本文選題：對蝦早期死亡綜合征 + 菌種分離和鑒定�。� 參考：《上海海洋大學》2017年博士論文

【摘要】：自2009年開始,亞洲蝦類養(yǎng)殖開始爆發(fā)一種未知的新興疾病,養(yǎng)殖的對蝦在養(yǎng)殖初期(35天左右)就以極快的速度死亡,因此得名早期死亡綜合征(EMS),由于其可以使患病對蝦的肝胰腺出現(xiàn)急性壞死的癥狀,被更形象的命名為急性肝胰腺壞死綜合征(AHPNS)。該疾病在之后幾年內(nèi)迅速蔓延至越南、馬來西亞、泰國和墨西哥。2013年初,研究人員基本確認了一種特殊的副溶血性弧菌為該病的主要病因,這種特別的副溶血性弧菌中含有的獨特的質(zhì)粒,可以編碼出的類殺蟲毒素的毒素蛋白,會引起蝦類的肝胰腺壞死。本論文從中國廣東西南沿海爆發(fā)過EMS的養(yǎng)殖池塘中對病原菌進行了分離和深入地鑒定。通過構(gòu)建實驗室南美白對蝦動物模型,對獲得的EMS病原菌進行了攻毒驗證,對其毒力基因表達和毒力表型的規(guī)律進行了探索研究。主要結(jié)果如下:1.對蝦早期死亡綜合征病原菌的分離以副溶血弧菌食品衛(wèi)生微生物學檢驗GB/T 4789.7-2013和美國FDA細菌分析手冊為參照標準,在疾病爆發(fā)區(qū)域的對蝦養(yǎng)殖場中,采集健康、瀕死、死亡對蝦、池塘水樣、泥土樣,以副溶血性弧菌為主要分離對象,進行細菌分離工作。在8個不同位置的養(yǎng)殖池塘中共分離出81株常見的海洋細菌,其中39株為副溶血性弧菌。使用三對較權(quán)威的引物對81株細菌進行鑒定,選取三對引物均為陽性的菌株作為對蝦早起死亡綜合征的疑似病原菌株�？偣埠Y選出12株疑似病原菌。2.對蝦早期死亡綜合征病原菌的鑒定對篩選出的12株疑似病原菌進行分子生物學鑒定。常規(guī)毒力基因鑒定結(jié)果顯示這些菌株均含有tlh基因,不含有tdh和trh基因,因此不會對人類健康產(chǎn)生太大的威脅。通過API20E微生物鑒定條對菌株進行了菌種鑒定,并通過對16S rRNA基因測序做了系統(tǒng)發(fā)育分析,結(jié)果顯示這些菌株全部為副溶血性弧菌。通過ERIC-PCR和質(zhì)粒指紋圖譜對菌株的多樣性進行了分析,結(jié)果表明這12株菌的多樣性較為單一。對毒素基因pirA的全長進行了擴增和測序,發(fā)現(xiàn)此12株菌均含有該毒素基因并具有很高的同源性。提取了這些菌株的總RNA并測定了毒力基因pirA的表達量,結(jié)果顯示毒力基因的表達情況在菌株之間存在較大差異,表達量最高和最低相差約13倍。使用SDS-PAGE和Western Blotting的方法對蛋白表型和毒素蛋白進行了鑒定,結(jié)果表明與已經(jīng)確證的病原菌株3HP相比,本研究分離獲得的12株菌株菌有特異性的毒素蛋白分泌。由此分別從菌株的菌種信息、多樣性分析、質(zhì)粒指紋圖譜、毒素基因的多樣性、轉(zhuǎn)錄和蛋白表達的層面對疑似病原菌株進行了深入地鑒定。3.南美白對蝦動物模型的建立及攻毒驗證實驗本研究構(gòu)建了可用于實驗室攻毒驗證實驗的小型南美白對蝦動物模型。對蝦養(yǎng)殖、攻毒設(shè)備可控溫度范圍為室溫至34℃,監(jiān)控的水質(zhì)指標主要為pH(6.7-8.5)、亞硝酸鹽0.1 mM、總氨氮含量0.35 mM,每組實驗最大水體體積為50L。首先使用了3HP菌株對攻毒驗證實驗的感染方式和感染濃度進行了確定,最終選擇以小體積菌液浸泡的方式感染對蝦,感染用菌液濃度為106CFU/m L,感染時間15 min。攻毒驗證實驗以無毒的S02和無菌的TSB+培養(yǎng)基為陰性對照,分別驗證副溶血性弧菌和培養(yǎng)基對于實驗的影響。感染后每12 h觀察記錄一次,并收取死亡對蝦尸體,進行組織病理學切片觀察確癥。結(jié)果表明,12株菌株均具有使對蝦患早期死亡綜合征的毒力,除F5和F18號菌株,其余10株均可達到100%致死率,其中多數(shù)菌株可以在48 h以內(nèi)使感染對蝦迅速大量死亡。通過組織切片鑒定也可發(fā)現(xiàn)這些對蝦的肝胰腺組織呈現(xiàn)壞死癥狀,具有典型的對蝦早期死亡綜合征的病癥特點。以無毒的S02菌株作為陰性對照的實驗組只有1只對蝦死亡,(TSB+培養(yǎng)基對照組無對蝦死亡),組織切片結(jié)果顯示并未出現(xiàn)肝胰腺壞死的癥狀。這些結(jié)果都與分子生物學鑒定結(jié)果相吻合。通過上述實驗可以確認,此12株副溶血性弧菌為對蝦早起死亡綜合征的致病菌株。4.毒素基因表達和毒力對應情況的討論以及溫度對其的影響研究中發(fā)現(xiàn),在分離獲得的12株對蝦早期死亡綜合征的病原菌中,毒素基因的表達與毒力的對應情況并不好。對此發(fā)現(xiàn)初步分析后認為可能是由于毒素基因表達的溫度與毒素感染對蝦的溫度不同,溫度的差異導致對蝦對于毒素的應激反應不同導致了該現(xiàn)象。因此設(shè)計了不同溫度對菌株感染能力影響的實驗。實驗選取37℃下毒力基因表達最強的F12號菌株和表達最弱的G10號菌株為研究對象。選取對蝦較為適宜生長的20-32℃為溫度范圍,每3℃設(shè)置一個梯度,對兩株菌在5個不同溫度下的毒力進行攻毒測試。結(jié)果表明,在實驗設(shè)置的5個溫度梯度范圍內(nèi),致病菌株的毒力隨著溫度的升高而增強,兩株菌株都在32℃時最早出現(xiàn)死亡對蝦和最早達到100%致死率。菌株間的橫向比較發(fā)現(xiàn)F12菌株在每個溫度梯度下的致死速度都強于G10號菌株,這一結(jié)果與毒素基因表達結(jié)果相符,說明在一定的溫度范圍內(nèi),菌株之間的確存在毒力強弱的差異,毒素基因表達的差異是導致這一現(xiàn)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.

【學位授予單位】：上海海洋大學
【學位級別】：博士
【學位授予年份】：2017
【分類號】：S945.46

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2 隋R，

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