2013-2016年沿海地区虾源副溶血弧菌的特性分析及致病性研究
发布时间：2019-05-27 06:42
【摘要】：第一部分:副溶血弧菌(Vibrio parahaemolyticus)是最早被发现的对虾病原弧菌,也是危害最严重的病原之一。研究证明V.parahaemolyticus是近年来全球性暴发疾病对虾急性肝胰腺坏死病(Acute Hepatopancreatic Necrosis Disease,AHPND)的主要病原。本研究对实验室2013-2016年分离自我国沿海地区发病养殖场对虾体内的细菌进行分析,鉴定出V.parahaemolyticus 151株,按地域分类,分离自河北省34株,天津市13株,山东省21株,江苏省7株,浙江省29株,广东省18株,广西省24株,海南省5株;按感染宿主分类,分离自中国明对虾(Fenneropenaeus chinensis)3株,日本囊对虾(Marsupenaeus japonicus)7株,凡纳滨对虾(Litopenaeus vannamei)141株。对其进行AHPND的相关毒力蛋白基因pir AVP和pir BVP检测,阳性检出率为33.11%。血清分型研究结果表明,151株虾源V.parahaemolyticus中,有149株可以鉴定出O抗原血清型,其鉴定率为98.68%,其中以O1,O8,O3,O2为主;有62株可以鉴定出K抗原血清型,其鉴定率仅为41.06%,且型别较为分散,除K25群较多(22.58%)外无明显趋势;有60株可以同时被O和K两种抗原血清型分型,完全分型率为39.74%,形成24个O:K血清组合,出现最多的是O1:K25,占23.33%(14/60),其余型别较为分散,无明显优势组合,且没有出现O3:K6人类“大流行菌群”菌株。药物敏感性分析结果显示:151株虾源V.parahaemolyticus对哌拉西林、庆大霉素、环丙沙星、阿莫西林/克拉维酸、哌拉西林/他唑巴坦以及奈替米星这6种药物完全敏感,对其余14种药物表现出了不同程度的耐药性,其中对替卡西林、头孢噻吩具有最广泛的耐药性,其耐药率分别为53.64%和39.74%,对阿莫西林、头孢呋辛和复方新诺明的耐药也较为严重。多重耐药现象十分突出,有84株(55.63%)V.parahaemolyticus对2种以上药物表现出耐药性,34株(22.52%)对3种以上药物表现出耐药性,10株(6.62%)对4种以上的药物存在耐药性,2株同时对8种药物存在耐药性。此外,V.parahaemolyticus耐药性还存在明显的地区差异。第二部分:AHPND是当前严重危害对虾产业的流行疫病,自2010年首次报道以来,已对全球对虾养殖造成了极大的损失。已报道凡纳滨对虾和斑节对虾是AHPND的易感品种,中国明对虾对该病的易感性尚不清楚。菌株20140723005分离自疑似患AHPND的中国明对虾体内,通过急性浸泡感染实验研究该菌对中国明对虾的致病性。结果发现,感染后3 h对虾不再摄食,6 h肠胃变空,9 h肝胰腺呈浅白色。感染后10 h感染组中国明对虾开始出现死亡,12 h大量死亡,18 h内累积死亡率达100%。组织病理研究发现,感染组对虾肝胰腺小管变薄、崩塌,上皮细胞严重脱落,呈现典型的AHPND病理特征。经16S r RNA和分子伴侣蛋白gro EL基因序列分析,结合细菌生理生化特征以及致病基因的PCR检测,鉴定该菌为V.parahaemolyticus,且携带AHPND的相关毒力蛋白基因pir AVP和pir BVP,但不携带耐热直接溶血毒素(Thenmostable Direct Hemolysin,tdh)和相对耐热直接溶血毒素(TDH-Related Hemolysin,trh)毒力基因。该分离菌的血清型被鉴定为O3:K31。药敏试验显示,该菌对头孢噻吩、头孢呋辛表现为耐药,对阿莫西林、庆大霉素、环丙沙星等19种药物敏感。本研究证实中国明对虾也可以被副溶血弧菌感染而罹患AHPND,是该病的易感品种。第三部分:2016年3月山东潍坊某凡纳滨对虾养殖场下塘38天虾苗(2~4 cm)出现大规模急性死亡。发病虾表现为空肠空胃,肝胰腺颜色变浅或发白,伴有肌肉轻微浑浊等症状,与AHPND临床特征相似。本研究从患病的凡纳滨对虾肝胰腺中分离得到一株优势菌,编号为20160303005-1。人工感染实验表明,该菌对凡纳滨对虾具有较强的致病性,浸泡感染的半数致死剂量(LD50)为7.96×103 CFU/ml。对虾急性感染后,6 h肝胰腺颜色变浅,肠胃变空;9 h肝胰腺呈浅白色,萎缩变小,死亡数过半;24 h感染组对虾全部死亡。组织病理学分析显示,感染组对虾的鳃、肝胰腺和肠出现不同程度的病理损伤,其中肝胰腺小管崩塌,上皮细胞严重脱落,呈现出典型的AHPND病理症状。通过16S r RNA和分子伴侣蛋白gro EL基因序列分析,并结合生理生化特征,将该细菌鉴定为V.parahaemolyticus,其血清型为O1:KUT(K untypeable)。该菌株携带可引起对虾AHPND的相关毒力蛋白基因pir AVP和pir BVP,但不携带tdh和trh基因。菌株药敏试验结果显示,该菌对庆大霉素、环丙沙星和头孢他啶等16种药物敏感,对阿莫西林、替卡西林和头孢噻吩等5种药物表现为耐药。第四部分:2015年11月广西北海某日本囊对虾养殖场下塘90余天的日本囊对虾(5~6 cm)出现大量不明原因死亡。从pir AVP和pir BVP基因PCR检测结果呈阳性的病虾肝胰腺组织中,分离得到一株优势菌20151116002-3。另外,从河北沧州某养殖场AHPND病原弧菌检测结果呈阳性的日本囊对虾样品中,分离到菌株20140722001-1。通过急性浸泡感染实验研究这两株菌对日本囊对虾的致病性,20151116002-3感染组9 h后对虾体色变浅,肝胰腺颜色变浅,开始出现死亡,30 h后对虾死亡数过半,72 h后对虾死亡率达到100%。而20140722001-1感染组30 h后对虾开始出现死亡,肝胰腺呈浅白色,明显萎缩,96 h后对虾死亡率达到100%。组织病理研究发现,20151116002-3感染对虾的肝胰腺小管崩塌,上皮细胞严重脱落,与AHPND病理特征一致。20140722001-1感染对虾的肝胰腺小管上皮细胞大部分已变性坏死,整体消溶,肝胰腺小管结构出现崩解,成为一个大空泡,没有体现出AHPND的典型特征。菌株20140722001-1能导致日本囊对虾患病致死,但是否为AHPND还有待进一步研究。经鉴定菌株20151116002-3和20140722001-1均为携带编码毒力蛋白pir AVP和pir BVP基因的V.parahaemolyticus,但不携带临床菌株毒力基因tdh和trh,其血清型分别为O1:KUT和O3:K31。药敏试验显示,菌株20151116002-3对替卡西林、头孢噻吩、头孢西丁3种药物表现为耐药,而菌株20140722001-1只对头孢噻吩表现为耐药,对阿莫西林、庆大霉素等20种药物敏感。
[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.
【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】：S945.4
本文编号：2485962
[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.
【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】：S945.4
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