Antibacterial abilities of intestinal bacteria from larval and juvenile Japanese flounder against fish pathogens

The present study was undertaken to examine the antibacterial abilities of intestinal bacteria isolated from juveniles and larvae of Japanese flounder Paralichthys olivaceus . Newly hatched larvae of flounder were held in a 100 L plastic circular tank and fed rotifiers, Artemia nauplii and commercial feeds, depending on the developmental stage of the fish. Genera Aeromonas , Moraxella and Vibrio were predominantly isolated from the intestinal tracts of Japanese flounder at larval and juvenile stages, whereas Aeromonas , Bacillus , coryneforms, Moraxella , Pseudomonas and Vibrio were detected at high densities in live diets and artificial feeds. Antibacterial bacteria accounted for 1.7–24.3% of the intestinal isolates against Lactococcus garvieae , Pasteurella piscicida , Vibrio anguillarum and V. vulnificus . In particular, as much as 53.3% of Vibrio spp. other than Vibrio -swarmer isolated from 197-day-old juveniles inhibited the growth of P. piscicida . These results suggest that intestinal bacteria having antibacterial activity play a role in the prevention of infectious diseases.     

亚硝基胍诱变选育丁二酮高产菌株

以乳酸乳球菌乳酸亚种丁二酮变种为出发菌株，采用亚硝基胍进行诱变选育，得到高产丁二酮菌株，用邻苯二胺比色法检测突变株丁二酮，其产量达到0．72mg／L，比出发菌株产量0．056mg/L提高12．9倍，且遗传性质稳定。     

柔嫩艾美耳球虫TA4抗原基因cDNA在乳酸乳球菌中的克隆与表达

用PCR技术特异扩增了柔嫩史美耳球虫TA4抗原基因cDNA序列，克隆至质粒pMD18-T中，获得重组质粒pMD18-T-TA4，采用KpnⅠ／SacⅠ双酶切法及PCR确认正确后，将TA4基NcDNA目的片段亚克隆到乳酸乳球菌表达载体pMG36n中，电穿孔法转化乳酸乳球菌LM0230，获得重组质粒pMG36n-TA4，采用KpnⅠ／SacⅠ双酶切法及DNA测序证明cDNA序列完全正确。获得TA4乳酸乳球菌表达株，经SDS—PAGE电泳分析，结果表明表达产物与预期大小的TA4蛋白分子量一致。     

磷脂酰肌醇特异性磷脂酶C基因在乳酸乳球菌中的异源表达

根据乳酸乳球菌密码子的偏好性,在不改变编码蛋白的基础上,优化设计并合成枯草芽孢杆菌磷脂酰肌醇特异性磷脂酶C基因,将其与大肠埃希菌 乳酸乳球菌穿梭质粒pAMJ399连接,构建重组质粒pAMJ399 PIPLC,并电转化至乳酸乳球菌中进行诱导表达。SDS PAGE分析显示：重组蛋白以可溶性蛋白的形式分泌于胞外,分子质量约35 ku,与预期蛋白大小一致。重组蛋白在PI 李斯特氏菌显色平板上显现明显的乳白色晕圈,证明重组蛋白具有酶活性,磷脂酰肌醇特异性磷脂酶C（PI PLC）在重组乳酸乳球菌中成功获得表达。通过优化培养条件,以2%转接量,在含有1%红霉素抗性的GM17液体培养基中,于32 ℃静止培养24 h,测得培养基上清液中PI PLC的浓度为1.092 μg·mL-1。     

乳酸片球菌产谷氨酸脱羧酶的相关酶学性质研究

为研究乳酸片球菌谷氨酸脱羧酶（GAD）的性质,采用比色法测其酶活。结果表明：当底物L-MSG的浓度为100 mmol.L-1时,GAD的活力达到最大。GAD的浓度为3.16%,最适温度为37℃,最适pH为5.0,PLP浓度为0.1 mo.lL-1时,GAD酶活力最大。Mg2＋和Mn2＋使GAD活力提高10%左右,KCl...     

重组植物甜蛋白马宾灵的食品级乳酸乳球菌诱导表达系统的构建

为了将马宾灵(MabinlinⅡ)直接应用于功能性食品中,通过对MabinlinⅡ基因进行有目的的剪切重组,将重组MabinlinⅡ基因插入到食品级表达载体中,经电击转化和乳糖筛选获得重组的食品级乳酸乳球菌,构建了重组植物甜蛋白马宾灵(MBL-ABH)的食品级乳酸乳球菌诱导表达系统。结果表明,该食品级诱导表达系统经乳酸链球菌素(Nisin)诱导后所表达的目的蛋白的含量较低,Western-blot检测表明MBL-ABH成功的在食品级乳酸乳球菌中表达。本研究扩展了植物甜蛋白马宾灵在食品方面的应用,有望开发出能够避免添加食糖类物质的功能性食品。     

Use of different microbial probiotics in the diet of rohu,Labeo rohita fingerlings: effects on growth,nutrient digestibility and retention,digestive enzyme activities and intestinal microflora

Six iso‐nitrogenous (350 g protein kg^?1) and iso‐caloric (4100 kcal kg^?1) diets with or without probiotics supplementation namely T₁ (Basal feed (BF) without probiotics; control), T₂ (BF + Bacillus subtilis and Lactococcus lactis), T₃ (BF + L. lactis and Saccharomyces cerevisiae), T₄ (BF + B. subtilis and S. cerevisiae), T₅ (BF + B. subtilis, L. lactis and S. cerevisiae) and T₆ (BF + heat‐killed bacteria of B. subtilis, L. lactis and S. cerevisiae) were fed to Labeo rohita fingerlings (6.0 ± 0.06 g) for 60 days in triplicate tanks (30 fish per tank). In all probiotic‐supplemented diets, the probiotic concentration was maintained at 10¹¹ cfu kg^?1 feed. After 60 days of culture, the fish fed combination of three probiotics at equal proportion (T₅) had higher (P < 0.05) growth, protein efficiency ratio, nutrient retention and digestibility and lower (P > 0.05) feed conversion ratio over other treatment groups. Total heterotrophic bacterial population in intestine was drastically reduced on 15th and 30th days of sampling than the initial value (0 day of sampling) for T₃, T₄ and T₅ groups. Except T₆, the gut colonization of respective probiotics, which were supplemented through the diets, was also increased up to 30 days of culture of fish and thereafter remained constant.     

乳酸菌对Caco-2细胞分泌APRIL和IL-10的影响

【目的】通过检测促炎细胞因子APRIL和抗炎细胞因子IL-10的分泌水平来观察屎肠球菌和乳酸乳球菌对肠上皮细胞先天性免疫应答的调节作用。【方法】Caco-2细胞分别和PBS（CT组,阴性对照组）、Escherichia coli K88（EC组,阳性对照组）,Enterococcus faecium（EF组）或Lactococcus lactis（LL组）共孵育2 h,以及先分别和Enterococcus faecium或Lactococcus lactis共孵育1 h,再和Escherichia coli K88共孵育2 h（EF-EC组和LL-EC组）。试验结束时,用ELISA方法检测细胞培养上清中APRIL和IL-10的含量。【结果】结果表明,2株乳酸菌都促进正常状态下的肠上皮细胞分泌促炎细胞因子APRIL和抗炎细胞因子IL-10,抑制Escherichia coli K88对APRIL分泌的诱导作用,促进Escherichia coli K88感染的肠上皮细胞分泌IL-10。【结论】体外试验条件下,屎肠球菌和乳酸乳球菌能够诱导肠上皮细胞产生先天免疫应答,分泌APRIL和IL-10,抑制大肠杆菌K88引起的促炎反应,表现出抗炎作用。     

Development of a selective and differential medium for capsulated Lactococcus garvieae

A selective and differential medium termed ‘LG agar’ was developed for the isolation and presumptive identification of Lactococcus garvieae that results in black colonies with red halos. In this study, all 14 strains of L. garvieae and only 9 of the 148 strains representing 38 other species were able to grow on the LG agar. The nine viable strains on LG agar plates (including Enterococcus faecalis, Enterococcus faecium, Lactococcus lactis, Vibrio fluvialis, Vibrio furnissii, Vibrio mimicus and Vibrio salmonicida) were further differentiated from L. garvieae by various colours or colony features. Colonies isolated from the mixing culture and the infected giant sea perch using LG agar plates were all positively identified as L. garvieae by conventional tests and 16S rDNA sequencing. Furthermore, LG agar discriminated capsulated strains of L. garvieae, which were believed to be correlated with pathogens of fish and shellfish, from non‐capsulated ones by colony appearances. The specificity and differentiating ability of LG agar suggest that this medium displays considerable potential for primary isolation and presumptive identification of L. garvieae from pathological and environmental samples.     

Experimental Lactococcus garvieae infection in rainbow trout,Oncorhynchus mykiss,Walbaum 1792: a comparative histopathological and immunohistochemical study

The aim of this study was to induce Lactococcus garvieae infection in young and adult fish through different routes [intraperitoneal (IP) and immersion (IM)] and to investigate the pathogenesis and histopathological and immunohistochemical findings comparatively. For this purpose, a total of 180 rainbow trout (90 young, 20 ± 5 g and 90 adult, 80 ± 10 g) obtained from a commercial fish farm were used. The fish were divided into eight groups, four experimental groups (Young‐Adult IP groups and Young‐Adult IM groups, each contain 30 fish) and four control groups (Young‐Adult IP Control groups and Young‐Adult IM control groups, each contain 15 fishes). The experimental study was conducted using L. garvieae, and confirmatory identification was performed by PCR. The sequence result of the PCR amplicon of 16S rDNA from isolate L. garvieae LAC1 was determined and deposited in the GenBank database under accession number KC883976 . Fish in the IP groups were intraperitoneally administered an inoculate containing 10⁶ cfu mL^?1 bacteria 0.1 mL. In the IM groups, fish were kept in inoculated water containing 10⁸ cfu mL^?1 bacteria for 20 min. Mortality as well as clinical and pathological findings was recorded daily, and significant differences in macroscopic and microscopic results were observed between the IP and IM administration groups. All tissue samples were immunohistochemically stained by the avidin‐biotin‐peroxidase complex and immunofluorescence (IF) methods using polyclonal antibody to detect L. garvieae antigens. In immunoperoxidase staining in the IP groups, positive reactions to bacterial antigens were most commonly seen in the spleen, kidney, heart, liver, peritoneum and swim bladder. In the IM groups, bacterial antigens were most commonly found in the eye, gill, spleen and kidney. In the IF method, the distribution of antigens in tissue and organs was similar to the reactions with immunoperoxidase staining. Finally, in this experimental study, an important correlation was seen between the distribution of L. garvieae antigens and lesions developing in many organ and tissues.