【作者】 于鸽；

【导师】 邱德全；

【作者基本信息】 广东海洋大学 ， 水产养殖， 2011， 硕士

【摘要】 本论文从自然环境中分离,筛选了一株致病弧菌,经鉴定为溶藻弧菌,并同时从环境当中分离了它的噬菌体。本文研究了噬菌体的生理特性,制作溶藻弧菌菌影。并利用全菌疫苗,菌影疫苗及噬菌体注射对虾,比较它们对南美白对虾免疫功能的影响和对对虾溶藻弧菌弧菌病的防治效果。具体研究结果如下:1.本文从虾塘水体中分离了一株溶藻弧菌,对该菌进行了常规的生理生化鉴定,确认该菌为溶藻弧菌;2.以分离到的溶藻弧菌为宿主菌从虾塘水体中分离了一株溶藻弧菌噬菌体。平板上噬菌斑特征:噬菌斑较为透明,圆整,边缘清楚,无晕环。噬菌斑较小,直径为1.0～1.2mm。进行扩增之后该噬菌体的最高效价可达10¹⁰pfu/mL;3.测定了溶藻弧菌噬菌体的生理生化特性,实验结果表明:噬菌体的RTD为106pfu/mL。最适pH为8.0,太酸或者太碱的环境都不适合噬菌体的生存;最适温度为35℃,具有有较好的耐热性,对60℃以上的温度敏感;对紫外线敏感,溶藻弧菌和噬菌体混合时间影响出斑数量;该噬菌体对氯仿不敏感,但其对非离子去垢剂Triton-X100敏感,经处理后滴度剩余17.8%;Mg²⁺有利于噬菌体的吸附,比不添加的出斑率高出55～71个百分点。在盐度为25‰的情况下,对溶藻弧菌的裂解能力最强,这可能是因为这株噬菌体是在海水环境下分离出来的有关,所以对于高盐度的耐受性比较好;该噬菌体的最佳感染复数为在0.1¹0之间;噬菌体的潜伏期为35min;4.本文探索了如何能用噬菌体尽量完全的裂解宿主,由实验结果可以看出:溶藻弧菌的培养时期对于噬菌体的除菌效果是有影响的,噬菌体对于培养了2小时到8小时之间的宿主菌的裂解能力相当,宿主菌数量的变化规律也一致,都是刚开始有微弱的升高,到了4小时以后开始下降,6小时后下降更加明显,当达到一个最低点后,又有升高;噬菌体对于培养了10个小时的菌的裂解能力相比培养了2小时到8小时之的菌的裂解能力有所降低,在噬菌体和宿主菌反应时间到10小时时,菌的数量就达到了最低值,但这个最低值高于噬菌体和培养了2小时到8小时之间的宿主菌相互反应所达到的最低值;用培养时期在12小时以后的溶藻弧菌作为宿主时,结果发现噬菌体对菌的数量变化作用比较小。菌数量的变化曲线接近于菌的生长曲线;但当噬菌体数和溶藻弧菌数之间的比不同时,弧菌的数量变化规律也不同,当弧菌数量比噬菌体数多时,菌数量是先呈现上升趋势,然后下降,再上升;当噬菌体数量比较多时,菌的数量先下降,后缓慢上升,当MIO=10时,除菌效果最好,噬菌体数和溶藻弧菌数之间的比对除菌效果影响不是很大,只是会导致到达菌数量最少的时间点不同;5.实验结果发现在实验室中,噬菌体不能完全裂解细菌,使用100μL的大蒜液或浓度为100U/mL青霉素、100μg/mL链霉素的PBS就可以把经噬菌体裂解剩下的菌除干净;6.用溶藻弧菌对南美白对虾进行攻毒实验。结果表明,肌注射1×10⁶cfu/mL的溶藻弧菌可以使对虾的死亡率达到30%,而1×10⁷cfu/mL的溶藻弧菌可导致成虾红体病,1×10⁸cfu/mL的溶藻弧菌可使对虾的死亡率达到90%。各实验组凡纳滨对虾的各部分的弧菌含量都远比对照组高,肝胰脏和肠内的含菌量尤其多;对虾的血细胞数目呈升高趋势;7.本实验给南美白对虾注射甲醛处理的全菌疫苗,噬菌体处理的菌影疫苗和噬菌体,比较三者对对虾的免疫学指标的影响。经噬菌体处理过的菌影疫苗注射的对虾的NOS活力最高,注射噬菌体对NOS活力的影响不是很明显,甚至低于阴性对照组;注射了10⁸pfu/mL全菌疫苗的南美白对虾在12h的PO值最高。注射了10⁸pfu/mL噬菌体的南美白对虾的血蓝蛋白在6h的含量最高;8.免疫后攻毒实验结果可以看出,10⁸pfu/mL噬菌体对南美白对虾的免疫保护率最好,可以达到88.2%,随着浓度的降低,免疫保护率也随之下降,但10⁶pfu/mL的噬菌体对南美白对虾仍有70.6%的免疫保护率,高于10⁸cfu/mL的全菌疫苗。10⁸cfu/mL菌影疫苗的免疫保护率也有76.5%高于10⁸的全菌疫苗。更多还原

【Abstract】 A new Vibrio alginolyticus phage was isolated and screened from the polluted water Vibrio alginolyticus is one of important pathogenic to aquatic and human beings. In the present Vibrio alginolyticus study aims at the biophysical chemistry characters of the phage and the produce of bccterial Vibrio alginolyticus ghosts. Compares the whole cell vaccine, bacterial ghosts vaccine and phage immune function and preventing the prawn vibrio disease. Our works coverd:1. A new Vibrio alginolyticus was isolated form the pond. The isolation was identified as Vibrio alginolyticus by physiologicaland and biochemical characteristic analysis;2. In this experiment a new phage of Vibrio alginolyticus was isolated from sewage by using Vibrio alginolyticus as its host bacteria. The plaques of the phage which was screen were clear in the center and with haloy around.The plaques were about 1.0～1.2mm diameter in the flat. The potency of the phages was 10¹⁰pfu/mL after amplification.3 .The physiological and biochemical characteristic of the phages indicated that:the RTD of the phage is 106pfu/mL ;the optimum PH value of the phage is about 8.0; Overacidity or overalkalinity environment are not suitable for the survival of phage . The optimum temperature of the phage infected to Vibrio parahaemolyticus is 35℃; the phage has good heat tolerance; the phages were sensitive to upper 60℃. The phages were sensitive to ultraviolet radiation and Triton-X100. The mixed time of phages and hosts affects quantity of plaques. The phages were resistent to chloroform; titer of the phage is remin 17.8% after treatment. It’s favorable to the attachment of the phage when Mg²⁺ is in the culture medium.The ability of cleavage of the phages was strong when the salinity of the water is about 25‰. It adapt to the living entironment of phages. The best infect proportion was 0.1¹. The incubation period of the phage is 35min.4. This paper study in how to use the phage to completely pyrolysis hosts. They can be seen from the experimental results: the cultivation period of Vibrio alginolyticus affect the disinfection of phages, between 2 hours and 8 hours the effection is not significant and the change of the amount of Vibrio alginolyticus is uniform that began with a faint rise, four hours later began to fall, after six hours fall more evident and rised after reached the lowest point the splitted ability of phage is reduce to the 10 hours Vibrio alginolyticus .The number of bacteria in 10 hours to reach the lowest ,but is higher than before. In 12 hours with training period after as the host of dissolving algae vibrio, found the quantity change of bacteria phage role smaller. Phages play a smaller role to the change of the bacteria amount when the hosts have growed more than 12 hours. The curve of the number of bacteria is similar with the bacteria growth curve;The change of the amount of Vibrio alginolyticus is different when the amount of the phage is different from the amount of the hosts.When the number of the hosts is larger than the number of the phage , the mumber of hosts is first showed a rising trend, then down and up ;In contrast,the number of bacteria slowly increased but dropped later.When MIO=10, the disinfection dffect is best.The amount of the phages and the ratio of the amount of the hosts to the disinfection effect is not very big, but will lead to reach the minimum amount of bacteria in different time.5. The results of Experiments have found phage can’t completely cracking bacteria in laboratory. Using 50μL garlic liquid or 10μLconcentration for 100U/mL of penicillin and 100 muon g/mL streptomycin of PBS can clean the rest of the bacteria.6. The virulence test show: 1×10⁶cfu/mL Vibrio alginolyticus can make the prawn mortality 30%, and 1×10⁷cfu/mL of Vibrio alginolyticus can cause red body disease to the shrimp, 1×10⁸cfu/mL of Vibrio alginolyticus make the prawn mortality achieve 90%. The experiment of bacteria burden of the tissue and apparatus of Litopanaeus vannamei show that the amount of vibrios in the body of prawn is bigger than the control group expecially in hepatopancreas and intestines.7. This experiment showed effects of the immunological indexes after injected whole cell vaccine, bacterial ghosts and phage.The NOS value is the highest when injected bacterial ghosts, and the influence of phage is not obvious,even lower than negative control; PO value of the Litopanaeus vannamei Given 10⁸ the whole cell vaccine is the highest. Hemocyanin is the highest of Litopanaeus vannamei Given 10⁸ phage.8. The virulence test after immunization showed: the immune protective rate of Litopanaeus vannamei injected 10⁸pfu/mL phage is better than others and up to 88.2%. With the concentration decreasing the immune protective rate also fall subsequently but the immune protective rate of Litopanaeus vannamei which injected 10⁶pfu/mL phages is remin 70.6% and higher than 10⁸cfu/mL whole cell vaccines. The immune protective rate of Litopanaeus vannamei injected 10⁸cfu/mL bacterial ghosts is remin 76.5% and higher than 10⁸cfu/mL whole cell vaccine.更多还原