoxins in Kscherichia coli strains isolated from piglets in Poland [J].Comp Immunol Microbiol Infect Dis, 1992, 15(4):285～292.
[14] Nakazawa M, Sugimoto C, Isayama Y, et al. Virulence factors in Escherichia coli isolated from piglets with neonatal and post-weaning diarrhea in Japan[J].Vet microbial, 1987, 13(4):291～300.
[1
[15] Choi C, Chae C. Genotypic prevalence of F4 variants (ab, ac, and ad) in Escherichia coli isolated from diarrheic piglets in Korea[J].Vet Microbiol, 1999, 67(4): 307～10.
[16] Ha SK, Choi C, Jung K, et al. Genotypic prevalence of the adhesin involved in diffuse adherence in Escherichia coli isolates in pre-weaned pigs with diarrhoea in Korea[J]. J. Vet Med Infect Dis Vet Public Health, 2004, 51(4):166～8.
[17] Supar, Hirst RG, Patten BE. The importance of enterotoxigenic Escherichia coli containing the 987P antigen in causing neonatal colibacillosis in piglets in Indonesia[J]. Vet Microbiol, 1991, 26(4):393～400.
[18] HTTP://WWW.CPST. NET. CN/KXJ/ZGKXJSZJ/CX/NXB/PE/YZ08033001. HTM.
[19] 何明清，廖德惠，谢镜怀，等．仔猪白痢与黄痢的相关性研究[J]．家畜传染病，1983，18(4)：5～8．
[20] 毛盛英，郭秀琦，王友田，等．仔猪腹泻致病性大肠埃希氏菌血清型及其日龄分布的调查研究[J]．中国兽医科技，1983，8：6～8．
[21] 辛英伊，丁洪明，李元芳，等．新生仔猪大肠杆菌病致病性大肠杆菌的分离和鉴定[J]．吉林畜牧兽医，1990，6：30～32．
[22] 蔡葵蒸，靳国琴，柴君秀，等．仔猪大肠杆菌性腹泻病的调查及病原学特性的研究[J]．动物医学进展，2002，23(5)：101～103．
[23] 陈祥，高崧，王雷，等．华东地区致初生仔猪腹泻大肠杆菌的O血清型和毒力因子[J]．微生物学报，2004，44(1)：96～100．
[24] 陆承平．兽医微生物学(第三版)[M]．北京：中国农业出版社，2001，215～223．
[25] 王开功，周碧君，潘仕文等．仔猪大肠杆菌流行病学调查研究[J]．贵州畜牧兽医，1997，21(2)：3～5．
[26] 李常纯．应加强仔猪黄痢的预防[J]．黑龙江畜牧兽医，1999，4：48．
[27] 陈螺眉，郭金森，黄瑜．福州地区哺乳仔猪腹泻研究 Ⅰ．福州地区哺乳仔猪腹泻流行病学调查[J]．福建畜牧兽医，2000，22(5)：2．
[28] 周碧君，王开功，金志强．贵州省仔猪大肠杆菌病病原学的调查研究[J]．中国兽医科技，2001，31(1)： 15～17．
[29] 王红宁，刘世贵，刘书亮，等．规模化猪场仔猪黄、白痢的防治研究[J]．中国预防兽医学报，1999，21(4)：264～267．
[30] 王琛，方英．仔猪黄痢的诊断和预防[J]．中国兽医杂志，2001，37(7)：22～23．
[31] 张永红，崔德凤，沈永舟，等．北京地区猪致病性大肠埃希氏菌的分离鉴定．中国兽医科技，1999，29(4)：29～30．
[32] 韩庆彦，杨小玲，高生智，等．甘肃省集约化养猪场猪大肠杆菌的调查及防治试验[J]．甘肃畜牧兽医，2001，5：10～12．
[33] 刘书亮，王红宁，陶勇，等．规模化猪场仔猪黄白痢大肠杆菌的药敏区系调查[J]．中国兽医杂志，2001，37(9)：18(20)．
[34] 李文刚，杨治田，兰尊海，等．河南省猪致病性大肠杆菌分离株的鉴定[J]．河南农业科学，2001，11：28～29．