中华绒螯蟹幼体副溶血性弧菌病的病原分离与药敏试验

受副溶血性弧菌感染的河蟹蚤状幼体的症状为：幼体游动无力，头胸甲发红，色素加深，背刺常折断，摄食减少，肠胃空；感染副溶血性弧菌大眼幼体的症状为：幼体游动无力，不能逆水游动、甚至平游，上下翻“筋头”，体表发红，有时附肢也红，色素加深，眼柄上色素细胞扩大呈树枝状。一、材料与实验方法１．病原菌分离及培养基直接从育苗场采得发病的中华绒螯蟹蚤状幼体，在验室内将中华绒螯蟹幼体用载玻片压碎，在显微镜下见大量的病原菌，用接种环挑取病原菌在培养基上划培养２４小时后再进一步进行分离、提纯病原菌，置于温箱（２５℃）中培…     

三株AHPND致病型副溶血性弧菌烈性噬菌体的分离鉴定及其生物学特性

为分离获得AHPND致病型副溶血性弧菌烈性噬菌体,并鉴定及研究其生物学特性,本研究以AHPND致病型副溶血性弧菌为宿主菌,采用双层平板法分别从中国福建及墨西哥海水样本中对其噬菌体进行分离;通过噬菌斑形态、限制性内切酶、全基因组测序构建系统发育树及透射电镜观察对所获噬菌体进行分类鉴定;同时对供试噬菌体裂解谱及其生物学特性进行测定,包括最佳感染复数(MOI)、pH稳定性、热稳定性、过氧乙酸稳定性及不同储存温度下的存活稳定性等。结果显示,本研究分离获得3株AHPND致病型副溶血性弧菌烈性噬菌体P46、P48及VP7,3株噬菌体的噬菌斑均呈透亮圆形,P46噬菌斑直径4～5 mm,P48与VP7噬菌斑直径5～6 mm。3株噬菌体核酸均为双链DNA,电镜下观察其头部均呈二十面立体结构,直径约60 nm,尾部宽约8 nm,长约18～20 nm,属于短尾噬菌体科。3株供试噬菌体对AHPND型副溶血性弧菌裂解率达91.5%,对非AHPND型副溶血性弧菌裂解率为92.3%,且无法识别除副溶血性弧菌外其他种属的细菌;噬菌体P46、P48及VP7感染AHPND致病型副溶血性弧菌的最佳MOI分别为0.001、0.1及0.001;P46与VP7最适pH为6～8,P48最适pH为6～9;3株噬菌体对60°C的温度耐受力较强,在4°C条件下存放50周后仍具有较高的存活率;对通用杀菌浓度的过氧乙酸具有一定耐受性。将噬菌体P46、P48及VP7保守蛋白序列在NCBI数据库中比对后发现,3株供试噬菌体与其他噬菌体同源性较低,因此,噬菌体P48与P46及VP7很可能为3株新发现的短尾科噬菌体,这是中国首次报道AHPND致病型副溶血性弧菌短尾科噬菌体。本研究丰富了AHPND致病型副溶血性弧菌噬菌体的菌种资源,并为其应用于噬菌体生物防治制剂的开发奠定了理论基础。     

应用电子鼻技术检测南美白对虾副溶血性弧菌试验

为了将电子鼻技术应用于水产品中副溶血性弧茵等食源性致病菌的快速无损检测,试验利用电子鼻对从南美白对虾(Litopenaeus vannamei)中分离的一株副溶血性弧菌(Vibrio parahaemolyticus)进行检测.试验采用具有18个金属氧化物传感器的电子鼻对副溶血性弧茵经纯培养所得的挥发性代谢产物进行检测,同时用空白3.5%氯化钠TSB培养液作为对照.电子鼻所得的数据用主成分分析(PCA)、判别因子分析(DFA)、单类成分判别分析(SMICA)等多元统计方法进行分析.结果显示,副溶血性弧菌经纯培养后产生了明显不同于空白培养液气味的挥发性代谢产物.电子鼻可以应用于纯培养微生物的检测.     

连云港海域副溶血性弧菌的分布状况及危害性评估

对连云港周边不同海域的海水及海产品中的副溶血性弧菌（Ｖｉｂｒｉｏｐａｒａｈａｅｍｏｌｙｔｉｃｕｓ,Ｖｐ）进行分离,对检出菌株进行溶血性及耐药性检测。菌株检出率７８％,溶血素阳性率６８％,文蛤浸养感染率及死亡率２０％ ～３０％;耐药性检测结果表明,其对ＧＮ、ＳＸＴ、ＴＥ、ＣＡＺ、ＣＴＸ、ＣＲＯ、ＣＩ、ＤＯ和ＮＯＲ较为敏感,耐药率低于1０％;对ＦＲ和ＡＭＰ的耐药率在６０％以上。连云港海域内及海产品中均存在副溶血性弧菌的污染,其中ＴＲＨ型毒素分布较多。对海水养殖行业及人体健康具有潜在危险性。     

蚤状幼体副溶血性弧菌病病原体的鉴定及防治

从患病河蟹蚤状幼体体内分离出的病原菌为副溶血性弧菌;人工回感染实验表明副溶血性弧菌是导致河蟹蚤状幼体发病的病原;其对氧哌嗪青霉素、呋哺西林高度敏感。副溶血性弧菌可以通过食物和水体感染河蟹蚤状幼体。加强消毒措施有利于对该病的预防。     

连云港海域副溶血性弧菌的分布状况及危害性评估

对连云港周边不同海域的海水及海产品中的副溶血性弧菌(Vibrio parahaemolyticus,Vp)进行分离,对检出菌株进行溶血性及耐药性检测.菌株检出率78%,溶血素阳性率68%,文蛤浸养感染率及死亡率20%～30%;耐药性检测结果表明,其对GN、SXT、TE、CAZ、CTX、CRO、CI、DO和NOR较为敏感,耐药率低于10%;对FR和AMP的耐药率在60%以上.连云港海域内及海产品中均存在副溶血性弧菌的污染,其中TRH型毒素分布较多.对海水养殖行业及人体健康具有潜在危险性.     

市售仔虾中副溶血性弧菌的检测

应用常规细菌分离法对西宁市某几个农贸市场采集的70份仔虾样品进行了副溶血性弧菌的检测,经细菌形态观察、嗜盐性试验、细胞色素氧化酶试验和生化特性测定,鉴定为副溶血性弧菌。结果检出阳性菌株4株,阳性率为5.7%(4/70),同时通过致病性试验证明4株分离菌对小白鼠有致死效应。     

连云港周边海域副溶血性弧菌的污染及耐药性研究

为了了解江苏连云港周边沿海不同区域海水及海产品中副溶血性弧菌的分布情况并研究它们的毒性及耐药性,对连云港周边沿海不同区域海水及海产品中的副溶血性弧菌进行分离,并对检出菌株进行溶血性及耐药性检测.结果显示,菌株的检出率为78%;溶血素阳性率68%;文蛤的浸养感染率和死亡率为20%～30%;副溶血性弧菌对GN,SXT,TE,CAZ,CTX,CRO,CI,DO和NOR较为敏感,耐药率低于10%;对FR和AMP的耐药率在60%以上.说明连云港周边沿海不同区域海水及海产品中均存在副溶血性弧菌的污染,TRH型毒素分布较多,对海水养殖行业及人体健康具有潜在的影响.     

副溶血性弧菌增菌培养基及培养条件的改进

对副溶血性弧菌增菌培养基及培养条件进行了改进和优化,建立一种更适用于副溶血弧菌增菌的方法。针对能够决定副溶血弧菌综合营养需求的2个最为关键的因素:混合蛋白胨、酵母浸膏以及两者交互作用,进行试验设计,结合统计学软件对结果进行直观分析和方差分析,验证最佳培养基和培养条件。直观分析表明,副溶血弧菌增殖过程中,混合蛋白胨是最主要因素,其次为酵母浸膏。方差分析表明,混合蛋白胨与酵母浸膏对副溶血弧菌生长的影响均显著。最佳培养基配方为:混合蛋白胨2%(胰蛋白胨1%、鱼蛋白胨1%)、酵母浸膏0.2%、NaCl 3.5%、pH8.6;最佳培养条件为:(36±1)℃+摇床120 r/min+2层牛皮纸封口。本优化培养基与培养条件用于副溶血弧菌增菌培养,在相同培养时间内,其增菌菌液浓度为GB/T4789.7-2008方法的(1.8±0.6)倍。     

副溶血弧菌养殖对虾分离株耐药性及耐药基因分析

文章采用琼脂稀释法检测从养殖患病对虾中分离的36株副溶血弧菌(Vibrio parahaemolyticus)对16种药物的耐药性,并用PCR法检测喹诺酮类耐药基因qnrA、qnrB、qnrS和qnrVC,酰胺醇类耐药基因cat、optrA、floR和cfr,四环霉素类耐药基因tetA、tetB和tetM,磺胺类耐药基因sul1、sul2和sul3,氨基糖苷类耐药基因strA、strB、aadA和aacA,利福霉素类耐药基因arr,β-内酰胺类耐药基因bla CARB和大环内酯类耐药基因erm的携带状况,分析其耐药表型和基因型之间的相关性。结果显示,36株副溶血弧菌对β-内酰胺类药物氨苄西林耐药率最高(88.9%),其次为磺胺类药物磺胺甲噁唑(66.7%),硫酸新霉素、庆大霉素、头孢曲松和美罗培南呈现100%敏感。多重耐药副溶血弧菌比例高达61.1%(22/36),其中1株对6类抗菌药耐药。喹诺酮类耐药基因qnrVC检出率最高达72.2%(26/36);其次为氨基糖苷类耐药基因srtB,检出率58.3%(21/36);大环内酯类、利福霉素类耐药基因均未检测到。耐药基因检出率与耐药表型没有表现出一一对应的关系,提示副溶血弧菌耐药的复杂性。     

养殖大菱鲆肠道中大菱鲆弧菌的分离鉴定及药敏试验

2014年11月,大连地区某大菱鲆养殖场发生病害并伴有死亡,主要症状为吻部下颌明显出血,腹腔积水,肝脏充血,肠道出血有白便。从患病大菱鲆肠道中分离出1株可在硫代硫酸盐—柠檬酸盐—胆盐—蔗糖琼脂培养基上生长的菌株HZ-C1,人工回接感染试验证明其对健康大菱鲆具有较强致病性。通过形态学观察、16SrDNA序列同源性分析及生理生化试验确定菌株HZ-C1为大菱鲆弧菌。药敏检测结果显示,大菱鲆弧菌HZ-C1对氯霉素、丁胺卡那及头孢曲松等11种抗生素敏感,但对青霉素、四环素及诺氟沙星等15种抗生素耐药。本研究为大连地区养殖大菱鲆细菌性病害防治提供了一定病原学依据。     

一株副溶血弧菌的分离和鉴定

从患病的凡纳滨对虾体内分离了一株致病菌。对该菌进行了常规的生理生化鉴定,确认该菌为副溶血弧菌;该菌在第6 h就进入对数早期,对数期一直持续到22 h;用该菌腹腔注射小白鼠,对小白鼠半数致死量LD50为7.6×106cfu/g。     

一株副溶血弧菌噬菌体的分离与鉴定

分离到2株副溶血弧菌噬菌体,筛选出其中具有强裂解性的1株.该噬菌体的噬菌斑中心清亮,边缘有晕环.12 h观察,噬菌斑直径约3 mm,效价为1011pfu/ml.该噬菌体的核酸为线型双链DNA,大小约4 kb.电镜观察显示,头部为正二十面体,直径约110 nm,有尾,尾长约270 nm,底部有3个刺突.按国际病毒分类委员会第五次报告的分类标准,该噬菌体具肌尾噬菌体科的特征,与其他副溶血弧菌噬菌体比较显示,该噬菌体可能是一种新型的副溶血弧菌噬菌体.     

乳酸菌胞外产物对副溶血弧菌抑制作用的研究

分离到对副溶血弧菌有抑制作用的3株乳酸菌,命名为A-1、A-2、A-3。对其胞外产物的抑制作用进行研究,结果表明:3株乳酸菌胞外产物对副溶血弧菌有较强抑制作用,抑菌环直径分别可达18.6mm、20.3 mm1、9.5 mm;抑菌物质对热处理不敏感:在60℃、80℃抑菌活性基本不变,在121℃处理20min条件下抑菌效果分别是原液的67%,83%,74%,蛋白酶K处理对其活性有较大影响,抑菌成分可能是细菌素。进一步研究了细菌素抑菌活性与乳酸菌培养时间的关系。根据形态特征和常规生理生化反应进行鉴定,A-1、A-2鉴定为嗜热链球菌,A-3鉴定为嗜酸乳杆菌。     

Occurrence of Vibrio cholerae and Vibrio parahaemolyticus among milkfish farms in Zanzibar

ABSTRACT

Fishing is among the main economic activities of the people of Zanzibar. Few fish dealers are transforming this sector into mariculture. Among the farmed fish is milkfish. Diseases are among the limiting factors in the development of the mariculture industry. Among other zoonotic diseases, vibriosis is caused by bacteria from the genus Vibrio. This study aimed to establish the occurrence of Vibrio cholerae and Vibrio parahaemolyticus among milkfish farms in Zanzibar. A total of 380 milkfish were sampled. Swabs were collected from gills, intestine, and kidney of each sampled milkfish. Preliminary identification of V. cholerae and V. parahaemolyticus was done by biochemical tests. PCR was run on 16S rRNA, outer membrane protein W, and collagenase genes to confirm Vibrio species, V. cholerae, and V. parahaemolyticus respectively. Almost one-third (32.1%) of all sampled milkfish were found to contain targeted Vibrio; 18% and 29.5% of the sampled milkfish were positive for V. cholerae and V. parahaemolyticus respectively.     

Effect of Sargassum wightii fucoidan on growth and disease resistance to Vibrio parahaemolyticus in Penaeus monodon post‐larvae

The polysaccharide – fucoidan was extracted from brown seaweed Sargassum wightii and its antibacterial activity was screened by agar well diffusion method. The maximum zone of inhibition observed was 15.66 mm in 20 mg mL^?1 concentration against Vibrio parahaemolyticus. The Minimum Inhibitory Concentration (MIC) of the fucoidan was 12 mg mL^?1 against V. parahaemolyticus. The fucoidan was then enriched with Artemia nauplii at four different concentrations such as 100, 200, 300 and 400 mg L^?1 for 12 h. The enriched Artemia nauplii were fed to Penaeus monodon post‐larvae for 20 days and the growth performance was assessed. The weight gain and SGR of the control group were 0.2432 g and 15.78%, respectively. But, in experimental groups fed with fucoidan enriched Artemia nauplii, the weight gain and SGR were increased and were respectively ranged from 0.2602 to 0.3161 g and from 16.11 to 17.05%. The P. monodon post‐larvae were challenged with V. parahaemolyticus for a period of 30 days showed a reduction in mortality percentage of experimental groups over the control group and it was ranged between 36.97 and 89.86%. During the challenge test, the V. parahaemolyticus load was also enumerated from the infected shrimp at every 10 day intervals. In the control group, the Vibrio load showed a linear increase in hepatopancreas and muscle tissues from 10th to 30th days of challenge test, whereas in the experimental groups, the Vibrio load established a declining trend with the advancement of challenged test.     

低氧胁迫对九孔鲍免疫防御因子的影响

为了解低氧胁迫条件下九孔鲍免疫防御因子的变化及机体抗病力变化, 将九孔鲍置于不同的溶解氧环境中, 观测相关免疫因子变化。结果显示, 在水温为(22.1?1.3) oC, 盐度为30.72?0.54, pH值为8.20?0.14的海水环境中, 溶解氧含量由(7.49?0.14) mg/L下调至(2.53?0.16) mg/L后120 h内, 平均体质量为(14.25±2.21) g的九孔鲍未见死亡, 而每只鲍注射5.0?10⁵ CFU 副溶血弧菌后120 h内实验鲍的累计死亡率高达91.11%?7.70%, 比对照组(11.11%?3.83%)累计死亡率高出80%, 血淋巴抑菌清除率下降至?(3 340.47%?298.57%), 显而易见, 低氧胁迫使得九孔鲍拮抗副溶血弧菌感染的抵抗力显著下降。低氧(2.53?0.16) mg/L胁迫下, 九孔鲍血淋巴细胞数量(THC)下降30.62%?4.87%、血淋巴细胞吞噬率下降62.77%?5.79%、呼吸爆发产生的超氧阴离子数量下降22.21%?5.89%。低氧胁迫下九孔鲍血淋巴细胞内MPO活性上升(9.63%?7.59%)~(22.90%?13.73%)、CAT活性提高(7.68%?6.83%)~(56.28%?13.96%), 反映出低氧胁迫下九孔鲍体内应激反应产生的物质氧化加剧, 血淋巴细胞内产生大量的活性氧自由基。溶解氧含量由(7.49?0.14) mg/L下调至(4.51?0.12) mg/L后九孔鲍已明显处于低氧胁迫和应激反应状态, 120 h内的累计死亡率达到37.78%?3.85%, 血淋巴抑菌率下降至883.56%?123.22%; THC、血淋巴细胞吞噬率、呼吸爆发产生的超氧阴离子O₂^- 数量等最大降幅分别达到12.51%?6.59%、21.90%?15.84%、12.93%?5.74%; MPO活性在原有水平的?(10.61%?4.20%)~(7.13%?6.45%)之间波动, CAT活性在?(5.17%?18.08%)~(16.26%?10.85%)之间变化。     

Characterization and pathogenicity studies of Vibrio parahaemolyticus isolated from diseased freshwater prawn, Macrobrachium rosenbergii (de Man)

Vibrio parahaemolyticus, an important pathogen of freshwater prawn, Macrobrachium rosenbergii causes high mortality. Four strains of V. parahaemolyticus were isolated from the gill, haepatopancreas, hemolymph and carapace of diseased prawn. The species were characterized based on biochemical and serological tests. On performing an antibiotic assay, these strains were found to be highly susceptible to cephalaxin, tetracycline and erythomycin. During the in vitro pathogenicity test, all the strains were found to be positive to the Congo red binding assay and to be haemolytic in nature, whereas an in vivo pathogenicity test revealed that 2 × 10⁹ CFUs mL^?1of bacteria induced disease symptoms such as black colouration on the carapace, red discolouration of the exoskeleton and loss of appendages within 6 days and 80% mortality. In histopathological studies, a prominent necrosis was seen in gill lamellae, and branchial arches were thickened at places due to hyperplasia and haemocytic infiltration. Hepatopancreatic tissue showed dilation of tubules, vacuolation of hepatocytes and marked necrosis in acinar cells. Development of an immunological technique for the detection and screening of V. parahaemolyticus infection and its treatment is highly important in freshwater prawn aquaculture.