再谈抗菌肽及其在水产养殖上的应用(中)

正3.抗菌肽的屏障功能及机制对于水产动物来说,非特异性免疫的重要性远高于特异性免疫,水产动物的屏障作用是保护水产动物生命安全的第一道防线。近年来研究表明,很多水产动物的上皮细胞均可表达抗菌肽,它可以通过多种信号途径参与机体免疫调节,提高角质形成细胞的功能,从而提高机体对病原微生物等致病生物的抵抗能力,抗菌肽在动物黏膜和皮肤防御方面起重要作用。其作用机制在于抗菌肽可以调控紧     

抗菌肽在水产动物中的应用及前景

抗菌肽是存在于生物体内的,具有抗菌活性的碱性多肽,是机体免疫系统的重要组成成分。抗菌肽具有广泛的抑制性,在农业、医学等领域有良好的应用前景。本文综合近年来的研究,概述抗菌肽对水产动物生长性能及免疫能力的影响,展望抗菌肽的应用前景,分析抗菌肽在水产动物应用上存在的问题,为抗菌肽在水产动物中的应用提供技术支撑,也为抗菌肽的深入研究和合理应用提供理论基础。     

水产动物基因工程抗菌肽研究及其应用前景的研究

抗菌肽是水产动物体内非特异性免疫系统的第一道防线,在抵御外来病原微生物的过程中发挥着重要作用.来源于鱼、虾、蟹等水产动物的抗菌肽杀菌作用更强,溶血效应小,种类多样化,显示出比其它种类更具优势的应用前景.介绍了一些水产动物抗菌肽的研究近况、基因工程研究进展以及转抗菌肽基因在水产动物中的研究进展,并展望了其在水产领域的应用前景.     

抗菌肽的研究进展

抗菌肽是广泛存在于自然界生物中的具有广谱抗菌、抗病毒、抑制杀伤肿瘤细胞等作用的多肽。本文介绍了抗菌肽的结构,抗菌肽的生物学活性,抗菌肽的作用机理和作用机制,以及抗菌肽的应用和前景。     

抗菌肽及其在水生动物中的研究现状

面对滥用抗生素所带来的诸多负面影响,抗菌肽以其独特的抗菌机制、广谱、高效的抗菌活性以及靶菌株难以产生抗性等优点而有望成为抗生素的替代品。水产动物抗菌肽是其自身先天免疫的重要组成部分,对抗菌肽的深入研究有望通过充分调动开发动物自身的防御潜力达到抗病的目的,是实现养殖业可持续发展的重要方向。综述了水生动物抗菌肽的类型、结构及抗菌机制,分析了抗菌肽研究中存在问题,并展望其应用前景。     

水产动物抗菌肽的研究进展

抗菌肽是一种具有抗菌活性的生物短肽，广泛存在于生物界中。综述了抗菌肽的几种类型及作用机理，介绍了水产动物抗菌肽的研究状况及其在水产养殖上的应用前景。     

中国对虾中抗菌短肽的分离纯化与功能分析

以水产病害的病原菌为目标菌，从中国对虾（Penaeuschinesis）体液中分离筛选抗菌肽，通过Sephadex G-50、RP-HPLC等分离纯化技术，得到一种抑制水产病原菌的抗菌肽PC-V-2。经初步鉴定，该抗菌肽具有广谱抗性，不仅抗鲁克氏耶尔森氏菌（Yersinia ruckeri），而且对金黄色葡萄球菌（Staphyloccus auleus）、枯草杆菌（Bacillus subttilis）等革兰氏阳性菌，大肠杆菌（Escherichia coli）等革兰氏阴性菌和白色念珠菌（Candida albicans）等真菌也有较强的抗性。另外，该抗菌肽还有一定的抑制凝血的活性，但没有检测到溶血和丝氨酸蛋白酶抑制活性。经MALDI-TOF质谱分析，其分子量为1848D，但其序列及其他生物特性还有待进一步研究。     

传统抗生素替代物抗菌肽在水产养殖中的应用前景

抗菌肽(antibacterial peptides)是具有抗菌活性基因编码的一类短肽,目前发现抗菌肽或类似抗菌肽的小分子肽类广泛存在于昆虫、两栖类、水产动物及哺乳动物中,这种内源性抗菌肽经诱导而合成,在机体抵抗病原的入侵方面起着重要的作用,更被认为是缺乏特异性免疫功能生物的重要防御成分.该文系统介绍了抗菌肽的定义、结构、作用机理、与传统抗生素的差异以及在水产养殖中的应用前景,以期为抗菌肽进一步深入研究提供参考.     

厚壳贻贝抗菌肽Mytilin的初步鉴定

厚壳贻贝是我国具有重大经济价值的水产养殖贝类,对其抗菌肽的研究有助于人们了解厚壳贻贝的免疫机制。为了解厚壳贻贝血清中抗菌肽Mytilin的分子组成和特性,采用多维高效液相色谱对厚壳贻贝血清进行分离纯化,从中获得3种对革兰氏阳性菌以及阴性菌均有抑制作用的抗菌肽分子,序列分析表明3种抗菌肽具有较高的序列相似性,均属于贻贝抗菌肽Mytilin家族,分别命名为Mytilin-1,Mytilin-2和Mytilin-3。其中,Mytilin-1为34个氨基酸残基构成的多肽,其分子量为3885.17u,含8个半胱氨酸,形成4对二硫键。根据所测Mytilin-1的氨基酸序列设计特异性引物,通过菌落PCR方法筛选厚壳贻贝cDNA文库,获得Mytilin-1的cDNA基因并进行了序列分析。以上研究结果表明,作为贻贝的主要抗菌肽家族,Mytilin在厚壳贻贝血清中具有较高丰度,对其蛋白质序列以及基因序列的研究为深入了解厚壳贻贝Mytilin抗菌肽的分子多样性奠定了基础。     

海洋鱼类和甲壳类抗菌肽的研究进展与应用前景

抗菌肽的发现与研究已有几十年的历史,抗菌肽是一类生物体中普遍存在并具有广谱抗微生物活性的小分子多肽,被科学家誉为"天然抗生素"。海洋动物抗菌肽是鱼、虾、蟹、贝等海洋动物的非特异性免疫防御系统的重要组成部分,在海洋环境中对于防御外源病原菌的入侵起到重要作用。海洋抗菌肽的研发与应用,不仅具有重要的医学开发价值,也是减少抗生素污染、实现海水健康养殖的重要保障。本文主要以鱼类抗菌肽和甲壳类抗菌肽为例,从抗菌肽的来源与分类、结构特点以及生物学活性等方面,简要概述近年来海洋鱼类和甲壳类抗菌肽的一些研究进展及其开发利用前景。     

Rare serotype occurrence and PFGE genotypic diversity of Streptococcus agalactiae isolated from tilapia in China

Previously, we reported 10 PEGE types of 85 tilapia Streptococcus agalactiae(GBS), which shifted from Streptococcus iniae in China, by using PEGE method. Presently, larger and more representative tilapia GBS were isolated, for the ?rst time in China, to characterize their serotypes and genetic diversities more precisely than had done before. 168 GBS strains were distributed in ?ve provinces of China, in which Guangdong, Guangxi and Hainan were the major ones, holding36.9%(62/168), 37.5%(63/168) and 19.6%(33/168), respectively. Serotypes, Ia, Ib and III, were observed in these strains and the most predominant one was Ia(95.2%), which mainly distributed in Guangdong, Guangxi and Hainan. Ia initially occurred in 2009, it shoot up to 32.1% in 2010,but decreased to 16.1% in 2011 before went up to 45.2% in 2012. Ib sporadically occurred during2007–2011, III onlyoccurred in 2012. 14 different PFGE types, including 4 new types(N, O,P and Q), were observed, in which B, D, F and G were the predominant types, holding 83.9%(141/168) of the total GBS strains. Ia corresponded to 11 PFGE types(A–H, N–P), in which type D predominated(51%). Ib represented 3 genotypes(I, J and Q) and III harbored only 2genotypes(N and F). Type N and Fsynchronously presented in Ia and III. In summary, the genetic diversity of tilapia GBS varied by serotypes and changed with geographical locations and years.Although Iastillpredominated, new rareserotypeIII alreadyoccurred in China.     

Growth hormone (GH) and reproduction: a review

Interaction between growth and reproduction occurs in many vertebrates and is particularly obvious at certain stages of the life cycle in fish. Endocrine interactions between the gonadotropic axis and the somatotropic axis are described, the potential role of GH being emphasised. A comparative analysis of these phenomena in mammals, amphibians and fish, suggests a specific role of GH in the physiology of puberty, gametogenesis and fertility. It also shows the original contribution made by studies on the fish model in this field of investigations.     

Purification and partial characterization of GtHs (cfLH and cfFSH) from Indian walking catfish (Clarias batrachus) (L.) and development of a homologous ELISA for cfLH

Two gonadotropins (GtH; Qa and Qb) were purified by gel filtration and ion exchange chromatography from the pituitaries of Indian walking catfish (Clarias batrachus). The presence of GtH during purification was assessed by in vitro oocyte maturation and in vivo steroidogenic activity, and their identities were determined by elution profiles, molecular weight, biological activities and yield. The molecular weights of Qa and Qb were 37 and 42 kDa, respectively, and composed of distinct subunits (Qa: 20 and 14 kDa and Qb: 26 and 18 kDa). Polyclonal antibodies raised against Qa immunostained Qa, Qb and pituitary GtH cells. A competitive Qa‐ELISA was developed whose sensitivity was 6.25 ng mL^?1 (1.25 ng well^?1) with intra‐ (3.5%) and inter‐ (12.4%) assay coefficients of variation. Displacement curves parallel to the standard were obtained with plasma and pituitary extracts of catfish, Qb and carp GtHII. The assay was validated by measuring the plasma Qa levels after LHRH treatment and in relation to ovarian growth in the female catfish during different reproductive phases. Based on the results, Qa and Qb corresponded to fish LH and FSH respectively. The findings will increase the knowledge of the mechanisms controlling fish reproduction and identification of sensitive phases in fish in captivity for hormonal manipulation.     

Controlled infection of Poecilia reticulata Peters (guppy) with Tetrahymena by immersion and intraperitoneal injection

Tetrahymena is a protozoan parasite, which infects guppy, Poecilia reticulata Peters, and causes substantial economical losses in commercial farms worldwide. Studies of guppy infected by Tetrahymena require standardized infection protocols. The LD50 for Tetrahymena infection of guppies by intraperitoneal (IP) injection was calibrated, and the level obtained was 946 parasites per fish. Guppy infection with Tetrahymena by immersion, imitating the natural route of infection via the integument, was studied under normal or stress conditions. Exposure to cold and netting (CNI) and to cold only (CI) followed by immersion exposure to 10 000 Tetrahymena per mL resulted in 22.5% and 19.2% mortality, respectively, as compared to 14.2% and 10% in groups that were netted only (NI) or non‐stressed (I). Histopathology revealed that immersion infection resulted in a systemic infection. Lysozyme levels, measured 3 weeks after infection, were significantly higher in the CNI group (288 μg per mg protein) compared with CI‐, NI‐ and I‐treated groups (94.5, 64 and 62.3 μg mg^?1, respectively). There was no evident parasite immobilization activity in body homogenates, suggesting no development of acquired immunity. Re‐infection by IP injection revealed no increase in protection in any of the treatment groups, mortality range of 56.3–75%, higher than in the non‐exposed control (40.6% mortality).     

Imported ornamental fish are colonized with antibiotic‐resistant bacteria

There has been growing concern about the overuse of antibiotics in the ornamental fish industry and its possible effect on the increasing drug resistance in both commensal and pathogenic organisms in these fish. The aim of this study was to carry out an assessment of the diversity of bacteria, including pathogens, in ornamental fish species imported into North America and to assess their antibiotic resistance. Kidney samples were collected from 32 freshwater ornamental fish of various species, which arrived to an importing facility in Portland, Oregon from Colombia, Singapore and Florida. Sixty‐four unique bacterial colonies were isolated and identified by PCR using bacterial 16S primers and DNA sequencing. Multiple isolates were identified as bacteria with potential to cause disease in both fish and humans. The antibiotic resistance profile of each isolate was performed for nine different antibiotics. Among them, cefotaxime (16% resistance among isolates) was the antibiotic associated with more activity, while the least active was tetracycline (77% resistant). Knowing information about the diversity of bacteria in imported ornamental fish, as well as the resistance profiles for the bacteria will be useful in more effectively treating clinical infected fish, and also potential zoonoses in the future.     

Effect of iodophor disinfection of non‐hardened Salmo trutta eggs on their bacterial and fungus load

This study investigated the efficiency of iodophor disinfection (135 ppm active iodine for 15–30 min) of non‐hardened Salmo trutta eggs against different groups of bacteria and against fungus. Egg samples were taken from non‐disinfected and from disinfected eggs, microorganisms were cultured on specific nutrient media and their mass was measured by turbidimetric methods. Bacteria and fungus mass of non‐hardened eggs could be reduced but not eliminated by iodophor disinfection with 135 ppm active iodine for 15 min. The extent of reduction was 47–65% (Experiment 1). The efficiency of disinfection increased with disinfection time as the reduction in bacteria and fungus mass was 40–55% after 15 min and 58–74% after 30 min (Experiment 2). Disinfection efficiency of iodophor solution diluted in water (reduction 49–57%) and of iodophor solution diluted in sodium chloride solution iso‐osmolar to the oocytes (reduction 52–61%) was similar (Experiment 3). The reduction in bacteria and fungus mass was persistent as it was 39–72% lower in embryos deriving from disinfected eggs than in embryos deriving from non‐disinfected ones (Experiment 4). In conclusion, the tested disinfection method is inadequate to eliminate pathogens completely but it could positively influence immune defence of eggs and embryos.     

Nitrogenous waste excretion and accumulation of urea and ammonia inChalcalburnus tarichi (Cyprinidae), endemic to the extremely alkaline Lake Van (Eastern Turkey)

The endemic, anadromous cyprinidChalcalburnus tarichi is the only fish species known to occur in alkaline Lake Van (Eastern Anatolia, Turkey). EightC. tarichi were maintained individually in Lake Van water (17 – 19°C; pH 9.8; 153 mEq·I^–1 total alkalinity; 22 total salinity) and tank water samples analyzed for 24 h in 2 to 4 h intervals. At zero time, < 1µM ammonia was present and urea was undetectable in the tank water; at 24 h, total ammonia and urea made up 114±32 and 35±25µM, respectively. Over the experimental period, ammonia-N and urea-N excretion averaged 1041±494 and 607±169moles·kg^–1 fish·h^–1, respectively. The extent of urea excretion was highly variable between specimens. Uric acid excretion was not detectable.Urea was present at high concentrations in all tissues and plasma (25 – 35moles·g^–1·ml^–1) of freshly caughtC. tarichi; total ammonia content of the tissues was by a factor of 1.9 (liver) to 3.0 (brain) lower. High arginase activity (2.4±0.2 U·min^–1·g^–1) was detected in the liver ofC. tarichi but ornithine carbamoylphosphate transferase, a key enzyme of the ornithine-urea-cycle, was absent. Ureagenesis is likely through degradation of arginine and/or uricolysis. High glutamine synthetase activity (11±0.6 U·min^–1·g^–1) and low ammonia content in brain suggest that, like other teleosts,C. tarichi has an efficient ammonia detoxification in the brain, but in no other tissue.Nitrogenous waste excretion at alkaline pH is discussed. The ability ofC. tarichi to excrete high levels of ammonia at extremely alkaline pH is unique among teleosts studied so far. The mechanism of ammonia excretion under Lake Van conditions remains to be elucidated.     

Influence of the antibacterial herbs, Solanum trilobatum, Andrographis paniculata and Psoralea corylifolia on the survival, growth and bacterial load of Penaeus monodon post larvae

The indiscriminate use of antibiotics and chemicals in shrimp hatcheries has led to biomagnification and that in turn could lead to rejection of a whole consignment. The application of the bioencapsulation technique as a tool for curative treatment in shrimp larvae was investigated. Herbs having antibacterial properties such as Solanum trilobatum, Andrographis paniculata and Psoralea corylifolia (methanolic extracts) were bioencapsulated in Artemia and fed to Penaeus monodon post larvae PL 1–25. The post larvae were reared in a medium inoculated with pathogenic bacteria such as Pseudomonas aeruginosa, Staphylococcus aureus, Salmonella typhi and Vibrio sp. Post larvae reared in the non-inoculated water and fed with non-enriched Artemia exhibited 90% survival, highest specific growth rate (12.43%) and reduced bacterial load. P. monodon reared in the bacterial inoculated water and fed with the non-enriched Artemia exhibited the lowest survival (10–30%), specific growth rate (8.42–9.1%) and increased bacterial load (2.86 × 10³ to 3.76 × 10⁵ cfu/g). The methanolic extracts of the herbs helped to increase survival and specific growth rate and reduced bacterial load in the P. monodon culture system. Among the three herbal extracts, P. corylifolia enriched Artemia fed post larvae showed the tendency to higher survival (>50%), growth rate (11.5 averaged) and low bacterial load (1.12 × 10⁵ cfu/g). This revised version was published online in August 2006 with corrections to the Cover Date.