黄鳝嗜水气单胞菌病和温和气单胞菌病防治研究
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摘要
黄鳝(Monopterus albus)不仅有丰富的营养价值,且有重要的药用价值,深受消费者青睐。目前已在除西藏、东三省以外的全国各地养殖,尤其以四川、湖北、江西等地最广。由于黄鳝的人工养殖历史较短,并且以前的研究多集中于繁殖,饲养等方面,因此对黄鳝疾病一直没有有效的防治措施。黄鳝嗜水气单胞菌和温和气单胞菌是危害黄鳝养殖业较为重要的两种疾病,且这两种疾病经常混合感染,因此研制能预防这两种疾病的二联疫苗和有效的复方中草药必将大大促进黄鳝养殖业的发展。本研究从二联疫苗和中药防治两方面对黄鳝疾病的防治进行研究,检测了二联疫苗的免疫效果,中药的体外抑菌效果。
1嗜水气单胞菌与温和气单胞菌最佳灭活条件的优化
选用不同的营养源,不同的培养温度,不同的pH值对嗜水气单胞菌,温和气单胞菌培养条件进行优化,结果表明嗜水气单胞菌最佳培养条件为：普通肉汤中加入1%胰蛋白胨,温度为37℃,pH值为7.5；温和气单胞菌最佳培养条件为：普通肉汤中加入1%胰蛋白胨,温度为28℃,pH值为7.5。将两种细菌扩大培养后按照1：1的比例混合,加入0.2%福尔马林(终浓度),灭活温度28℃,灭活36 h制成二联灭活苗的沉淀线最强。
2嗜水气单胞菌与温和气单胞菌二联疫苗对黄鳝的免疫效果
用嗜水气单胞菌、温和气单胞菌菌体制成二联疫苗,以腹腔注射的方式免疫黄鳝。检测免疫黄鳝血清抗体效价、IgM含量、溶菌酶活性、超氧化物歧化酶活性、补体C3以及对嗜水气单胞菌,温和气单胞菌攻毒的免疫保护率,评价该二联苗对黄鳝的免疫效果。结果表明二联疫苗免疫黄鳝后诱导的抗体效价在一免后的第4周达到最高,然后下降,加强免疫后在第6周达到最高,随后开始下降,抗嗜水气单胞菌和温和气单胞菌的效价均为26.67,而空白对照组在整个试验期间未能检测到抗嗜水气单胞菌、抗温和气单胞菌效价；免疫后黄鳝的IgM含量,血清溶菌酶和超氧化物岐化酶,补体C3分别为：0.5020±0.008mg/mL,12.27±0.17ug/mL,33.96±0.15U/mL,0.1054±0.0049 mg/mL。免疫8周后分别用两种病原菌攻毒,二联疫苗对黄鳝嗜水气单胞菌、温和气单胞菌攻毒的免疫保护率分别为56.67%和46.67%。试验表明该二联疫苗对黄鳝两种细菌病可起到一定的预防效果。
3复合佐剂对黄鳝的免疫效果
二联苗+复合佐剂组能更好地增强黄鳝的非特异性和特异性免疫,对嗜水气单胞菌与温和气单胞菌攻毒后的免疫保护率分别为63.67%,53.67%。与无佐剂组相比免疫保护率提高了7%。研究结果表明黄芪多糖和左旋咪唑能更好地增强嗜水气单胞菌和温和气单胞菌的免疫原性和免疫保护力。
4中草药对嗜水气单胞菌、温和气单胞菌抑菌效果
采用牛津杯法和二倍稀释法,选择五倍子、黄芩、杨树花、连翘、大黄等10种中草药对患病黄鳝上分离到的10株嗜水气单胞菌和1株温和气单胞菌进行了体外抑菌试验。在此基础上,按照中兽医配伍的特点初步筛选了5种中草药组成4个复方(A、B、C、D),并选择1株嗜水气单胞菌和1株温和气单胞菌进行体外抑菌试验。结果表明两种病原菌对五倍子、黄芩、连翘、杨树花、大黄较为敏感,其中五倍子的抑菌效果较显著,抑菌圈直径大于20mm；4个复方均具有较好的抑菌作用,且复方A效果最好。
Rice field eel (Monopterus albus) is rich not only in nutritional value but also in medicinal value, so it is popular among consumers. Currently it is bred around the country except Tibet and the Three Northeastern Provinces, especially in Sichuan, Hubei, Jiangxi. Because China has just made a start in Rice field eel farms, on the other hand, more attention has been paid for breeding and feeding, people failed to prevent disease in Rice field eel farming industry. Rice field eel Aeromonas hydrophila and Aeromonas sobria are two major pathogenic agents of Rice field eel, and the mixed infection of them is common in Rice field eel, so bivalent vaccines and Chinese herbal medicine which can prevent Rice field eel from both two pathogenic agents are urgent need for Rice field eel culture development. In this study, a bivalent vaccine against Aeromonas hydrophila and Aeromonas sobria was produced, and its immune efficacy was evaluated. In addition, the antibacterial activity of Chinese herbs against Aeromonas hydrophila and Aeromonas sobria was tested. 1 Optimization of inactivation of Aeromonas hydrophila and Aeromonas sobria
Chose different nutrient sources, different incubation temperature, different pH values to determine the optimal culture conditions of Aeromonas hydrophila and Aeromonas sobria.The results showed that optimal culture conditions of Aeromonas hydrophila were:Ordinary broth with 1% tryptone, the temperature was 37℃, pH= 7.5; the optimal culture conditions of Aeromonas hydrophila were:Ordinary broth with 1% tryptone, the temperature was 28℃, pH was 7.5. Mixed the two kinds of bacteria according to 1:1 propotrtion after expanding them, then joined 0.2% formalin (finally density), deactivated at 28℃for 36h to produce the two joint deactivation seedlings of which precipitation line was the strongest.
2 Immune responde of Rice field eel to Aeromonas hydrophila and Aeromonas sobria bivalent vaccine
The bivalent vaccine composed of Aeromonas hydrophila and Aeromonas sobria. The immunogenicity of bivalent vaccine was tested by examining the antibody titer,IgM, lysozyme activity, contents of alexine C3 and the challenge test of live pathogen was carried out, too. Results showed that:antibody titer in eel immunized with bivalent vaccine was induced, after 4 weeks came to a head, then declined. After strengthening immunization, the antibody titer reached the peak value (26.67 Against Aeromonas hydrophila and 26.67 Against Aeromonas sobria) after the first 6 weeks post-immunization, and then began to decline. IgM, lysozyme activity, contents of alexine C3 after immunization reached 0.5020±0.008mg/mL,12.27±0.17ug/mL, 33.96±0.15U/mL,0.1054±0.0049 mg/mL separately.The challenge test of both pathogen was also carried out 8 weeks after immunization, the relative survival to Aeromonas hydrophila and Aeromonas sobria challenge were 56.67% and 46.67% separately. Results indicated the bivalent vaccine can effectively protect eel from Aeromonas hydrophila and Aeromonas sobria infection.
3 Immunization efficacy of Compound Adjuvant for Rice field eel
Adding compound adjuvant enhanced the immunogenicity of vaccines. After the first immntity, the specific antibodies of the anti- Aeromonas hydrophila and Aeromonas sobria were tested in a week.At the same time, the antibody titers and the immune protective rate of the compound adjuvant+bivalent vaccine Group were the highest. The results indicated that APS and LMS could protect eel from the infection of Aeromonas hydrophila and Aeromonas sobria.
4 antibacterial activity of Chinese herbal against Aeromonas hydrophila and Aeromonas sobria
Adopting the Oxford cup method and dilution method, Selected Rhus chinesis, Scutellaria baicalensis, Populus canadensis Mocnch, Forsythia suspense, Pheum officinale and other kinds of Chinese herbal, in vitro, to inhibite the growth of the 10 strains of Aeromonas hydrophila and 1 strain of Aeromonas sobria which were isolated from the sick eel. On this basis, selected five Chinese herbal to compound four prescriptions (A, B, C, D) according to the characteristics of Veterinary compatibility and selected one strain of Aeromonas hydrophila and one strain of Aeromonas sobria to make the vitro antibacterial test. The results showed that the two pathogens were sensitive to the Rhus chinesis, Scutellaria baicalensis, Populus canadensis Mocnch, Forsythia suspense, Pheum officinale and the inhibitory effect of Rhus chinesis was the most significant. The inhibition zone diameter was more than 20mm; The four complex prescriptions had the effect on antimicrobial.The effect of A complex prescription on inhibitting the growth of Aeromonas hydrophila and Aeromonas sobria was better than the others.
1嗜水气单胞菌与温和气单胞菌最佳灭活条件的优化
选用不同的营养源,不同的培养温度,不同的pH值对嗜水气单胞菌,温和气单胞菌培养条件进行优化,结果表明嗜水气单胞菌最佳培养条件为：普通肉汤中加入1%胰蛋白胨,温度为37℃,pH值为7.5；温和气单胞菌最佳培养条件为：普通肉汤中加入1%胰蛋白胨,温度为28℃,pH值为7.5。将两种细菌扩大培养后按照1：1的比例混合,加入0.2%福尔马林(终浓度),灭活温度28℃,灭活36 h制成二联灭活苗的沉淀线最强。
2嗜水气单胞菌与温和气单胞菌二联疫苗对黄鳝的免疫效果
用嗜水气单胞菌、温和气单胞菌菌体制成二联疫苗,以腹腔注射的方式免疫黄鳝。检测免疫黄鳝血清抗体效价、IgM含量、溶菌酶活性、超氧化物歧化酶活性、补体C3以及对嗜水气单胞菌,温和气单胞菌攻毒的免疫保护率,评价该二联苗对黄鳝的免疫效果。结果表明二联疫苗免疫黄鳝后诱导的抗体效价在一免后的第4周达到最高,然后下降,加强免疫后在第6周达到最高,随后开始下降,抗嗜水气单胞菌和温和气单胞菌的效价均为26.67,而空白对照组在整个试验期间未能检测到抗嗜水气单胞菌、抗温和气单胞菌效价；免疫后黄鳝的IgM含量,血清溶菌酶和超氧化物岐化酶,补体C3分别为：0.5020±0.008mg/mL,12.27±0.17ug/mL,33.96±0.15U/mL,0.1054±0.0049 mg/mL。免疫8周后分别用两种病原菌攻毒,二联疫苗对黄鳝嗜水气单胞菌、温和气单胞菌攻毒的免疫保护率分别为56.67%和46.67%。试验表明该二联疫苗对黄鳝两种细菌病可起到一定的预防效果。
3复合佐剂对黄鳝的免疫效果
二联苗+复合佐剂组能更好地增强黄鳝的非特异性和特异性免疫,对嗜水气单胞菌与温和气单胞菌攻毒后的免疫保护率分别为63.67%,53.67%。与无佐剂组相比免疫保护率提高了7%。研究结果表明黄芪多糖和左旋咪唑能更好地增强嗜水气单胞菌和温和气单胞菌的免疫原性和免疫保护力。
4中草药对嗜水气单胞菌、温和气单胞菌抑菌效果
采用牛津杯法和二倍稀释法,选择五倍子、黄芩、杨树花、连翘、大黄等10种中草药对患病黄鳝上分离到的10株嗜水气单胞菌和1株温和气单胞菌进行了体外抑菌试验。在此基础上,按照中兽医配伍的特点初步筛选了5种中草药组成4个复方(A、B、C、D),并选择1株嗜水气单胞菌和1株温和气单胞菌进行体外抑菌试验。结果表明两种病原菌对五倍子、黄芩、连翘、杨树花、大黄较为敏感,其中五倍子的抑菌效果较显著,抑菌圈直径大于20mm；4个复方均具有较好的抑菌作用,且复方A效果最好。
Rice field eel (Monopterus albus) is rich not only in nutritional value but also in medicinal value, so it is popular among consumers. Currently it is bred around the country except Tibet and the Three Northeastern Provinces, especially in Sichuan, Hubei, Jiangxi. Because China has just made a start in Rice field eel farms, on the other hand, more attention has been paid for breeding and feeding, people failed to prevent disease in Rice field eel farming industry. Rice field eel Aeromonas hydrophila and Aeromonas sobria are two major pathogenic agents of Rice field eel, and the mixed infection of them is common in Rice field eel, so bivalent vaccines and Chinese herbal medicine which can prevent Rice field eel from both two pathogenic agents are urgent need for Rice field eel culture development. In this study, a bivalent vaccine against Aeromonas hydrophila and Aeromonas sobria was produced, and its immune efficacy was evaluated. In addition, the antibacterial activity of Chinese herbs against Aeromonas hydrophila and Aeromonas sobria was tested. 1 Optimization of inactivation of Aeromonas hydrophila and Aeromonas sobria
Chose different nutrient sources, different incubation temperature, different pH values to determine the optimal culture conditions of Aeromonas hydrophila and Aeromonas sobria.The results showed that optimal culture conditions of Aeromonas hydrophila were:Ordinary broth with 1% tryptone, the temperature was 37℃, pH= 7.5; the optimal culture conditions of Aeromonas hydrophila were:Ordinary broth with 1% tryptone, the temperature was 28℃, pH was 7.5. Mixed the two kinds of bacteria according to 1:1 propotrtion after expanding them, then joined 0.2% formalin (finally density), deactivated at 28℃for 36h to produce the two joint deactivation seedlings of which precipitation line was the strongest.
2 Immune responde of Rice field eel to Aeromonas hydrophila and Aeromonas sobria bivalent vaccine
The bivalent vaccine composed of Aeromonas hydrophila and Aeromonas sobria. The immunogenicity of bivalent vaccine was tested by examining the antibody titer,IgM, lysozyme activity, contents of alexine C3 and the challenge test of live pathogen was carried out, too. Results showed that:antibody titer in eel immunized with bivalent vaccine was induced, after 4 weeks came to a head, then declined. After strengthening immunization, the antibody titer reached the peak value (26.67 Against Aeromonas hydrophila and 26.67 Against Aeromonas sobria) after the first 6 weeks post-immunization, and then began to decline. IgM, lysozyme activity, contents of alexine C3 after immunization reached 0.5020±0.008mg/mL,12.27±0.17ug/mL, 33.96±0.15U/mL,0.1054±0.0049 mg/mL separately.The challenge test of both pathogen was also carried out 8 weeks after immunization, the relative survival to Aeromonas hydrophila and Aeromonas sobria challenge were 56.67% and 46.67% separately. Results indicated the bivalent vaccine can effectively protect eel from Aeromonas hydrophila and Aeromonas sobria infection.
3 Immunization efficacy of Compound Adjuvant for Rice field eel
Adding compound adjuvant enhanced the immunogenicity of vaccines. After the first immntity, the specific antibodies of the anti- Aeromonas hydrophila and Aeromonas sobria were tested in a week.At the same time, the antibody titers and the immune protective rate of the compound adjuvant+bivalent vaccine Group were the highest. The results indicated that APS and LMS could protect eel from the infection of Aeromonas hydrophila and Aeromonas sobria.
4 antibacterial activity of Chinese herbal against Aeromonas hydrophila and Aeromonas sobria
Adopting the Oxford cup method and dilution method, Selected Rhus chinesis, Scutellaria baicalensis, Populus canadensis Mocnch, Forsythia suspense, Pheum officinale and other kinds of Chinese herbal, in vitro, to inhibite the growth of the 10 strains of Aeromonas hydrophila and 1 strain of Aeromonas sobria which were isolated from the sick eel. On this basis, selected five Chinese herbal to compound four prescriptions (A, B, C, D) according to the characteristics of Veterinary compatibility and selected one strain of Aeromonas hydrophila and one strain of Aeromonas sobria to make the vitro antibacterial test. The results showed that the two pathogens were sensitive to the Rhus chinesis, Scutellaria baicalensis, Populus canadensis Mocnch, Forsythia suspense, Pheum officinale and the inhibitory effect of Rhus chinesis was the most significant. The inhibition zone diameter was more than 20mm; The four complex prescriptions had the effect on antimicrobial.The effect of A complex prescription on inhibitting the growth of Aeromonas hydrophila and Aeromonas sobria was better than the others.
引文
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