Structural characterization of the trisaccharide chain from the skin mucus glycoprotein of the rainbow trout, Salmo gairdneri

Skin mucus glycoprotein (RGP) was extracted from the skin mucus of rainbow trout, Salmo gairdneri. Alkaline borohydride treatment of RGP yielded a major oligosaccharide alditol NeuAcα2 to6GalNAc-ol and several minor oligosaccharide alditols. A trisaccharide alditol (OS-I) was purified by Sephadex G-25 gel filtration and high-performance liquid chromatography (HPLC). The amount of OS-I was estimated to be 7% in RGP from the amount of GalNAc-ol. Structural determination of the OS-I was examined. Structure of OS-I was identified to be NeuAcα2 to6 (GalNAcα1 to3) GalNAc-ol by α-N-acetylgalactosaminidase digestion, methylation analysis, and ¹H-NMR spectroscopy. This revised version was published online in August 2006 with corrections to the Cover Date.     

Purification and characterization of two novel cysteine protease inhibitors, Eel-CPI-2 and Eel-CPI-3, in the skin mucus of the Japanese eel Anguilla japonica

We have discovered multiple acidic cysteine protease inhibitors, in addition to the known Eel-CPI-1, in the skin mucus extract of the Japanese eel Anguilla japonica by using the two-dimensional gel system of gelatin reverse zymography. Two of the acidic inhibitors, which we have named Eel-CPI-2 and Eel-CPI-3, were purified to homogeneity by anion exchange chromatography on a column of DEAE-Sepharose CL-6B, followed by fast protein liquid chromatography on Superdex 75 10/300 GL and HiTrap Q HP columns. The amino acid compositions of Eel-CPI-2 and Eel-CPI-3 were found to be almost identical and closely similar to that of the eel galectin AJL1. The molecular masses of Eel-CPI-2 and Eel-CPI-3 were elucidated to be 16,089.080 and 16,089.137 Da, respectively, by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The apparent dissociation constant of Eel-CPI-2 and Eel-CPI-3 for cysteine protease papain was determined to be 1.79 × 10⁻⁷ and 1.05 × 10⁻⁷ M, respectively, by a quartz crystal microbalance technique.     

Changes in skin mucus of common carp,Cyprinus carpio L., after exposure to water with a high bacterial load

Water in aquaculture systems may contain a high load of microorganisms. Reduction in overall bacterial tank water load improves fish health and growth parameters. In this study, the effect of an increase of overall bacterial load in tank water on carp skin mucus was assessed. Intracellular and released high molecular weight glycoproteins (HMGs) of carp skin mucus were analysed for changes using histological, histochemical and biochemical techniques. Increase of bacterial load did not induce obvious clinical responses in carp, but the skin of exposed carp responded quickly. The amount of skin mucus HMGs isolated increased as well as their total glycosylation. An increased goblet cell number was observed for all carbohydrate stainings, but most clearly for acidic glycoconjugates. A change in the terminal presence of some sugars was also seen. After the initial response of carp, an adaptation to the higher bacterial load in the water appeared to occur as mucins had a higher glycosylation. The changes observed suggest that these skin mucus adaptations are part of a primary defence mechanism of mucosal epithelia, even at a low pathogenic pressure.     

Competition for attachment of aquaculture candidate probiotic and pathogenic bacteria on fish intestinal mucus

Probiotics for aquaculture are generally only selected by their ability to produce antimicrobial metabolites; however, attachment to intestinal mucus is important in order to remain within the gut of its host. Five candidate probiotics (AP1-AP5), isolated from the clownfish, Amphiprion percula (Lacepéde), were examined for their ability to attach to fish intestinal mucus and compete with two pathogens, Aeromonas hydrophila and Vibrio alginolyticus. Two different radioactive isotopes were used to quantify competition between pathogens and probionts. Attachment of the pathogens was enhanced by the presence of the candidate probiotics. However, the addition of the candidate probiotics after the pathogens resulted in reduced pathogen attachment. Only AP5 caused lower attachment success of V. alginolyticus when added before the pathogen. When AP5 was added first, the average attachment change was 41% compared with 72% when added after V. alginolyticus, suggesting that the probiotic is displaced but that enhanced attachment of the pathogen does not occur. Conversely, when V. alginolyticus was added first, followed by AP5, attachment change was 37% while AP5 had 92% attachment change when added second. This implies that the pathogen was displaced by the candidate probiotic and therefore it appeared that, based on the ability of probiont AP5 to attach to mucus, the growth of the pathogen in the digestive tract might be suppressed by the candidate probiont's presence.     

黄鳝皮肤黏液抗菌肽的分离纯化及其部分特性研究

以黄鳝皮肤黏液为材料,通过Sephadex G-50凝胶层析和DEAE Sepharose Fast Flow离子交换层析,分离纯化了具有抗菌和镇痛活性的多肽MAS-Ⅳ-4,用Tricine-SDS-PAGE测得其分子量为10.4 kD,用显微熔点仪测得其熔点为208～211℃.用抑菌圈法测定其抑菌活性,结果表明,MAS-Ⅳ-4对嗜水气单胞菌、金黄色葡萄球菌、枯草芽孢杆菌有较强的抑菌效果.此外,每只注射0.75 ml以上的MAS-Ⅳ-4样品液能显著提高小白鼠的痛阈值.MAS-Ⅳ-4还具有微弱的溶血活性和丝氨酸蛋白酶抑制活性.     

间接ELISA法研究嗜水气单胞菌对鳗鲡表皮黏液的黏附特性

采用间接ELISA法研究菌浓度、孵育时间、温度、pH、阳离子及碳源等因子对嗜水气单胞菌(Aeromonas hydrophila)黏附鳗鲡(Anguilla anguilla)表皮黏液的影响。结果表明, 改良后的间接ELISA法的检测灵敏度约为9.92×10⁴ CFU, 细菌的黏附量随菌浓度的升高而逐渐增大并符合饱和黏附动力学方程: y=0.135ln(x)-0.936(R²= 0.986); 嗜水气单胞菌黏附鳗鲡表皮黏液的最佳条件为: 温度20~28℃, pH 6.2~6.6, NaCl、MgCl₂质量浓度分别为15~25 g/L和3 g/L, 孵育时间为150 min。碳源对嗜水气单胞菌的黏附作用有不同程度的影响, 葡萄糖和麦芽糖能显著提高嗜水气单胞菌的黏附量(P<0. 05), 果糖则显著降低嗜水气单胞菌的黏附量(P<0. 05)。以上结果说明, 嗜水气单胞菌对鳗鲡表皮黏液具有较强的黏附作用, 且其黏附作用具有可控性。

     

Purification and characterization of a 630 kDa bacterial killing metalloprotease (KilC) isolated from plaice Pleuronectes platessa (L.), epidermal mucus

Antibacterial chemicals in the mucus of fish such as lysozyme, lectins, peptides and proteases provide an efficient first line of defence against pathogens. This study shows that there are at least three antibacterial proteins in plaice skin mucus in addition to lysozyme. One of these proteins is responsible for approximately 74% of the antibacterial activity and is a 630 kDa protease complex designated KilC (bacterial killing metalloprotease C). Purified KilC kills the bacteria Staphylococcus aureus, Escherichia coli, Bacillus subtilis and Pseudomonas aeruginosa efficiently. The protease activity of KilC is dependent upon the divalent cation Mg(2+) and shows pH dual optima of 5.0 and 8.0. The enzyme has a temperature optimum of 25 degrees C and is made up of at least five different sized peptides. Studies with protease inhibitors show that the catalytic site of KilC may be cysteine- or serine protease-like. KilC may kill bacterial cells by acting directly upon the bacteria or by producing low molecular weight bioactive compounds such as peptides.     

Evaluation of antibacterial activity and innate immune components in skin mucus of Indian major carp,Cirrhinus mrigala

The present work has been undertaken to analyse the antibacterial activity and innate immune components in the skin mucus of Indian major carp, Cirrhinus mrigala. Skin mucus was extracted separately in triple‐distilled water (TDW), 3% acetic acid (3% AA) or 0.1% trifluoroacetic acid (1% TFA). All mucus extracts exhibited different spectrum of the antibacterial activity against different groups of pathogenic bacteria. Protein profiling by polyacrylamide gel electrophoresis revealed a series of protein bands in the TDW extract, four major protein bands in the AA extract and two protein bands in the TFA extract. Tandem mass spectrometry analysis of distinct protein bands identified potential innate immune factors – histone H2A, histone H3, histone H4, haemoglobin, cofilin and nucleoside diphosphate kinase in the TDW extract, and ubiquitin and histone H2B isoforms in acidic extracts of skin mucus of C. mrigala. The presence of these innate immune molecules suggests that skin mucus play an important role in the protection of the fish against microbial invasion.     

鳜浸泡嗜水气单胞菌全菌疫苗后皮肤黏液抗体的变化

应用间接ELISA方法,研究经嗜水气单胞菌(Aeromonas hydrophila)疫苗浸泡免疫后鳜(Siniperca chuatsi)皮肤黏液中抗体消长规律,以揭示鳜局部黏膜免疫在免疫保护中的作用及浸泡免疫的保护效果。结果表明,鳜皮肤黏液中抗体滴度在免疫后第7天达到峰值211,抗体从开始形成到消失持续21 d;添加佐剂(IMS1312、葡聚糖、莨菪碱、食盐)可以提高鳜皮肤黏液及血清中抗体滴度和相对免疫保护率,其中添加IMS1312组免疫保护率最高,达77.8%,免疫保护与抗体滴度成正相关(R2=0.79,P<0.05);以溶菌酶为代表的非特异性免疫在免疫后1周内即抗体未形成时,对鱼体起主要保护作用。此外,通过比较鳜血清与皮肤黏液中的抗体消长规律,发现血清中抗体滴度峰值出现时间较迟(第14天),抗体持续时间较长(42 d),初步推断鳜皮肤黏膜中可能存在相对独立于全身免疫系统的局部黏膜免疫系统。     

牡蛎及其酶解产物抗皮肤光老化的初步研究

文章探讨了香港牡蛎(Crassostrea hongkongensis)肉粉(Oyster meat,OM)及其酶解产物(Oyster hydrolysis,OH)对紫外线诱导的小鼠(Mus musculus)皮肤光老化的保护作用。紫外线(UVA+UVB)每天照射构建小鼠皮肤光老化模型并灌胃OM和OH,分为低、中、高三组(30、90、180 mg·kg^?1)给药,持续8周。结果表明,OM和OH可减轻小鼠皮肤皱纹,提高皮肤弹性,减少皮下血管的萎缩,防止血管通透性增加;皮肤HE染色结果表明,中高剂量的OM和OH可显著减缓表皮层过度角化增厚(P<0.05);Masson染色和醛品红染色结果表明,OM和OH可减少胶原纤维的卷曲和降解,避免弹性纤维的异常增生,恢复真皮细胞外基质网络的排列;与模型组相比,OM和OH可使皮肤中超氧化物歧化酶(SOD)和谷胱甘肽过氧化氢酶(GSH-Px)的活力显著提高,同时使丙二醛(MDA)和8-羟基脱氧鸟苷(8-OHDG)的浓度降低(P<0.05);OM和OH可抑制基质金属蛋白酶(MMP-3、MMP-9)的表达,显著降低真皮中羟脯胺酸(Hyp)的降解;此外,高剂量的OH可显著降低皮肤中炎症因子细胞白介素6(IL-6)和肿瘤坏死因子(TNF-α)的表达,并通过提高转化生长因子(TGF-β)来提高皮肤抗炎能力。     

牙鲆皮肤黏液免疫球蛋白的纯化及部分特性分析

采用饱和硫酸铵分步盐析结合Sephacryl S-300凝胶过滤层析及DEAE Sepharose离子层析法,对牙鲆皮肤黏液中的免疫球蛋白(Ig)进行了分离纯化,并通过SDS-PAGE及Western-blotting技术对纯化蛋白的部分特性进行了分析。结果表明,30%、50%饱和硫酸铵分步盐析可以去除牙鲆黏液中除Ig外的很多杂蛋白,得到粗提黏液Ig;再经Sephacryl S-300纯化,Ig纯度较高,其中含有72和26 ku的条带;DEAE Sepharose层析法可进一步纯化Sephacryl S-300柱层析的产物,所提取黏液Ig经SDS-PAGE检测,只含有72和26 ku两个条带,初步认为是牙鲆黏液Ig的重链和轻链。Western-blotting结果显示,抗牙鲆血清Ig单克隆抗体可与黏液Ig重链72 ku条带发生发应。     

Identification and characterization of outer membrane vesicles from the fish pathogen Vibrio ordalii

Vibriosis outbreaks due to Vibrio ordalii occur globally, but Chilean salmon aquaculture, in particular, has suffered significant monetary losses in the last 15 years. Little is known about the virulence mechanisms employed by V. ordalii. However, most Vibrio pathogens (e.g., Vibrio anguillarum, a very close taxonomic species) present outer membrane vesicles (OMVs) that are released extracellularly and implicated in the delivery of virulence factors to host cells. This study provides the first reported evidence of the fish pathogen V. ordalii producing and releasing OMVs under normal growth conditions. Analyses were conducted with the V. ordalii strain Vo-LM-18 and the type strain ATCC 33509^T. For comparative purposes, the reference strain V. anguillarum ATCC 43307 was employed. The average size for the three Vibrio strains was 0.215 ± 0.6 µm (via scanning electron microscopy) or between 0.19 and 1.8 µm (via dynamic light scattering), with each bacterium presenting a wide range. SDS-PAGE revealed similarities in OMV patterns, but neither total nor external proteins were identical. Comparing V. ordalii ATCC 33509^T and Vo-LM-18, bands were most evident in the total proteins, and the greatest degree of similarity in OMV profiles was between 37 and 50 kDa. The purified OMVs demonstrated haemolytic enzyme activity, which could play a role during V. ordalii infection. These data represent an initial step towards gaining new insights into this virulence factor, of which a lot is known in other pathogenic microorganisms.     

一株感染深水网箱养殖许氏平鲉的 病原菌分离与鉴定

2016年7月,山东省长岛县深水网箱养殖许氏平鲉(Sebastes schlegeli)暴发严重皮肤溃疡症。作者对病鱼进行病原菌分离。通过形态学观察、常规生理生化试验、gyrB和16S rDNA基因克隆测序等方法对分离菌株进行鉴定。在病灶溃疡处分离到一株绝对优势菌BZ01,该菌株在TSB固体培养基上呈半透明菌落,在TCBS选择性培养基上菌落呈绿色。透射电镜观察为短棒状,具有单根极生鞭毛。人工回接感染证明,该菌株对许氏平鲉具有较强的致病力,可以引起皮肤溃疡等症状,且与自然感染症状一致,其LD50为2.07×106 CFU/ml。通过gyrB和16S rDNA基因序列测定并构建系统发育树显示,菌株BZ01与弧菌属同源性最高,并在系统发育树中与轮虫弧菌(Vibrio rotiferianus)聚为一枝,结合形态及生理生化表型测定结果,将该菌株鉴定为轮虫弧菌(V. rotiferianus)。药敏试验结果显示,该菌株对四环素类、喹诺酮类、香豆素类、肽酰转移酶类高度敏感,而对大环内酯类、多肽类、磺胺类、β-内酰胺类、氨基糖苷类中度敏感或不敏感。     

一株感染深水网箱养殖许氏平鲉的病原菌分离与鉴定

2016年7月,山东省长岛县深水网箱养殖许氏平鲉(Sebastes schlegeli)暴发严重皮肤溃疡症。作者对病鱼进行病原菌分离。通过形态学观察、常规生理生化试验、gyrB和16S rDNA基因克隆测序等方法对分离菌株进行鉴定。在病灶溃疡处分离到一株绝对优势菌BZ01,该菌株在TSB固体培养基上呈半透明菌落,在TCBS选择性培养基上菌落呈绿色。透射电镜观察为短棒状,具有单根极生鞭毛。人工回接感染证明,该菌株对许氏平鲉具有较强的致病力,可以引起皮肤溃疡等症状,且与自然感染症状一致,其LD₅₀为2.07×10~6 CFU/ml。通过gyrB和16S rDNA基因序列测定并构建系统发育树显示,菌株BZ01与弧菌属同源性最高,并在系统发育树中与轮虫弧菌(Vibrio rotiferianus)聚为一枝,结合形态及生理生化表型测定结果,将该菌株鉴定为轮虫弧菌(V.rotiferianus)。药敏试验结果显示,该菌株对四环素类、喹诺酮类、香豆素类、肽酰转移酶类高度敏感,而对大环内酯类、多肽类、磺胺类、β-内酰胺类、氨基糖苷类中度敏感或不敏感。     

Chemotaxis towards, adhesion to, and growth in carp gut mucus of two Aeromonas hydrophila strains with different pathogenicity for common carp, Cyprinus carpio L

Characteristics that promote bacterial colonization of the intestinal mucosal surface were examined in two strains of the common fish pathogen Aeromonas hydrophila, with different pathogenicity. The characteristics examined were chemotactic activity towards mucus, bacterial adherence to mucus and growth in mucus. Intestinal gut mucus of healthy common carp was used. The results indicate that chemotaxis is not necessary for a bacterium to become pathogenic, but it may be a necessary parameter for a bacterium to be an obligate pathogen. Adhesion also seems to be a factor influencing pathogenicity. The results suggest that higher adhesion to mucus and subsequent growth is associated with differences in pathogenicity.     

2株海水鱼源潜在益生菌的分离、鉴定及特性分析

为筛选用于微生态制剂研发的海水鱼源益生菌,本研究对海捕野生许氏平鲉(Sebastes schlegelii)和大泷六线鱼(Hexagrammos otakii)的消化道内壁黏膜样品进行细菌分离纯化,获得80株可培养细菌。使用选择性培养基对菌株的产酶能力进行测定,选取海水鱼常见病原菌为指示菌测定分离菌株产物的抑菌活性,筛选出2株潜在益生菌TS2和TH8,并进行菌株的生理生化检测、16S rDNA序列分析、生长特性及其对宿主安全性的研究。结果显示,TS2产蛋白酶(protease)、淀粉酶(amylase)和脂肪酶(lipase),其无菌培养产物可显著抑制鳗弧菌(Vibrio anguillarum)、副溶血弧菌(Vibrio parahaemolyticus)、哈维氏弧菌(Vibrio harvey)和假交替单胞菌(Pseudoalteromonas nigrifaciens)的生长。TH8产蛋白酶和脂肪酶,其无菌培养产物可显著抑制鳗弧菌、溶藻弧菌(Vibrio alginolyticus)、副溶血弧菌、假交替单胞菌、嗜水气单胞菌(Aeromonas hydrophila)、金黄色葡萄球...