一例日本对虾暴发性死亡的病原分析及益生菌联用对对虾免疫增强作用
发布时间：2018-10-20 06:58
【摘要】：第一部分:一例日本囊对虾暴发性死亡的病原分析为了阐明山东省潍坊市1对虾养殖场发生日本囊对虾(Marsupenaeus japonicus)暴发性死亡的原因,采用分子生物学检测方法,对发病对虾进行了白斑综合征病毒(White spot syndrome virus,WSSV)、桃拉综合征病毒(Taura syndrome virus,TSV)、黄头病毒(Yellow head virus,YHV)、传染性皮下及造血组织坏死病毒(Infectious hypodermal and hematopoietic necrosis virus,IHHNV)、传染性肌肉坏死病毒(Infectious myonecrosis virus,IMNV)、偷死野田村病毒(Covert mortality nodavirus,CMNV)及急性肝胰腺坏死病(Acute hepatopancreatic necrosis disease,AHPND)7种病原的检测,且对发病对虾进行了常规组织病理学观察。同时采用16SrDNA细菌鉴定方法及浸泡回接感染实验对分离自发病对虾体内的可疑病原菌进行了分子鉴定及毒力测试。结果显示,发病对虾样品核酸检测呈现WSSV强阳性,IHHNV和CMNV为弱阳性,其他4种病原为阴性。组织病理学观察发现,在对虾的胃、鳃等上皮组织中存在WSSV包涵体,头部肌肉纤维出现离散。对分离的编号为2901、2902、2903的3株优势可疑病原菌鉴定结果表明,3株菌分别与印度格里蒙菌(Grimontia indica)、交替单胞菌(Alteromonas sp.)及溶藻弧菌(Vibrio alginolyticus)相似,相似度分别为99%、99%及100%。攻毒结果显示3株可疑病原菌的LC50分别为9.8×107CFU/m L、1.1×108 CFU/mL与2.3×108CFU/mL,各细菌毒力均较弱,非导致对虾出现暴发性死亡的病原。综合上述结果推断,导致本次日本囊对虾暴发死亡的病因与混合感染病原WSSV、IHHNV、CMNV有关,其中WSSV感染是造成日本囊对虾暴发死亡的主因,研究结果可为解析当前养殖日本囊对虾疾病暴发及其成因提供参考。第二部分:益生菌联用对对虾免疫增强作用为了研究有益菌防控对虾AHPND的效果,将假交替单胞菌(Pseudoalteromonas.sp.)、盐单胞菌(Halomonas.sp)、地衣芽孢杆菌(Bacillus licheniformis)、枯草芽孢杆菌(B.subtitles)、肠膜明串珠菌(Leuconostocmesemteroides)、酿酒酵母菌(Saccharomyces cerevisiae)、复合菌1(盐单胞菌、地衣芽孢杆菌、枯草芽孢杆菌、肠膜明串珠菌、酿酒酵母菌)及复合菌2(假交替单胞菌、盐单胞菌、肠膜明串珠菌、酿酒酵母菌)分别添加到凡纳滨对虾养殖水体及饲料中,养殖15d时,取部分实验虾人工感染副溶血弧菌(V.parahaemolyticus,VPAHPND),分析评价对虾的抗感染能力,养殖30d后统计实验对虾存活率。同时,分析对虾血清样品及组织样品免疫及消化相关酶活。并对各实验组养殖水样及实验对虾肠道微生物样品进行多样性分析。结果显示,各组对虾人工感染VPAHPND后,枯草芽孢杆菌、假交替单胞菌、复合1、复合2、地衣芽孢杆菌、盐单胞菌、肠膜明串珠菌、酿酒酵母菌各组的死亡率分别为37.5%、45.8%、50.0%、41.6%、54.1%、58.3%、62.5%及70.8%,均显著低于对照组的死亡率80.0%;养殖期间统计各组的养殖存活率显示,复合1组、复合2组、枯草芽孢杆菌组、假交替单胞菌组、地衣芽孢杆菌组、盐单胞菌组、肠膜明串珠菌组、酿酒酵母菌组的存活率分别为65.0%、60.4%、54.7%、50%、53.8%、52.8%、49.9%、53.0%,均高于对照组的存活率40.0%。对各实验组血清样品进行溶菌酶、酚氧化酶(PO)、超氧化物歧化酶(SOD)和肝胰腺、肠道样品进行脂肪酶(LBS)、淀粉酶(AMS)的活力分析。结果显示,复合2组(69.2)、地衣芽孢杆菌组(39.2)、枯草芽孢杆菌组(34.7)、肠膜明串珠菌组(65.6)、假交替单胞菌组(56.9)的PO活力显著高于对照组(8.0)(P0.05);复合1组的LBS活力(9.38)显著高于对照组(0.4)(P0.05)。本实验所用有益菌对血清中的SOD活力及肝胰腺、肠道样品中的AMS活力不具有增强作用,对血清中的溶菌酶活力作用不大。对各实验组养殖水样及实验对虾肠道样品进行微生物多样性分析,结果显示,实验组与对照组养殖水体中微生物多样性组成存在差异,养殖水体中主要菌群为拟杆菌门(Bacteroidetes)、变形菌门(Proteobacteria)、防线菌门(Actinobacteria)、蓝藻门(Cyanobacteria)、疣微菌门(Verrucomicrobia)、浮霉门(Planctomycetes)、绿菌门(Chlorobi),其中优势菌为拟杆菌门,占复合1组的54.5%、复合2组的55.0%、地衣芽孢杆菌组的34.8%、枯草芽孢杆菌组的55.0%、假交替单胞菌组的34.0%、肠膜明串珠菌组的32.5%,且实验组均低于对照组的69.1%;对虾肠道中微生物区系主要组成为变形菌门(Proteobacteria)、拟杆菌门(Bacteroidetes)、防线菌门(Actinobacteria)、疣微菌门(Verrucomicrobia),且实验组与对照组中的菌群组成比例相似。其中变形菌门与拟杆菌门为主要菌群,二者占菌群的80%以上。研究结果表明,有益菌的联用对防控对虾AHPND具有一定的效果,研究结果可为对虾健康养殖提供参考。
[Abstract]:The first part: An analysis of the causes of fulminating death of shrimp (Marsupenaeus japonicus) in the shrimp farms of Shandong Province in order to clarify the causes of fulminating death of shrimp (Marsupenaeus japonicus) in one prawn farm in Shandong Province, and adopt the method of molecular biology detection. White Spot syndrome virus (WSSV), Taura syndrome virus (TSV), yellow head virus (YHV), infectious subcutaneous and hematopoietic tissue necrosis virus (IHNV), infectious bovine myonnecrosis virus, IMNV, the detection of 7 pathogens of Covert mportality novovirus (CMNV) and acute liver pancreatic necrosis disease (AHPND), and the routine histopathological observation of prawn were carried out. At the same time, 16SrDNA bacterial identification method and soaking-back infection experiment were used to identify and test suspicious pathogenic bacteria isolated from prawn. The results showed that the detection of nucleic acid in shrimp samples showed WSSV strong positive, IHHNV and CMNV were weak positive, and the other four pathogens were negative. Histopathological observation showed that there were WSSV inclusion bodies in the upper gastrointestinal tract of prawn, and the head muscle fibers were dispersed. The results of identification of 3 strains of isolates numbered 29001, 2902 and 2903 indicated that 3 strains were associated with Grimontia indica, Alternaria sp., respectively.) The similarity was 99%, 99% and 100%, respectively. The results showed that the virulence of the three suspicious pathogenic bacteria were 9. 8, 107 CFU/ m L, 1. 1, 108 CFU/ mL and 2. 3/ 108CFU/ mL, respectively, and the virulence of each bacterium was weak, which did not lead to the occurrence of fulminating death of prawn. According to the above results, the cause of the outbreak of prawn in this Japanese capsule is related to the mixed infection pathogen WSSV, IHHNV and CMNV, among which WSSV infection is the cause of the outbreak of shrimp in Japan. The results of the study could provide a reference for the analysis of the outbreak of shrimp disease and its cause. The second part: Probiotics combined with probiotics to study the effect of shrimp AHPND on the immune enhancement of penaeus vannamei, and Pseudomonas sp.), Pseudomonas sp., Bacillus Lichensis, Bacillus subtilis, Enterobacteriaceae, Saccharomyces cerevisiae, composite bacteria 1 (Zymomonas mobileticus, Bacillus subtilis, Bacillus subtilis, Enterobacteriaceae), Saccharomyces cerevisiae (Saccharomyces cerevisiae) and compound bacteria 2 (Pseudomonas fluorescens, Pseudomonas sp., Enterobacteriaceae, Saccharomyces cerevisiae) were added to the water body and feed of Litopenaeus vannamei respectively, and when cultured for 15d, the vibrio paraolyticus (VPAHND) was artificially infected with some experimental shrimp. The anti-infective ability of penaeus vannamei was evaluated, and the survival rate of prawn was counted after 30 days. At the same time, the serum samples and tissue samples of prawn were analyzed to immunize and digest related enzymes. The diversity of intestinal microbial samples was analyzed in each experimental group. The results showed that after artificial infection of VPAHND, the mortality rates of Bacillus subtilis, Pseudomonas aeruginosa, Compound 1, Compound 2, Bacillus Licbacillus, Pseudomonas, Enterobacteriaceae, and Saccharomyces cerevisiae were 37. 5%, 45. 8%, 50. 0%, 41. 6%, 54. 1%, 58. 3%, respectively. 62. 5% and 70. 8%, all significantly lower than that of the control group, the mortality rate was 80. 0%, the culture survival rate of each group was shown in the cultivation period, the compound 1 group, the compound 2 group, the bacillus subtilis group, the pseudomonas fluorescens group, the lichen bacillus group, the salt pseudomonas group and the intestinal membrane Mingzhu group, The survival rate of Saccharomyces cerevisiae group was 65. 0%, 60. 4%, 54. 7%, 50%, 53. 8%, 52. 8%, 49. 9%, 53. 0%, all higher than the survival rate of the control group was 40. 0%. Lysozyme, phenoloxidase (PO), superoxide dismutase (SOD) and liver pancreas were carried out in serum samples of each experimental group, and the activity of lipase (LBS) and amylase (AMS) in intestinal samples were analyzed. The results showed that the PO activity was significantly higher in the group 2 (69.2), B.lichen group (39. 2), Bacillus subtilis group (34. 7), Enterobacteriaceae group (66.5), Pseudomonas aeruginosa (56. 9), and the PO activity was significantly higher than that in the control group (P0.05). The LBS activity (9.38) in the compound 1 group was significantly higher than that in the control group (0.4) (P0.05). In this experiment, the activity of SOD in serum and the activity of AMS in liver pancreas and intestinal samples were not enhanced, and the activity of lysozyme in serum was not significant. The results showed that there was a difference in microbial diversity composition between experimental group and control group, and the main bacteria in cultured water body were Bacteroidites and Proteobacter. (Acinetobacter), Cyanobacteria (Cyanobacteria), Microbacterium (Verrucomicrobia), Panctaceae, and Chloboi, among which the dominant bacteria were Bacteria, 55.5% of compound 1, 55.0% of compound 2, 34. 8% of B.lichen group and 55.0% of Bacillus subtilis group. 34. 0% of pseudomonas fluorescens group, 32. 5% of intestinal membrane and conostoc group, and the experimental group was lower than 69.1% of the control group; the microbial area in the intestinal tract of the prawn was mainly composed of Proteobacteria, Bacteroidites, Acinetobacter, Verrucomicrobnia. and the composition ratio of the experimental group to the flora in the control group is similar. wherein the deformation bacteria gate and the pseudo-bacillus door are the main bacteria groups, and the two strains account for more than 80% of the bacterial population. The results showed that the combination of quercetin had a certain effect on the prevention and control of prawn AHPND, and the research results could provide reference for prawn health cultivation.
【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S945.45
[Abstract]:The first part: An analysis of the causes of fulminating death of shrimp (Marsupenaeus japonicus) in the shrimp farms of Shandong Province in order to clarify the causes of fulminating death of shrimp (Marsupenaeus japonicus) in one prawn farm in Shandong Province, and adopt the method of molecular biology detection. White Spot syndrome virus (WSSV), Taura syndrome virus (TSV), yellow head virus (YHV), infectious subcutaneous and hematopoietic tissue necrosis virus (IHNV), infectious bovine myonnecrosis virus, IMNV, the detection of 7 pathogens of Covert mportality novovirus (CMNV) and acute liver pancreatic necrosis disease (AHPND), and the routine histopathological observation of prawn were carried out. At the same time, 16SrDNA bacterial identification method and soaking-back infection experiment were used to identify and test suspicious pathogenic bacteria isolated from prawn. The results showed that the detection of nucleic acid in shrimp samples showed WSSV strong positive, IHHNV and CMNV were weak positive, and the other four pathogens were negative. Histopathological observation showed that there were WSSV inclusion bodies in the upper gastrointestinal tract of prawn, and the head muscle fibers were dispersed. The results of identification of 3 strains of isolates numbered 29001, 2902 and 2903 indicated that 3 strains were associated with Grimontia indica, Alternaria sp., respectively.) The similarity was 99%, 99% and 100%, respectively. The results showed that the virulence of the three suspicious pathogenic bacteria were 9. 8, 107 CFU/ m L, 1. 1, 108 CFU/ mL and 2. 3/ 108CFU/ mL, respectively, and the virulence of each bacterium was weak, which did not lead to the occurrence of fulminating death of prawn. According to the above results, the cause of the outbreak of prawn in this Japanese capsule is related to the mixed infection pathogen WSSV, IHHNV and CMNV, among which WSSV infection is the cause of the outbreak of shrimp in Japan. The results of the study could provide a reference for the analysis of the outbreak of shrimp disease and its cause. The second part: Probiotics combined with probiotics to study the effect of shrimp AHPND on the immune enhancement of penaeus vannamei, and Pseudomonas sp.), Pseudomonas sp., Bacillus Lichensis, Bacillus subtilis, Enterobacteriaceae, Saccharomyces cerevisiae, composite bacteria 1 (Zymomonas mobileticus, Bacillus subtilis, Bacillus subtilis, Enterobacteriaceae), Saccharomyces cerevisiae (Saccharomyces cerevisiae) and compound bacteria 2 (Pseudomonas fluorescens, Pseudomonas sp., Enterobacteriaceae, Saccharomyces cerevisiae) were added to the water body and feed of Litopenaeus vannamei respectively, and when cultured for 15d, the vibrio paraolyticus (VPAHND) was artificially infected with some experimental shrimp. The anti-infective ability of penaeus vannamei was evaluated, and the survival rate of prawn was counted after 30 days. At the same time, the serum samples and tissue samples of prawn were analyzed to immunize and digest related enzymes. The diversity of intestinal microbial samples was analyzed in each experimental group. The results showed that after artificial infection of VPAHND, the mortality rates of Bacillus subtilis, Pseudomonas aeruginosa, Compound 1, Compound 2, Bacillus Licbacillus, Pseudomonas, Enterobacteriaceae, and Saccharomyces cerevisiae were 37. 5%, 45. 8%, 50. 0%, 41. 6%, 54. 1%, 58. 3%, respectively. 62. 5% and 70. 8%, all significantly lower than that of the control group, the mortality rate was 80. 0%, the culture survival rate of each group was shown in the cultivation period, the compound 1 group, the compound 2 group, the bacillus subtilis group, the pseudomonas fluorescens group, the lichen bacillus group, the salt pseudomonas group and the intestinal membrane Mingzhu group, The survival rate of Saccharomyces cerevisiae group was 65. 0%, 60. 4%, 54. 7%, 50%, 53. 8%, 52. 8%, 49. 9%, 53. 0%, all higher than the survival rate of the control group was 40. 0%. Lysozyme, phenoloxidase (PO), superoxide dismutase (SOD) and liver pancreas were carried out in serum samples of each experimental group, and the activity of lipase (LBS) and amylase (AMS) in intestinal samples were analyzed. The results showed that the PO activity was significantly higher in the group 2 (69.2), B.lichen group (39. 2), Bacillus subtilis group (34. 7), Enterobacteriaceae group (66.5), Pseudomonas aeruginosa (56. 9), and the PO activity was significantly higher than that in the control group (P0.05). The LBS activity (9.38) in the compound 1 group was significantly higher than that in the control group (0.4) (P0.05). In this experiment, the activity of SOD in serum and the activity of AMS in liver pancreas and intestinal samples were not enhanced, and the activity of lysozyme in serum was not significant. The results showed that there was a difference in microbial diversity composition between experimental group and control group, and the main bacteria in cultured water body were Bacteroidites and Proteobacter. (Acinetobacter), Cyanobacteria (Cyanobacteria), Microbacterium (Verrucomicrobia), Panctaceae, and Chloboi, among which the dominant bacteria were Bacteria, 55.5% of compound 1, 55.0% of compound 2, 34. 8% of B.lichen group and 55.0% of Bacillus subtilis group. 34. 0% of pseudomonas fluorescens group, 32. 5% of intestinal membrane and conostoc group, and the experimental group was lower than 69.1% of the control group; the microbial area in the intestinal tract of the prawn was mainly composed of Proteobacteria, Bacteroidites, Acinetobacter, Verrucomicrobnia. and the composition ratio of the experimental group to the flora in the control group is similar. wherein the deformation bacteria gate and the pseudo-bacillus door are the main bacteria groups, and the two strains account for more than 80% of the bacterial population. The results showed that the combination of quercetin had a certain effect on the prevention and control of prawn AHPND, and the research results could provide reference for prawn health cultivation.
【学位授予单位】：上海海洋大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S945.45
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