發(fā)布時(shí)間：2018-05-03 10:19

  本文選題：對(duì)蝦早期死亡綜合征 + 菌種分離和鑒定��； 參考：《上海海洋大學(xué)》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(duì)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(duì)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(lái)西亞、泰國(guó)和墨西哥。2013年初,研究人員基本確認(rèn)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(dú)特的質(zhì)粒,可以編碼出的類殺蟲毒素的毒素蛋白,會(huì)引起蝦類的肝胰腺壞死。本論文從中國(guó)廣東西南沿海爆發(fā)過(guò)EMS的養(yǎng)殖池塘中對(duì)病原菌進(jìn)行了分離和深入地鑒定。通過(guò)構(gòu)建實(shí)驗(yàn)室南美白對(duì)蝦動(dòng)物模型,對(duì)獲得的EMS病原菌進(jìn)行了攻毒驗(yàn)證,對(duì)其毒力基因表達(dá)和毒力表型的規(guī)律進(jìn)行了探索研究。主要結(jié)果如下:1.對(duì)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(xué)檢驗(yàn)GB/T 4789.7-2013和美國(guó)FDA細(xì)菌分析手冊(cè)為參照標(biāo)準(zhǔn),在疾病爆發(fā)區(qū)域的對(duì)蝦養(yǎng)殖場(chǎng)中,采集健康、瀕死、死亡對(duì)蝦、池塘水樣、泥土樣,以副溶血性弧菌為主要分離對(duì)象,進(jìn)行細(xì)菌分離工作。在8個(gè)不同位置的養(yǎng)殖池塘中共分離出81株常見的海洋細(xì)菌,其中39株為副溶血性弧菌。使用三對(duì)較權(quán)威的引物對(duì)81株細(xì)菌進(jìn)行鑒定,選取三對(duì)引物均為陽(yáng)性的菌株作為對(duì)蝦早起死亡綜合征的疑似病原菌株。總共篩選出12株疑似病原菌。2.對(duì)蝦早期死亡綜合征病原菌的鑒定對(duì)篩選出的12株疑似病原菌進(jìn)行分子生物學(xué)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(huì)對(duì)人類健康產(chǎn)生太大的威脅。通過(guò)API20E微生物鑒定條對(duì)菌株進(jìn)行了菌種鑒定,并通過(guò)對(duì)16S rRNA基因測(cè)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(guò)ERIC-PCR和質(zhì)粒指紋圖譜對(duì)菌株的多樣性進(jìn)行了分析,結(jié)果表明這12株菌的多樣性較為單一。對(duì)毒素基因pirA的全長(zhǎng)進(jìn)行了擴(kuò)增和測(cè)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(cè)定了毒力基因pirA的表達(dá)量,結(jié)果顯示毒力基因的表達(dá)情況在菌株之間存在較大差異,表達(dá)量最高和最低相差約13倍。使用SDS-PAGE和Western Blotting的方法對(duì)蛋白表型和毒素蛋白進(jìn)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(dá)的層面對(duì)疑似病原菌株進(jìn)行了深入地鑒定。3.南美白對(duì)蝦動(dòng)物模型的建立及攻毒驗(yàn)證實(shí)驗(yàn)本研究構(gòu)建了可用于實(shí)驗(yàn)室攻毒驗(yàn)證實(shí)驗(yàn)的小型南美白對(duì)蝦動(dòng)物模型。對(duì)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(biāo)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(shí)驗(yàn)最大水體體積為50L。首先使用了3HP菌株對(duì)攻毒驗(yàn)證實(shí)驗(yàn)的感染方式和感染濃度進(jìn)行了確定,最終選擇以小體積菌液浸泡的方式感染對(duì)蝦,感染用菌液濃度為106CFU/m L,感染時(shí)間15 min。攻毒驗(yàn)證實(shí)驗(yàn)以無(wú)毒的S02和無(wú)菌的TSB+培養(yǎng)基為陰性對(duì)照,分別驗(yàn)證副溶血性弧菌和培養(yǎng)基對(duì)于實(shí)驗(yàn)的影響。感染后每12 h觀察記錄一次,并收取死亡對(duì)蝦尸體,進(jìn)行組織病理學(xué)切片觀察確癥。結(jié)果表明,12株菌株均具有使對(duì)蝦患早期死亡綜合征的毒力,除F5和F18號(hào)菌株,其余10株均可達(dá)到100%致死率,其中多數(shù)菌株可以在48 h以內(nèi)使感染對(duì)蝦迅速大量死亡。通過(guò)組織切片鑒定也可發(fā)現(xiàn)這些對(duì)蝦的肝胰腺組織呈現(xiàn)壞死癥狀,具有典型的對(duì)蝦早期死亡綜合征的病癥特點(diǎn)。以無(wú)毒的S02菌株作為陰性對(duì)照的實(shí)驗(yàn)組只有1只對(duì)蝦死亡,(TSB+培養(yǎng)基對(duì)照組無(wú)對(duì)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(xué)鑒定結(jié)果相吻合。通過(guò)上述實(shí)驗(yàn)可以確認(rèn),此12株副溶血性弧菌為對(duì)蝦早起死亡綜合征的致病菌株。4.毒素基因表達(dá)和毒力對(duì)應(yīng)情況的討論以及溫度對(duì)其的影響研究中發(fā)現(xiàn),在分離獲得的12株對(duì)蝦早期死亡綜合征的病原菌中,毒素基因的表達(dá)與毒力的對(duì)應(yīng)情況并不好。對(duì)此發(fā)現(xiàn)初步分析后認(rèn)為可能是由于毒素基因表達(dá)的溫度與毒素感染對(duì)蝦的溫度不同,溫度的差異導(dǎo)致對(duì)蝦對(duì)于毒素的應(yīng)激反應(yīng)不同導(dǎo)致了該現(xiàn)象。因此設(shè)計(jì)了不同溫度對(duì)菌株感染能力影響的實(shí)驗(yàn)。實(shí)驗(yàn)選取37℃下毒力基因表達(dá)最強(qiáng)的F12號(hào)菌株和表達(dá)最弱的G10號(hào)菌株為研究對(duì)象。選取對(duì)蝦較為適宜生長(zhǎng)的20-32℃為溫度范圍,每3℃設(shè)置一個(gè)梯度,對(duì)兩株菌在5個(gè)不同溫度下的毒力進(jìn)行攻毒測(cè)試。結(jié)果表明,在實(shí)驗(yàn)設(shè)置的5個(gè)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(qiáng),兩株菌株都在32℃時(shí)最早出現(xiàn)死亡對(duì)蝦和最早達(dá)到100%致死率。菌株間的橫向比較發(fā)現(xiàn)F12菌株在每個(gè)溫度梯度下的致死速度都強(qiáng)于G10號(hào)菌株,這一結(jié)果與毒素基因表達(dá)結(jié)果相符,說(shuō)明在一定的溫度范圍內(nèi),菌株之間的確存在毒力強(qiáng)弱的差異,毒素基因表達(dá)的差異是導(dǎo)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(xué)位授予單位】：上海海洋大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：S945.46

【相似文獻(xiàn)】

相關(guān)期刊論文 前10條

1 鄒婉虹;福建省牡蠣食用中感染副溶血性弧菌的風(fēng)險(xiǎn)評(píng)估[J];中國(guó)水產(chǎn);2003年01期

2 王洪斌;閻斌倫;邵營(yíng)澤;徐加濤;;連云港海域副溶血性弧菌的分布狀況及危害性評(píng)估[J];水生態(tài)學(xué)雜志;2008年05期

3 楊松;;副溶血性弧菌分離鑒定[J];中國(guó)畜牧獸醫(yī);2011年08期

4 顧冬花;;市售仔蝦中副溶血性弧菌的檢測(cè)[J];山東畜牧獸醫(yī);2012年04期

5 馬月姣;孫曉紅;趙勇;盧瑛;吳啟華;潘迎捷;;不同樣品副溶血性弧菌多樣性分析[J];江蘇農(nóng)業(yè)科學(xué);2012年12期

6 劉伯義,陳昌;首次從進(jìn)口魚粉中分離到副溶血性弧菌[J];動(dòng)物檢疫;1989年02期

7 王文,陳貴連;生物素-親和素斑點(diǎn)酶聯(lián)免疫吸附試驗(yàn)快速檢測(cè)魚源副溶血性弧菌的研究[J];獸醫(yī)大學(xué)學(xué)報(bào);1991年01期

8 王晉鏞;李波峰;葉軍;胡士元;何其茂;姜雪詠;胡薇紅;;副溶血性弧菌與宿主——中國(guó)對(duì)蝦的微生態(tài)學(xué)研究[J];中國(guó)微生態(tài)學(xué)雜志;1992年02期

9 趙月蘭,秦建華;副溶血性弧菌對(duì)動(dòng)物性食品的污染及檢測(cè)[J];湖北畜牧獸醫(yī);1994年03期

10 許如蘇;蔡穎;林彩華;楊奇志;陳冠武;;實(shí)時(shí)熒光PCR快速檢測(cè)水產(chǎn)品中副溶血性弧菌的研究[J];中國(guó)動(dòng)物檢疫;2007年12期

相關(guān)會(huì)議論文 前10條

1 張凡非;杉山寬治;西尾智裕;鄉(xiāng)田淑明;秋山真人;;檢測(cè)環(huán)境樣品及食品中病原性副溶血性弧菌的研究[A];新世紀(jì)預(yù)防醫(yī)學(xué)面臨的挑戰(zhàn)——中華預(yù)防醫(yī)學(xué)會(huì)首屆學(xué)術(shù)年會(huì)論文摘要集[C];2002年

2 廖玉學(xué);謝旭;扈慶華;;2012年深圳市副溶血性弧菌感染危險(xiǎn)因素病例對(duì)照研究[A];2012深圳市預(yù)防醫(yī)學(xué)會(huì)學(xué)術(shù)研討會(huì)論文匯編[C];2012年

3 劉佛民;劉禮平;譚海玲;馬聰;陳文勝;羅建波;;食品中副溶血性弧菌檢測(cè)能力驗(yàn)證研究[A];2010廣東省預(yù)防醫(yī)學(xué)會(huì)學(xué)術(shù)年會(huì)資料匯編[C];2010年

4 馮立芳;孫偉杰;何珊珊;勵(lì)建榮;;基于基因組水平發(fā)掘副溶血性弧菌的物種特異性檢測(cè)靶點(diǎn)[A];中國(guó)食品科學(xué)技術(shù)學(xué)會(huì)第八屆年會(huì)暨第六屆東西方食品業(yè)高層論壇論文摘要集[C];2011年

5 程蘇云;張俊彥;王贊信;童貴忠;石亞素;;海水產(chǎn)品副溶血性弧菌污染定量檢測(cè)分析[A];應(yīng)對(duì)突發(fā)公共衛(wèi)生事件論壇論文集[C];2005年

6 李純宗;王秋娥;連珠春;薛月華;;一起副溶血性弧菌引起的食物中毒[A];華東地區(qū)第二次急診醫(yī)學(xué)學(xué)術(shù)研討會(huì)論文摘要匯編[C];1993年

7 張淑紅;吳清平;張菊梅;;副溶血性弧菌特異性顯色生化快速檢測(cè)方法的研究[A];2006中國(guó)微生物學(xué)會(huì)第九次全國(guó)會(huì)員代表大會(huì)暨學(xué)術(shù)年會(huì)論文摘要集[C];2006年

8 方平楚;;副溶血性弧菌研究進(jìn)展[A];2007年浙江省醫(yī)學(xué)病毒學(xué)、醫(yī)學(xué)微生物與免疫學(xué)學(xué)術(shù)年會(huì)論文匯編[C];2007年

9 周桂蓮;王淑真;楊寶蘭;;鑒別副溶血性弧菌和溶藻弧菌致病與非致病性的動(dòng)物試驗(yàn)方法[A];北京食品學(xué)會(huì)1982年年會(huì)論文（摘要）[C];1982年

10 馬聰;;廣東地區(qū)副溶血性弧菌暴發(fā)分離優(yōu)勢(shì)血清型菌株的分子特征[A];新發(fā)傳染病研究熱點(diǎn)研討會(huì)論文集[C];2012年

相關(guān)重要報(bào)紙文章 前10條

1 衣曉峰;副溶血性弧菌最愛夏秋作亂[N];中國(guó)醫(yī)藥報(bào);2007年

2 鄒雪芹邋逄春展;龍口局：進(jìn)口凍魚中檢出副溶血性弧菌[N];中國(guó)國(guó)門時(shí)報(bào);2008年

3 通訊員 謝林 記者 江麓;生吃金槍魚會(huì)中毒[N];醫(yī)藥導(dǎo)報(bào);2007年

4 山東省萊州市慢性病防治院 郭旭光;雕花碟菜最不衛(wèi)生[N];健康時(shí)報(bào);2009年

5 全國(guó)12320管理中心副主任 李蓉;防中毒 吃海鮮時(shí)多用醋[N];健康報(bào);2010年

6 中國(guó)消費(fèi)者報(bào) 顧艷偉 李青山;海水魚查出致病性副溶血性弧菌[N];中國(guó)消費(fèi)者報(bào);2005年

7 本報(bào)記者 肖玉保 實(shí)習(xí)生 吳鐸思;貝類食品成為餐桌隱憂[N];工人日?qǐng)?bào);2004年

8 張博;科技尖兵[N];中國(guó)國(guó)門時(shí)報(bào);2011年

9 本報(bào)記者 王峰;生吃海鮮防中毒[N];中國(guó)消費(fèi)者報(bào);2003年

10 中國(guó)消費(fèi)者報(bào) 孫燕明;副溶血性弧菌導(dǎo)致79名老人群體性食物中毒[N];中國(guó)消費(fèi)者報(bào);2005年

相關(guān)博士學(xué)位論文 前9條

1 馮博;引起對(duì)蝦早期死亡綜合征的副溶血性弧菌的分離鑒定及發(fā)病機(jī)理研究[D];上海海洋大學(xué);2017年

2 劉雪飛;副溶血性弧菌拮抗菌的分離及其抑菌機(jī)理研究[D];沈陽(yáng)農(nóng)業(yè)大學(xué);2016年

3 韓海紅;生食貝類中副溶血性弧菌污染水平調(diào)查、定量風(fēng)險(xiǎn)評(píng)估和分離菌株特征分析[D];中國(guó)疾病預(yù)防控制中心;2015年

4 Shimaa Samir El-Malah;副溶血性弧菌及其毒力相關(guān)因子誘導(dǎo)細(xì)胞毒性的分子與細(xì)胞機(jī)理研究[D];揚(yáng)州大學(xué);2015年

5 馬月姣;酸耐受副溶血性弧菌生物學(xué)特性及轉(zhuǎn)錄組、蛋白組分析[D];上海海洋大學(xué);2016年

6 唐曉陽(yáng);水產(chǎn)品中副溶血性弧菌風(fēng)險(xiǎn)評(píng)估基礎(chǔ)研究[D];上海海洋大學(xué);2013年

7 姬華;對(duì)蝦中食源性弧菌預(yù)測(cè)模型建立及風(fēng)險(xiǎn)評(píng)估[D];江南大學(xué);2012年

8 巢國(guó)祥;副溶血性弧菌傳播特征、O3：K6流行克隆分子生物學(xué)特性及多位點(diǎn)序列種群遺傳研究[D];揚(yáng)州大學(xué);2010年

9 王麗;副溶血性弧菌中密度感應(yīng)系統(tǒng)依賴的T3SS1和T6SS2調(diào)控機(jī)制研究[D];重慶醫(yī)科大學(xué);2014年

相關(guān)碩士學(xué)位論文 前10條

1 盧曉鳳;蛤肉中副溶血性弧菌風(fēng)險(xiǎn)評(píng)估體系初探[D];山東農(nóng)業(yè)大學(xué);2007年

2 隋R，

本文編號(hào)：1838105