引起对虾早期死亡综合征的副溶血性弧菌的分离鉴定及发病机理研究

发布时间：2018-05-03 10:19

本文选题：对虾早期死亡综合征 + 菌种分离和鉴定　；参考：《上海海洋大学》2017年博士论文

【摘要】：自2009年开始,亚洲虾类养殖开始爆发一种未知的新兴疾病,养殖的对虾在养殖初期(35天左右)就以极快的速度死亡,因此得名早期死亡综合征(EMS),由于其可以使患病对虾的肝胰腺出现急性坏死的症状,被更形象的命名为急性肝胰腺坏死综合征(AHPNS)。该疾病在之后几年内迅速蔓延至越南、马来西亚、泰国和墨西哥。2013年初,研究人员基本确认了一种特殊的副溶血性弧菌为该病的主要病因,这种特别的副溶血性弧菌中含有的独特的质粒,可以编码出的类杀虫毒素的毒素蛋白,会引起虾类的肝胰腺坏死。本论文从中国广东西南沿海爆发过EMS的养殖池塘中对病原菌进行了分离和深入地鉴定。通过构建实验室南美白对虾动物模型,对获得的EMS病原菌进行了攻毒验证,对其毒力基因表达和毒力表型的规律进行了探索研究。主要结果如下:1.对虾早期死亡综合征病原菌的分离以副溶血弧菌食品卫生微生物学检验GB/T 4789.7-2013和美国FDA细菌分析手册为参照标准,在疾病爆发区域的对虾养殖场中,采集健康、濒死、死亡对虾、池塘水样、泥土样,以副溶血性弧菌为主要分离对象,进行细菌分离工作。在8个不同位置的养殖池塘中共分离出81株常见的海洋细菌,其中39株为副溶血性弧菌。使用三对较权威的引物对81株细菌进行鉴定,选取三对引物均为阳性的菌株作为对虾早起死亡综合征的疑似病原菌株。总共筛选出12株疑似病原菌。2.对虾早期死亡综合征病原菌的鉴定对筛选出的12株疑似病原菌进行分子生物学鉴定。常规毒力基因鉴定结果显示这些菌株均含有tlh基因,不含有tdh和trh基因,因此不会对人类健康产生太大的威胁。通过API20E微生物鉴定条对菌株进行了菌种鉴定,并通过对16S rRNA基因测序做了系统发育分析,结果显示这些菌株全部为副溶血性弧菌。通过ERIC-PCR和质粒指纹图谱对菌株的多样性进行了分析,结果表明这12株菌的多样性较为单一。对毒素基因pirA的全长进行了扩增和测序,发现此12株菌均含有该毒素基因并具有很高的同源性。提取了这些菌株的总RNA并测定了毒力基因pirA的表达量,结果显示毒力基因的表达情况在菌株之间存在较大差异,表达量最高和最低相差约13倍。使用SDS-PAGE和Western Blotting的方法对蛋白表型和毒素蛋白进行了鉴定,结果表明与已经确证的病原菌株3HP相比,本研究分离获得的12株菌株菌有特异性的毒素蛋白分泌。由此分别从菌株的菌种信息、多样性分析、质粒指纹图谱、毒素基因的多样性、转录和蛋白表达的层面对疑似病原菌株进行了深入地鉴定。3.南美白对虾动物模型的建立及攻毒验证实验本研究构建了可用于实验室攻毒验证实验的小型南美白对虾动物模型。对虾养殖、攻毒设备可控温度范围为室温至34℃,监控的水质指标主要为pH(6.7-8.5)、亚硝酸盐0.1 mM、总氨氮含量0.35 mM,每组实验最大水体体积为50L。首先使用了3HP菌株对攻毒验证实验的感染方式和感染浓度进行了确定,最终选择以小体积菌液浸泡的方式感染对虾,感染用菌液浓度为106CFU/m L,感染时间15 min。攻毒验证实验以无毒的S02和无菌的TSB+培养基为阴性对照,分别验证副溶血性弧菌和培养基对于实验的影响。感染后每12 h观察记录一次,并收取死亡对虾尸体,进行组织病理学切片观察确症。结果表明,12株菌株均具有使对虾患早期死亡综合征的毒力,除F5和F18号菌株,其余10株均可达到100%致死率,其中多数菌株可以在48 h以内使感染对虾迅速大量死亡。通过组织切片鉴定也可发现这些对虾的肝胰腺组织呈现坏死症状,具有典型的对虾早期死亡综合征的病症特点。以无毒的S02菌株作为阴性对照的实验组只有1只对虾死亡,(TSB+培养基对照组无对虾死亡),组织切片结果显示并未出现肝胰腺坏死的症状。这些结果都与分子生物学鉴定结果相吻合。通过上述实验可以确认,此12株副溶血性弧菌为对虾早起死亡综合征的致病菌株。4.毒素基因表达和毒力对应情况的讨论以及温度对其的影响研究中发现,在分离获得的12株对虾早期死亡综合征的病原菌中,毒素基因的表达与毒力的对应情况并不好。对此发现初步分析后认为可能是由于毒素基因表达的温度与毒素感染对虾的温度不同,温度的差异导致对虾对于毒素的应激反应不同导致了该现象。因此设计了不同温度对菌株感染能力影响的实验。实验选取37℃下毒力基因表达最强的F12号菌株和表达最弱的G10号菌株为研究对象。选取对虾较为适宜生长的20-32℃为温度范围,每3℃设置一个梯度,对两株菌在5个不同温度下的毒力进行攻毒测试。结果表明,在实验设置的5个温度梯度范围内,致病菌株的毒力随着温度的升高而增强,两株菌株都在32℃时最早出现死亡对虾和最早达到100%致死率。菌株间的横向比较发现F12菌株在每个温度梯度下的致死速度都强于G10号菌株,这一结果与毒素基因表达结果相符,说明在一定的温度范围内,菌株之间的确存在毒力强弱的差异,毒素基因表达的差异是导致这一现象的主要原因。
[Abstract]:Since 2009, the shrimp culture in Asia has begun to break out an unknown disease, and the cultured prawns died in the early period (about 35 days), so the early death syndrome (EMS) was named as acute hepatopancreanopancreas with the symptoms of acute necrosis of the hepatopancreas of the sick prawns. Death syndrome (AHPNS). The disease spread rapidly over the following years into Vietnam, Malaysia, Thailand, and Mexico, at the beginning of.2013, and researchers basically identified a special Vibrio parahaemolyticus as the main cause of the disease. This special Vibrio parahaemolyticus contains a unique plasmid that encodes a toxin like toxin. The protein, which causes the necrosis of the hepatopancreas in the shrimps, has been separated and deeply identified from the EMS culture pond in the southwest coast of Guangdong, China. By constructing the laboratory of the laboratory South American white Penaeus prawns, the EMS pathogenic bacteria were verified, the expression of virulence genes and the regularity of virulence phenotypes of the pathogenic bacteria were carried out. The main results were as follows: 1. the isolation of the pathogen of early death syndrome of 1. prawns was taken as reference standard by the microbiological microbiology test of Vibrio parahaemolyticus and the American FDA bacterial analysis manual. In the shrimp farm of the outbreak area, the health, the dying, the death prawns, the pond water and the soil samples were collected. With Vibrio parahaemolyticus as the main separation object, 81 common marine bacteria were isolated in 8 different aquaculture ponds, of which 39 were Vibrio parahaemolyticus. 81 strains of bacteria were identified by three of the more authoritative primers, and three strains were selected as positive for the early death of prawns. A total of 12 suspected pathogenic bacteria of the early death syndrome of.2. shrimp were identified by molecular biological identification of 12 suspected pathogenic bacteria. The results of conventional virulence gene identification showed that all of these strains contained TLH genes, and did not contain TDH and TRH genes, so they did not produce healthy human beings. The strain was identified by the API20E microorganism identification strip, and the phylogenetic analysis of the 16S rRNA gene was carried out. The results showed that all of these strains were Vibrio parahaemolyticus. The diversity of the strains was analyzed by ERIC-PCR and plasmid fingerprints, and the results showed that the diversity of the 12 strains was more diverse. The total length of the toxin gene pirA was amplified and sequenced. It was found that the 12 strains all contained the toxin gene and had high homology. The total RNA of the strains was extracted and the expression of the virulence gene pirA was measured. The results showed that the expression of the virulence gene was different between the strains, the highest expression and the highest expression of the virulence gene. The low phase difference was about 13 times. The protein phenotype and toxin protein were identified by SDS-PAGE and Western Blotting. The results showed that compared with the confirmed pathogen 3HP, the isolates obtained from this study had specific toxin protein secretion. From the strain information, diversity analysis and plasmid fingerprinting, respectively. The diversity, gene diversity, transcription and protein expression in the layer of the suspected pathogenic strain of.3. in depth identification of the animal model of Penaeus vannamei and the test of the test of attack on the virus, a miniature shrimp model of Penaeus prawns, which can be used in laboratory attack verification experiment, was constructed. At room temperature to 34 C, the monitoring water quality index is mainly pH (6.7-8.5), nitrite 0.1 mM, total ammonia nitrogen content 0.35 mM. The largest body of water body volume of each group is 50L.. First, 3HP strain is used to determine the infection mode and the infection concentration of the test. Finally, it is selected to infect prawns with small volume bacteria solution. The concentration of bacteria was 106CFU/m L, and the infection time 15 min. was tested with non-toxic S02 and aseptic TSB+ medium as negative control. The effects of Vibrio parahaemolyticus and culture medium on the experiment were verified respectively. After infection, every 12 h records were recorded, and the corpse of death prawn was collected, and the pathological sections were observed and confirmed. The results showed that All the 12 strains had the virulence of the early death syndrome of the shrimp. Except the F5 and F18 strains, the other 10 strains could reach 100% fatality rate. Most of them could make the infected prawns rapidly death in 48 h. The disease characteristics of early death syndrome. Only 1 shrimp died in the experimental group with nontoxic S02 strain as negative control group, and (TSB+ culture group no shrimp death). The results of tissue section showed no symptoms of hepatopancreas necrosis. These results were consistent with the results of molecular biological identification. The 12 strains of Vibrio parahaemolyticus were discussed in the study of gene expression and virulence corresponding to the pathogenic strain of the early death syndrome of prawns and the effect of temperature on it. It was found that in the 12 strains of early death syndrome of prawns, the correspondence between the toxin gene and the toxin was not good. The 12 strains of Vibrio parahaemolyticus were found to be not good with the virulence. The preliminary analysis suggests that the temperature of the toxin gene expression may be different from the temperature of the toxin infected prawns, and the difference in temperature leads to the difference of the stress response to the toxin in the shrimp. Therefore, the experiment on the influence of different temperature on the infection ability of the strain is designed. The experiment was made to select the F12 bacteria with the strongest expression of virulence gene at 37. The strain and the weakest strain G10 were selected as the research object. The temperature range was 20-32 C suitable for the growth of the shrimp. A gradient was set at 3 degrees C, and the toxicity of two strains at 5 different temperatures was tested. The results showed that the virulence of the pathogenic strain increased with the increase of temperature in the 5 temperature gradient range. Strong, two strains of strain all appeared early death prawns and the earliest death rate of 100% at 32. The lateral comparison between the strains found that the death rate of F12 strain was stronger than that of strain G10 at each temperature gradient. This result was consistent with the result of toxin gene expression, indicating that there was a strong virulence among the strains within a certain temperature range. The difference of toxin gene expression is the main reason for this phenomenon.

【学位授予单位】：上海海洋大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：S945.46

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