【摘要】：第一部分:副溶血弧菌(Vibrio parahaemolyticus)是最早被發(fā)現(xiàn)的對蝦病原弧菌,也是危害最嚴(yán)重的病原之一。研究證明V.parahaemolyticus是近年來全球性暴發(fā)疾病對蝦急性肝胰腺壞死病(Acute Hepatopancreatic Necrosis Disease,AHPND)的主要病原。本研究對實驗室2013-2016年分離自我國沿海地區(qū)發(fā)病養(yǎng)殖場對蝦體內(nèi)的細(xì)菌進(jìn)行分析,鑒定出V.parahaemolyticus 151株,按地域分類,分離自河北省34株,天津市13株,山東省21株,江蘇省7株,浙江省29株,廣東省18株,廣西省24株,海南省5株;按感染宿主分類,分離自中國明對蝦(Fenneropenaeus chinensis)3株,日本囊對蝦(Marsupenaeus japonicus)7株,凡納濱對蝦(Litopenaeus vannamei)141株。對其進(jìn)行AHPND的相關(guān)毒力蛋白基因pir AVP和pir BVP檢測,陽性檢出率為33.11%。血清分型研究結(jié)果表明,151株蝦源V.parahaemolyticus中,有149株可以鑒定出O抗原血清型,其鑒定率為98.68%,其中以O(shè)1,O8,O3,O2為主;有62株可以鑒定出K抗原血清型,其鑒定率僅為41.06%,且型別較為分散,除K25群較多(22.58%)外無明顯趨勢;有60株可以同時被O和K兩種抗原血清型分型,完全分型率為39.74%,形成24個O:K血清組合,出現(xiàn)最多的是O1:K25,占23.33%(14/60),其余型別較為分散,無明顯優(yōu)勢組合,且沒有出現(xiàn)O3:K6人類“大流行菌群”菌株。藥物敏感性分析結(jié)果顯示:151株蝦源V.parahaemolyticus對哌拉西林、慶大霉素、環(huán)丙沙星、阿莫西林/克拉維酸、哌拉西林/他唑巴坦以及奈替米星這6種藥物完全敏感,對其余14種藥物表現(xiàn)出了不同程度的耐藥性,其中對替卡西林、頭孢噻吩具有最廣泛的耐藥性,其耐藥率分別為53.64%和39.74%,對阿莫西林、頭孢呋辛和復(fù)方新諾明的耐藥也較為嚴(yán)重。多重耐藥現(xiàn)象十分突出,有84株(55.63%)V.parahaemolyticus對2種以上藥物表現(xiàn)出耐藥性,34株(22.52%)對3種以上藥物表現(xiàn)出耐藥性,10株(6.62%)對4種以上的藥物存在耐藥性,2株同時對8種藥物存在耐藥性。此外,V.parahaemolyticus耐藥性還存在明顯的地區(qū)差異。第二部分:AHPND是當(dāng)前嚴(yán)重危害對蝦產(chǎn)業(yè)的流行疫病,自2010年首次報道以來,已對全球?qū)ξr養(yǎng)殖造成了極大的損失。已報道凡納濱對蝦和斑節(jié)對蝦是AHPND的易感品種,中國明對蝦對該病的易感性尚不清楚。菌株20140723005分離自疑似患AHPND的中國明對蝦體內(nèi),通過急性浸泡感染實驗研究該菌對中國明對蝦的致病性。結(jié)果發(fā)現(xiàn),感染后3 h對蝦不再攝食,6 h腸胃變空,9 h肝胰腺呈淺白色。感染后10 h感染組中國明對蝦開始出現(xiàn)死亡,12 h大量死亡,18 h內(nèi)累積死亡率達(dá)100%。組織病理研究發(fā)現(xiàn),感染組對蝦肝胰腺小管變薄、崩塌,上皮細(xì)胞嚴(yán)重脫落,呈現(xiàn)典型的AHPND病理特征。經(jīng)16S r RNA和分子伴侶蛋白gro EL基因序列分析,結(jié)合細(xì)菌生理生化特征以及致病基因的PCR檢測,鑒定該菌為V.parahaemolyticus,且攜帶AHPND的相關(guān)毒力蛋白基因pir AVP和pir BVP,但不攜帶耐熱直接溶血毒素(Thenmostable Direct Hemolysin,tdh)和相對耐熱直接溶血毒素(TDH-Related Hemolysin,trh)毒力基因。該分離菌的血清型被鑒定為O3:K31。藥敏試驗顯示,該菌對頭孢噻吩、頭孢呋辛表現(xiàn)為耐藥,對阿莫西林、慶大霉素、環(huán)丙沙星等19種藥物敏感。本研究證實中國明對蝦也可以被副溶血弧菌感染而罹患AHPND,是該病的易感品種。第三部分:2016年3月山東濰坊某凡納濱對蝦養(yǎng)殖場下塘38天蝦苗(2~4 cm)出現(xiàn)大規(guī)模急性死亡。發(fā)病蝦表現(xiàn)為空腸空胃,肝胰腺顏色變淺或發(fā)白,伴有肌肉輕微渾濁等癥狀,與AHPND臨床特征相似。本研究從患病的凡納濱對蝦肝胰腺中分離得到一株優(yōu)勢菌,編號為20160303005-1。人工感染實驗表明,該菌對凡納濱對蝦具有較強的致病性,浸泡感染的半數(shù)致死劑量(LD50)為7.96×103 CFU/ml。對蝦急性感染后,6 h肝胰腺顏色變淺,腸胃變空;9 h肝胰腺呈淺白色,萎縮變小,死亡數(shù)過半;24 h感染組對蝦全部死亡。組織病理學(xué)分析顯示,感染組對蝦的鰓、肝胰腺和腸出現(xiàn)不同程度的病理損傷,其中肝胰腺小管崩塌,上皮細(xì)胞嚴(yán)重脫落,呈現(xiàn)出典型的AHPND病理癥狀。通過16S r RNA和分子伴侶蛋白gro EL基因序列分析,并結(jié)合生理生化特征,將該細(xì)菌鑒定為V.parahaemolyticus,其血清型為O1:KUT(K untypeable)。該菌株攜帶可引起對蝦AHPND的相關(guān)毒力蛋白基因pir AVP和pir BVP,但不攜帶tdh和trh基因。菌株藥敏試驗結(jié)果顯示,該菌對慶大霉素、環(huán)丙沙星和頭孢他啶等16種藥物敏感,對阿莫西林、替卡西林和頭孢噻吩等5種藥物表現(xiàn)為耐藥。第四部分:2015年11月廣西北海某日本囊對蝦養(yǎng)殖場下塘90余天的日本囊對蝦(5~6 cm)出現(xiàn)大量不明原因死亡。從pir AVP和pir BVP基因PCR檢測結(jié)果呈陽性的病蝦肝胰腺組織中,分離得到一株優(yōu)勢菌20151116002-3。另外,從河北滄州某養(yǎng)殖場AHPND病原弧菌檢測結(jié)果呈陽性的日本囊對蝦樣品中,分離到菌株20140722001-1。通過急性浸泡感染實驗研究這兩株菌對日本囊對蝦的致病性,20151116002-3感染組9 h后對蝦體色變淺,肝胰腺顏色變淺,開始出現(xiàn)死亡,30 h后對蝦死亡數(shù)過半,72 h后對蝦死亡率達(dá)到100%。而20140722001-1感染組30 h后對蝦開始出現(xiàn)死亡,肝胰腺呈淺白色,明顯萎縮,96 h后對蝦死亡率達(dá)到100%。組織病理研究發(fā)現(xiàn),20151116002-3感染對蝦的肝胰腺小管崩塌,上皮細(xì)胞嚴(yán)重脫落,與AHPND病理特征一致。20140722001-1感染對蝦的肝胰腺小管上皮細(xì)胞大部分已變性壞死,整體消溶,肝胰腺小管結(jié)構(gòu)出現(xiàn)崩解,成為一個大空泡,沒有體現(xiàn)出AHPND的典型特征。菌株20140722001-1能導(dǎo)致日本囊對蝦患病致死,但是否為AHPND還有待進(jìn)一步研究。經(jīng)鑒定菌株20151116002-3和20140722001-1均為攜帶編碼毒力蛋白pir AVP和pir BVP基因的V.parahaemolyticus,但不攜帶臨床菌株毒力基因tdh和trh,其血清型分別為O1:KUT和O3:K31。藥敏試驗顯示,菌株20151116002-3對替卡西林、頭孢噻吩、頭孢西丁3種藥物表現(xiàn)為耐藥,而菌株20140722001-1只對頭孢噻吩表現(xiàn)為耐藥,對阿莫西林、慶大霉素等20種藥物敏感。
[Abstract]:The first part: Vibrio parahaemolyticus (Vibrio parahaemolyticus) is the first to be found, and is one of the most serious pathogens. The results show that V. parhaemolyticus is the main pathogen of the acute hepatic pancreatic necrosis (AHPND) in the global outbreak of the disease. In this study, the bacteria isolated from the shrimp in the coastal area of China from 2013 to 2016 were analyzed, and the V. parhaemolyticus 151 strain was identified. According to the geographical classification, it was isolated from 34 strains of Hebei province,13 in Tianjin,21 in Shandong,7 in Jiangsu,29 in Zhejiang and 18 in Guangdong province. Among the 24 strains of Guangxi,5 strains of Hainan,3 strains of Fenneopenaeus chinensis,7 of Marsupenaeus japonica and 141 of Litopenaeus vannamei were isolated according to the classification of the infected host. The positive rate of pir AVP and pir BVP of the related virulence protein gene of AHPND was 33.11%. The results of serum typing showed that 149 of the 151 shrimp sources, V. parhaemolyticus, could identify the O-antigen serotype, and the identification rate was 98.68%, with O1, O8, O3 and O2 as the main;62 strains could identify the K-antigen serotype, the identification rate was 41.06%, and the type was not more dispersed. There was no obvious trend in the other than the K25 group (22.58%);60 strains can be classified by both O and K types, the total typing rate is 39.74%, and 24 O: K serum combinations are formed, the most of which are O1: K25, 23.33% (14/60), the remaining type is more dispersed, and no obvious advantages are combined. And no O3: K6 human "major epidemic flora" strain appeared. The results of the drug sensitivity analysis showed that V. parhaemolyticus of 151 shrimp was completely sensitive to 6 kinds of drugs, such as penethamate, gentamycin, ciprofloxacin, amoxicillin/ clavulanic acid, penethamate/ other sulbactam and netilmicin, and showed different drug resistance to the remaining 14 kinds of drugs. The drug-resistant rate was 53.64% and 39.74%, and the resistance to amoxicillin, ceftriaxine and compound neomycin was also serious. There were 84 strains (55.63%) V. paraxemoticus showed resistance to more than 2 drugs,34 (22.52%) had drug resistance to more than 3 drugs,10 (6.62%) had drug resistance to more than 4 drugs, and 2 strains were resistant to eight drugs at the same time. In addition, there are distinct regional differences in the resistance of V. parhaemolyticus. The second part: AHPND is the current epidemic disease which is seriously harmful to the prawn industry. Since the first report in 2010, it has caused great loss to the shrimp culture in the world. It has been reported that Litopenaeus vannamei and Penaeus monodon are susceptible to AHPNDs, and the susceptibility of prawns to the disease is not clear. Strain 20140723005 was isolated from Chinese penaeus chinensis suspected of being infected with AHPND, and the pathogenicity of the strain to the prawns in China was studied by acute soaking and infection experiment. The results showed that after infection, the penaeus vannamei was no longer fed, and the stomach and intestines were empty for 6 hours, and the pancreas in the 9 h was light white. In the 10-hour post-infection group, the death of the prawns began to occur and the cumulative mortality within 18 hours was 100%. The pathological study of the tissue found that the small, collapsed and epithelial cells of the hepatopancreatic tubules of the infected group showed typical AHPND pathological features. The bacterial physiological and biochemical characteristics and the PCR detection of the pathogenic gene were analyzed by 16S r RNA and molecular chaperone gro EL gene sequence, and the related virulence protein genes, pir AVP and pir BVP of the AHPND, were identified, but the heat-resistant direct hemolysin was not carried. Tdh) and a relative heat-resistant direct hemolysin (trh) virulence gene. The serotype of the isolated strain was identified as O3: K31. The drug sensitivity test shows that the bacteria are resistant to ceftriaxone and cefosinine, and are sensitive to 19 kinds of drugs such as amoxicillin, gentamicin, ciprofloxacin and the like. The study confirmed that the Chinese prawn could also be infected with Vibrio parahaemolyticus to develop the AHPND, which is the susceptible variety of the disease. The third part: In March,2016, a large-scale acute death occurred on the 38-day shrimp (2-4 cm) in the lower pond of the Litopenaeus vannamei farm in Shandong Province. The disease was manifested as the empty stomach of the jejunum, the color of the liver of the liver was light or whitish, accompanied by the slight turbidity of the muscle and the like, similar to the clinical characteristics of the AHPND. A dominant strain was isolated from the diseased liver of Litopenaeus vannamei. The number was 20160303005-1. The experiment of artificial infection showed that the bacterium has strong pathogenicity to the Litopenaeus vannamei, and the half lethal dose (LD50) of the soaking infection is 7.96-103 CFU/ ml. After acute infection of the prawns, the color of the pancreas of the liver in 6 hours was light and the intestines and stomach became empty; the pancreas in the liver of the 9 h was light white, the atrophy was small, the number of death was over half, and the 24 h infected group was all dead. Histopathological analysis showed that the liver, the pancreas and the intestine of the infected group had different degree of pathological damage, in which the pancreatic duct of the liver collapsed and the epithelial cells were severely dropped, presenting the typical pathological symptoms of AHPNDs. The bacteria were identified as V. parhaemolyticus and its serotype is O1: KUT by 16S r RNA and molecular chaperone gro EL gene sequence analysis and combined with the physiological and biochemical characteristics. The strain carries the related virulence protein gene pir AVP and pir BVP which can cause the prawn AHPND, but does not carry the tdh and trh genes. The results of drug sensitivity test showed that the bacteria were sensitive to 16 kinds of drugs such as gentamycin, ciprofloxacin and ceftriaxone, and were resistant to 5 kinds of drugs such as amoxicillin, tigecycline and ceftriaxone. The fourth part: There was a large number of unknown causes of death of the Penaeus japonicus (5-6 cm) over 90 days in the lower pond of a Japanese suppenaeus vannamei farm in the northwest of China in November 2015. A dominant bacterium,20151116002-3, was isolated from the tissue of the hepatopancreatic pancreas, which was positive by the PCR of the pir AVP and the pir BVP. In addition, the strain 20140722001-1 was isolated from the sample of the Japanese suppenaeus japonicus, which was positive for the detection of the Vibrio parahaemolyticus from a farm in Cangzhou, Hebei. The pathogenicity of the two strains to the Penaeus japonicus was studied by the experiment of acute soaking and infection. After 9 h of the 20151116002-3 infection group, the body color of the prawn was shallow, the color of the liver of the liver became light, the death was started, and the death rate of the prawns after 30 h was half, and the mortality of the prawns was 100% after 72 h. However, after 30 h of 20140722001-1 infection group, the death of the prawns began to occur, the pancreas of the liver was light white, and the mortality of the prawns was 100% after 96h. The pathological study of the tissue found that the pancreatic duct of the hepatopancreatic of the 20151116002-3 infected prawn is collapsed and the epithelial cells are seriously dropped, and the pathological characteristics of the AHPNDs are consistent with that of the AHPND. A typical feature of the AHPND is not shown. The strain 20140722001-1 can cause the disease to be fatal, but whether the AHPND has to be further studied. The identified strain,20151116002-3 and 20140722001-1, are the V. parhaemolyticus, which carries the pir AVP and the pir BVP gene, but does not carry the virulence genes tdh and trh of the clinical strains, and the serotypes are O1: KUT and O3: K31, respectively. The drug sensitivity test showed that the strain (20151116002-3) is resistant to the three drugs of tigecycline, ceftriaxone and cefoxitin, while the strain 20140722001-1 is only resistant to the ceftriaxone, and is sensitive to 20 drugs such as amoxicillin and gentamicin.
【學(xué)位授予單位】：上海海洋大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S945.4

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