Effect of in vitro exposure to Vibrio vulnificus on hydroelectrolytic transport and structural changes of sea bream (Sparus aurata L.) intestine

The everted gut sac technique has been used to investigate the effect of Vibrio vulnificus on water and electrolyte (Na⁺, K⁺, Cl⁻, HCO₃ ⁻) transport on the intestine of sea bream (Sparus aurata L.). Both the anterior and the posterior intestine were incubated in a medium containing 10⁸ V. vulnificus cells ml⁻¹ at 25°C for 2 h. The presence of V. vulnificus resulted in a significant reduction (P < 0.05) of water absorption in the anterior intestine, while sodium absorption in the anterior (P < 0.01) and posterior (P < 0.05) intestine was elevated. Chloride absorption was increased, but the changed was not significant, while potassium absorption decreased significantly (P < 0.05), but only in the posterior intestine. Incubation the sea bream intestine with V. vulnificus did not affect carbonate secretion in the anterior segment, whereas high secretion was stimulated in the posterior segment (P < 0.01). Histological evaluations demonstrated damage in the anterior intestine of sea bream that was characterized by the detachment of degenerative enterocytes, alterations in the microvilli, and the presence of a heterogenous cell population, indicating inflammation. Based on our results, we conclude that V. vulnificus caused cell damage to the intestine of sea bream and that the anterior intestine is more susceptible than the posterior part of the intestine. Several hypotheses are suggested to explain our observations, such as the presence of higher numbers of villosities in the anterior intestine than in the posterior one and/or the presence of endogenous bacteria in the posterior intestine which may have a protector role.     

霍乱弧菌封闭带毒素功能片段△G的原核表达及其免疫增强活性

用PCR扩增了MBP—ZOT质粒上的ZOT基因，该基因编码264～399位氨基酸残基C末端的15ku △G片段，并与经改造后的质粒P^BCX、连接。将重组后的质粒PMLAG在大肠杆菌BL21中表达，将表达的目的蛋白纯化，再将p^BCX-△G片段与传染性腔上囊炎病毒VP2包涵体蛋白通过滴鼻免疫途径对14日龄SPF鸡进行联合免疫试验，分别于免疫后第10、20d采集血清样品，进行ELISA试验，检测免疫后IBD抗体效价。动物免疫试验发现：融合蛋白P^BCX △G起到了明显免疫增强效果，与VP2重组蛋白联合免疫2次后，血清IBD的VP2 IgG抗体效价是单独用VP2免疫组的3倍，明显高于空白免疫组。     

鸡弧菌性肝炎的病理学研究

本文对产蛋母鸡发生鸡弧菌性肝炎后所表现出的临床症状及尸体剖检变化和病理组织学变化作了较为详细的观察与研究，为鸡弧菌性肝炎的进一步研究和临床诊断提供一定的理论依据     

Anethole inhibits growth of recently emerged multidrug resistant toxigenic Vibrio cholerae O1 El Tor variant strains in vitro

To search natural compounds having inhibitory effect on bacterial growth is important, particularly in view of growing multidrug resistant (MDR) strains of bacterial pathogens. Like other bacterial pathogens, MDR Vibrio cholerae, the causative agent of diarrheal disease cholera, is becoming a great concern. As an approach of searching new antimicrobial agents, here, we show that anethole, a well-studied natural component of sweet fennel and star anise seeds, could potentially inhibit the growth of MDR O1 El Tor biotype, the ongoing 7th cholera pandemic variant strains of toxigenic V. cholerae. The minimum inhibitory concentration (MIC) of anethole against diverse O1 El Tor biotype strains is evaluated as 200 µg/ml. Moreover, the effect of anethole is bactericidal and exerts rapid-killing action on V. cholerae cells. This study is the first report which demonstrates that anethole, purified from natural compound, is a potent inhibitor of growth of toxigenic V. cholerae. Our data suggest that anethole could be a potential antimicrobial drug candidate, particularly against MDR V. cholerae mediated infections.     

Isolation of a highly pathogenic Vibrio pelagius strain associated with mass mortalities of turbot,Scophthalmus maximus (L.), larvae

A bacterial strain, characterized as Vibrio pelagius (Hq 222), was isolated from a turbot, Scophthalmus maximus (L.), larvae mass mortality in a commercial fish farm in Spain. Turbot larvae, post-larvae (0.2 g) and juveniles (5 and 15 g) were experimentally infected. The bacterium appeared to be very virulent for larvae and post-larvae, LD50 being < 5 bacteria mL(-1) for larvae 1 week post-infection and 3.9 x 10(5) bacteria mL(-1) in post-larvae at day 12 post-infection. The bacterial strain was recovered in pure culture from the internal organs of infected fish. Histological lesions in post-larvae exhibited swelling and necrosis of gill secondary lamellae, sloughing of intestinal mucosa and necrosis of haematopoietic tissue in the kidney. Vibrio pelagius (Hq 222) was able to grow in sterile sea water when incubated at room temperature or at 15 degrees C. Vibrio pelagius (Hq 222) was more adherent to the turbot cell lines TV-1 and TF than Escherichia coli. In both cell lines, the number of adhered bacteria increased with incubation time.     

哈维氏弧菌Y91301菌株培养工艺的研究

用大黄鱼分离的哈维氏弧菌Y91301进行试验,研究接种浓度、培养瓶装液量、起始pH值、培养时间、培养温度和培养液配方等因素对菌株生长的影响,确定静置培养时最适条件为:接种母液浓度1×108～1×109cfu ml,接种量6%～8%(相对培养瓶装液量),装液量40～50ml 250ml,培养液初始pH值为6 0～6 5,培养温度28℃;使用Zobell2216E培养配方更适合大量培养和生产的需要;旋转培养能够获得更高的生物量。     

大菱鲆源鳗弧菌毒力基因的检测及序列分析

采用PCR方法,对分离于大菱鲆的30株致病性鳗弧菌进行了溶血素vah1基因、鞭毛蛋白flaA基因、金属蛋白酶empA基因及外膜蛋白ompU基因的检测,选取3个代表菌株的相应基因序列通过Blast与GenBank中公布的参考菌株进行核苷酸同源性比较分析。结果表明,所检测的30株鳗弧菌均携带vah1、flaA、empA、ompU基因,携带率为100%;所测代表菌株的vah1、flaA、empA基因同参考菌株间均具有较高的同源性（93.9%～99.9%）,而外膜蛋白ompU基因,3株代表菌间存在较大差异（86.0%～95.9%）,其中的S010610-1株与其他2株代表菌及参考菌株均存在较大差异,在进化树中单独聚为一支。3株代表菌的其他基因（vah1、flaA、empA）均具有较高的同源性（94.2%～99.5%）,但均存在一定的差异性。     

霍乱弧菌DPO-PCR特异性检测方法的建立与应用

采用一种新型的引物设计方法——双启动寡核苷酸引物（dual-priming oligonucleotide,DPO）,以霍乱弧菌mdh基因为靶序列,建立了霍乱弧菌DPO-PCR特异性检测方法,分析了DPO引物退火温度不敏感性、检测灵敏度及特异性,并对检测方法进行了初步应用。灵敏度结果显示,DPO-PCR方法对霍乱弧菌的最低检出限为1.07×10^2 CFU/mL。退火温度不敏感性试验中,与常规PCR引物相比,DPO引物在45～65℃退火温度均能够高效扩增出靶基因片段。特异性结果显示,DPO-PCR方法的特异性比常规PCR方法强,不产生任何非特异性扩增。利用建立的霍乱弧菌DPO-PCR检测方法对采集的550份样本进行检测,检出43份霍乱弧菌阳性样本,经行业标准法（SN/T2425-2010）复检,检测结果相同,表明所建立的DPO-PCR检测方法具有良好的实用性,为霍乱弧菌的快速准确检测提供了新途径。     

青虾“红体病”组织病理学观察

用连续切片的方法对患有红体病的青虾进行组织病理学研究，结果表明患病青虾鳃部、肌肉组织和肝胰腺部位病症比较明显，不同患病阶段组织变形差异显著。鳃片细胞在患病初期表现为胞质收缩，后期则细胞膨胀破裂。细胞内物质外溢，细胞边缘界限不完整；肌肉组织在后期出现肌纤维束之间间隙，且部分肌纤维有断裂的现象；中期肝小叶间隔出现空隙和腺细胞变形，在后期则表现为小叶溃烂。造成青虾因红体病死亡的原因，初期主要是因为鳃片的组织病变导致呼吸困难，进而造成青虾生理异常；后期主要是因为肝胰脏的溃烂造成消化功能全面丧失。     

乳山湾东流区细菌数量的分布及与环境因子关系的研究

根据1995年6～9月对乳山湾东流区的微生物调查资料，研究了乳山湾东流区海水及底泥中细菌和弧菌量的变化及其与环境因子的关系。结果表明，该海域海水中细菌和弧菌的数量变化与温度的变化相一致，6月中旬～8月中旬，细菌数量增长比较缓慢，8月中旬开始，细菌数量增长较快，到8月底，达到最高值，异养菌为5．71×106cell／mL，弧菌为2．1×104cell／mL。而后，随着温度的下降，水中的细菌量逐渐减少。滩涂底泥中，细菌和弧菌的数量均比海水中细菌高l～2个数量级。