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1.
广东省养殖罗非鱼、海鲈、尖吻鲈海豚链球菌感染调查 总被引：4，自引：0，他引：4
利用细菌分离培养方法结合特异PCR技术,对广东省珠三角地区养殖罗非鱼(Oreochromis spp.)、海鲈(Lateolabrax japonicus)及尖吻鲈(Lates calcarifer)的海豚链球菌(Streptococcus iniae)感染情况进行了周年调查。每月固定时间在特定养殖区域采集目标鱼的脑、肝、脾、肾和肌肉等组织,并对其进行海豚链球菌的细菌分离培养鉴定。仅从已经患病的尖吻鲈中分离到3株链球菌,经生理生化鉴定和16S rDNA测序确定为海豚链球菌。利用海豚链球菌特异PCR技术对上述养殖鱼类不同组织进行检测,发现罗非鱼、海鲈、尖吻鲈的海豚链球菌感染率分别为30.21%、23.53%、14.55%,其中罗非鱼脑和肌肉的感染率明显较其他组织高(P<0.05),分别为20.65%和23.75%;海鲈的脑部和肌肉感染率也较其他组织高(P<0.05),分别为12.1%和10%;而尖吻鲈各组织感染率没有较大差异(P>0.05)。另外,研究结果还表明采集样本的海豚链球菌感染率随着其体长的增加而呈现下降趋势。 相似文献
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广西罗非鱼链球菌病流行菌株PCR鉴定和PFGE基因型分析 总被引：4，自引：2，他引：2
为获知近年广西罗非鱼链球菌病流行菌种及其基因型变化信息,采用特异PCR方法对2006-2012年从广西发病罗非鱼分离获得的77株临床菌株进行鉴定,并通过脉冲电场凝胶电泳(PFGE)对2006-2011年分离获得的37株流行菌株进行基因型分析.结果显示,其中20株鉴定为海豚链球菌其余57株鉴定为无乳链球菌.2006-2007年获得的19株流行菌株中有18株为海豚链球菌(94.7％),仅1株无乳链球菌;2009-2012年分离的58株流行菌株中56株为无乳链球菌(96.6％),仅2株海豚链球菌.PFGE图谱聚类显示,海豚和无乳链球菌分别聚类为两个大分支,20株海豚链球菌共产生4种PFGE带型,带型相似度为83.9％～100％;17株无乳链球菌共产生5种PFGE带型,带型相似度为47.4％～100％.研究表明,广西罗非鱼链球菌病流行菌种已从过去(2008年前)以海豚链球菌为主转变为现在(2009-2012年)以无乳链球菌为主;流行菌株PFGE基因型存在多样性. 相似文献
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为获知近年广西罗非鱼链球菌病流行菌种及其基因型变化信息,采用特异PCR方法对2006~2012年从广西发病罗非鱼分离获得的77株临床菌株进行鉴定,并通过脉冲电场凝胶电泳(PFGE)对2006~2011年分离获得的37株流行菌株进行基因型分析。结果显示,其中20株鉴定为海豚链球菌(Streptococcus iniae,S.iniae)其余57株鉴定为无乳链球菌(Streptococcus agalactiae,S.agalactiae)。2006~2007年获得的19株流行菌株中有18株为海豚链球菌(94.7%),仅1株无乳链球菌;2009~2012年分离的58株流行菌株中56株为无乳链球菌(96.6%),仅2株海豚链球菌。PFGE图谱聚类显示,海豚和无乳链球菌分别聚类为两个大分支,20株海豚链球菌共产生4种PFGE带型,带型相似度在83.9%~100%之间;17株无乳链球菌共产生5种PFGE带型,带型相似度在47.4%~100%之间。研究表明,广西罗非鱼链球菌病流行菌种已从过去(2008年前)以海豚链球菌为主转变为现在(2009~2012年)以无乳链球菌为主;流行菌株PFGE基因型存在多样性。 相似文献
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为了研究铁调素调节蛋白(hemojuvelin,HJV)在硬骨鱼中抵御病原菌感染和维持自身铁稳态过程中的作用,实验扩增了尼罗罗非鱼铁调素调节蛋白基因(Onhjv)的开放阅读框(ORF),分析其在健康尼罗罗非鱼各组织中的分布模式及在抵御病原菌感染和调节铁稳态中的相关作用。结果显示,Onhjv的ORF全长由1 248个碱基组成,编码415个氨基酸,在不同物种之间具有一定的保守性。实时荧光定量PCR(qRT-PCR)结果显示,Onhjv在尼罗罗非鱼各组织中广泛分布,并在肝脏中的表达量最高。在无乳链球菌或嗜水气单胞菌感染后,Onhjv在肝脏、脾脏、肠和鳃中的表达量均显著上调。体外头肾单核/巨噬细胞和肝细胞中Onhjv表达量在受到这2种病原菌应激下也显著上调。此外,在1和10μmol/L FeCl_3溶液刺激后,Onhjv表达量在肝脏、脾脏、肠和鳃等组织,以及头肾单核/巨噬细胞和肝细胞中的表达量也呈显著上调。受重组罗非鱼IL-6蛋白[(r)OnIL-6]刺激后,头肾单核/巨噬细胞中Onhjv表达量显著上调,表明炎症因子可以促进Onhjv表达。研究表明,尼罗罗非鱼铁调素调节蛋白在宿主抵御病原菌感染和维持铁稳态的过程中发挥作用。本实验为探究HJV在硬骨鱼中的生物学功能提供了参考,同时为进一步研究铁代谢在宿主防御病原菌感染过程中的重要作用提供理论指导。 相似文献
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为检测斑点叉尾鮰源海豚链球菌兼职蛋白(fructose-1,6-bisphosphate aldolases,FBA)的抗原性和潜在的疫苗价值,本实验克隆得到斑点叉尾鮰源海豚链球菌DX09(基因组登陆号LXQF01)的fba基因序列(基因登录号A7N10_RS06935),对克隆序列进行生物信息学分析,并通过原核表达得到重组FBA蛋白(r FBA),制备了兔抗r FBA血清用于FBA蛋白抗原性检测,同时通过免疫保护实验评估重组蛋白的免疫保护效果。结果显示,海豚链球菌DX09 fba基因有1个882 bp的开放阅读框(ORF),编码293个氨基酸。生物信息学分析显示,其分子式为C_(1378)H_(2172)N_(368)O_(422)S_8,分子质量为30.9 ku,理论等电点为5.01,不具有信号肽和跨膜区域;具有保守的裂解酶结构域,且与其他来源的FBA蛋白同源性达100%;具有较高的抗原指数,表明其可形成多个抗原表位。SDS-PAGE检测发现,诱导表达的重组蛋白以包涵体的形式出现在沉淀中,大小约为47 ku。Western blot分析表明,兔抗r FBA血清能特异性结合菌体蛋白。同时免疫保护实验显示,重组蛋白对斑点叉尾鮰的相对保护率可达55%,免疫后鱼体抗体水平相对对照组显著升高。本研究表明,原核表达的斑点叉尾鮰源海豚链球菌DX09 rFBA具备较好的抗原性和免疫保护作用,具有研发斑点叉尾鮰海豚链球菌亚单位疫苗的潜在价值。 相似文献
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《淡水渔业》2020,(4)
为了探究不同添加水平的河豚(Takifugu rubripes)卵处理物对吉富罗非鱼(Oreochromis niloticus)生长性能、血液生化以及海豚链球菌感染后的存活率的影响,试验选取525尾平均体重为(0.66±0.01)g的吉富罗非鱼,随机分为7组,每组3个重复,每重复25尾鱼,依次饲喂河豚卵处理物添加水平为0、0.2%、0.4%、0.8%、1.6%、3.2%和6.4%的饲料,养殖8周。结果显示:随着河豚卵处理物添加水平的提高,试验鱼体增重、特定生长率、肝体比和脏体比均先上升后下降最后趋于稳定,饲料转化率显著降低,各组肝体比无显著差异,0.8%河豚卵处理物添加组罗非鱼的脏体比显著高于其他组。随着河豚卵处理物添加水平的提高,罗非鱼血液中红细胞、血红蛋白、红细胞比容含量显著下降,白细胞数量呈逐渐上升的趋势;罗非鱼血清中总胆固醇和血糖含量显著下降,血清中溶菌酶含量呈逐渐下降的趋势。罗非鱼腹腔注射5×10~7 CFU/mL海豚链球菌后,各组罗非鱼24 h内均未出现死亡;感染48～96 h,对照组罗非鱼累计存活率最低;感染192 h后,6.4%河豚卵处理物添加组罗非鱼累计存活率显著高于对照组。结果表明:饲料中添加河豚卵处理物对吉富罗非鱼的生长性能、血液生化指标产生影响,增强了罗非鱼抗海豚链球菌病感染的能力。 相似文献
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《南方水产科学》2019,(6)
为研制比无乳链球菌(Streptococcus agalactiae)单个蛋白抗原免疫效果更佳的多蛋白重组罗非鱼(Oreochromis sp.)链球菌病口服疫苗,该研究利用同源重组法构建表达无乳链球菌Sip-Pgk融合蛋白的pNZ8148-sippgk质粒,通过电转化乳酸乳球菌(Lactococcus lactis) NZ9000中获得L. lactis NZ9000 pNZ8148-sip-pgk重组乳酸菌,使用nisin诱导表达并进行Western blot鉴定,制备Sip-Pgk融合蛋白乳酸菌口服疫苗,通过不同免疫次数隔周免疫的方式灌胃罗非鱼。ELISA检测免疫后的血清抗体水平,在灌胃免疫结束后的第18天通过腹腔注射无乳链球菌攻毒获得相对免疫保护率。结果显示,构建的重组乳酸菌诱导表达的蛋白大小为92 kD,与目的蛋白大小一致。与2次免疫比较,3次免疫该融合蛋白乳酸菌疫苗能显著提高罗非鱼的血清抗体水平和对无乳链球菌的免疫保护效果。3次免疫Sip-Pgk融合蛋白乳酸菌疫苗的血清水平显著高于单一蛋白组和PBS组,其相对免疫保护率最高(45.56%)。 相似文献
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罗非鱼源无乳链球菌兼职蛋白EF-Tu的克隆、表达及其抗原性检测 总被引：1，自引：1，他引：0
为检测罗非鱼源无乳链球菌兼职蛋白EF-Tu(延伸因子Tu,Elongation Factor Tu)的抗原性,本实验克隆了罗非鱼源无乳链球菌HN0303的EF-Tu基因序列,并进行了蛋白相关性质的预测和系统发育树的构建。通过原核表达得到EF-Tu重组蛋白,同时利用纯化的蛋白免疫家兔获得多克隆兔抗EF-Tu重组蛋白血清以用于EF-Tu蛋白抗原性检测。结果显示,罗非鱼源无乳链球菌HN0303 EF-Tu基因有1个由1197个碱基组成的ORF,编码398个氨基酸。生物信息学分析显示其分子式为C_(1933)H_(3096)N_(532)O_(615)S_(11),分子质量为43.981 ku,理论等电点为4.749;具有多个磷酸化位点,不具有信号肽和跨膜区域;具有保守的EFTu结构域、EF-Tu-II结构域和EF-Tu-Ⅲ结构域,且与其他来源无乳链球菌的EF-Tu蛋白具有很高的同源性;具有较高的抗原指数,表明其可形成多个抗原表位。SDS-PAGE检测发现,诱导表达的重组蛋白以包涵体的形式出现在沉淀中,大小约为66.4 ku。Western Blot分析表明,兔抗EF-Tu重组蛋白血清能分别特异性结合菌体蛋白和EF-Tu重组蛋白。同时使用兔抗EF-Tu重组蛋白血清封闭罗非鱼源无乳链球菌HN0303表面的EF-Tu蛋白后,无乳链球菌HN0303粘附EPC(Epithelioma papulosum cyprini,鲤鱼上皮细胞)的能力下降了79.99%±2.43%。本研究表明,原核表达的罗非鱼源无乳链球菌EF-Tu重组蛋白具备较好的抗原性,用其制备的兔抗血清能够较好地抑制罗非鱼源无乳链球菌的粘附,推测其可能为罗非鱼源无乳链球菌亚单位疫苗的候选蛋白。 相似文献
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为获得具有单一克隆特性的能稳定传代培养的罗非鱼巨噬细胞系,本研究从尼罗罗非鱼腹腔中分离纯化巨噬细胞,采用EB病毒(Epstein-Barr virus,EBV)感染,筛选单克隆细胞的方法建立了尼罗罗非鱼巨噬细胞系,并对其进行了EBV感染鉴定、电镜观察、端粒酶活性检测、致癌性评估、核型分析以及分子生物学鉴定。研究表明,EBV已整合到尼罗罗非鱼巨噬细胞中且稳定表达,经30代稳定传代,该细胞系仍维持较好的增殖状态;该细胞系表面不平滑,有明显的钝圆形突起和细长的伪足,表现为典型的巨噬细胞形态;端粒酶活性显著高于未经感染的巨噬细胞,而与He La细胞差异不显著,且该细胞系不具有致癌性,说明永生化细胞系构建成功。核型分析结果发现,该细胞系具有44条染色体,其核型公式为2 n=2 x=44=4 sm+17 st+1 t。PCR检测发现,该细胞系存在CD33和CD205的转录本,这些都是单核巨噬细胞的标志物,经18S r RNA检测证明该细胞系来自尼罗罗非鱼巨噬细胞。永生化尼罗罗非鱼巨噬细胞系已被成功建立,该细胞系为研究罗非鱼链球菌HSP70-肽疫苗的高保护率,以及罗非鱼的免疫防御机制提供了工具。 相似文献
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The chemoattractant capabilities of Edwardsiella tarda extracellular products (ECP) were investigated from two isolates, the virulent FL6-60 parent and less virulent RET-04 mutant. Chemotaxis and chemokinesis were assayed in vitro using blind well chambers with peritoneal macrophages obtained from Nile tilapia, Oreochromis niloticus, 5 days following squalene injection. Non-purified ECP derived from both isolates stimulated predominantly chemokinetic migration of macrophages. Additionally, the ECP were semi-purified by high pressure liquid chromatography. The FL6-60 parent ECP yielded higher molecular weight components than did the ECP from the RET-04 mutant. The chemotactic activity of the macrophages for both the FL6-60 parent and RET-04 mutant semi-purified ECP was increased over the non-purified ECP and overall migration was primarily chemotactic. Exposure to ECP derived from virulent and less virulent E. tarda isolates promoted chemokinetic movement of macrophages that may be involved in inflammatory responses of Nile tilapia to E. tarda infection. 相似文献
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Nile tilapia were immunized by injecting extracellular products (ECP) of Mycobacterium spp. (strain TB40, TB267 or the type strain Mycobacterium marinum) into their swim bladders. A variety of adjuvants – Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA) and Titremax – were similarly injected into additional groups of tilapia. Phosphate-buffered saline (PBS) was used as a control. The number of nitroblue tetrazolium (NBT)-positive cells observed in the swim bladder of the immunized fish had significantly increased by the fourth day post-immunization. By day 8, the number of NBT-positive cells in fish immunized with ECP from mycobacteria strains TB40 or TB267 were fewer than in fish immunized with ECP from M. marinum or fish injected with FCA or FIA. The level of lysozyme activity detected in the serum of fish 4 days after being immunized with ECP from various Mycobacterium spp. was also significantly higher than that found in the serum of the control fish. Head kidney macrophages showed an enhanced reduction of NBT when cultured in vitro with 1 μg ml–1 of ECP. Concentrations greater than this (10 or 100 μg ml–1) were found to suppress the reduction of NBT by the macrophages. 相似文献
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为了更加了解草鱼B淋巴细胞吞噬活性,本研究采用实时荧光定量PCR(qRTPCR)检测了IgM在胚胎发育中的表达量,并且检测了IgM在不同组织中的分布以及细菌刺激下IgM的转录情况。结果显示,IgM在卵裂期到出膜前期的表达量变化不明显,在出膜后开始显著增加;IgM在检测的组织中均有分布,在头肾中表达量最高,并且其表达量在不同细菌的刺激下均显著上调。从草鱼外周血中分离纯化得到B淋巴细胞,并通过吉姆萨染色、qRT-PCR和间接免疫荧光进行了验证。细菌吞噬实验结果显示,B淋巴细胞对嗜水气单胞菌具有一定的吞噬能力,并随孵育时间增加而逐渐增强。细菌刺激B淋巴细胞后活性氧(reactive oxygen species, ROS)和一氧化氮(nitric oxide,NO)释放量显著上升;血清调理实验结果显示,通过血清调理可以显著促进B淋巴细胞ROS的释放,然而对NO的释放水平没有显著影响。研究表明,草鱼B淋巴细胞对细菌具有一定的吞噬能力,并且可以通过呼吸爆发等非特异性免疫的方式直接参与抗菌免疫。 相似文献
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The production of macrophage activation factor (MAF) by rainbow trout, Oncorhynchus mykiss (Walbaum) , head kidney leucocytes was examined after culturing in vitro with extracellular products (ECP) collected from Mycobacterium sp. Cultures of leucocytes were prepared from naive fish, or fish previously vaccinated with either the ECP or with formalin killed whole cell preparations (WC) of the bacterium. The cells were then incubated with the ECP in vitro and the ability of their supernatants to activate macrophages assessed. Macrophages from control fish were incubated with the supernatants, and their ability to reduce nitroblue tetrazolium (NBT) measured as an indicator of macrophage activation. Incubation of head kidney macrophages from naive fish directly with 1, 10 or 100 μg mL–1 of ECP for 48 h significantly enhanced macrophage activation compared with control macrophages. Vaccination of fish with either ECP or WC had no significant effect on the respiratory burst of control macrophages 4 weeks post-vaccination. By the eighth week, however, absorbance levels of respiratory burst reflecting both the primary (cells from vaccinated fish cultured in vitro with PBS) and the secondary (cells cultured in vitro with ECP) MAF responses of fish vaccinated with ECP and WC, had peaked and these were significantly different from the non-vaccinated controls. This activity had fallen to levels similar to control fish by week 12 for fish vaccinated with WC. 相似文献
16.
do Carmo Alves Angélica Priscila Peconick Ana Paula da Silva Cerozi Brunno Possebon Cyrino José Eurico 《Aquaculture International》2022,30(4):1905-1929
The development and intensification of Nile tilapia farming systems has happened together with ever increased use of antibiotics and therapeutants. Research on the use of probiotics has come along as alternative to routine use of antibiotics in fish diets. The manipulation of the gut microbiota through dietary probiotic inclusion targeting improved growth rate and disease resistance has been investigated in many farmed fish species. However, mechanisms underlying modulation of fish immune response remains unclear. This review summarizes and discuss recent findings in the role of probiotics on the immunity of Nile tilapia Oreochromis niloticus addressing available information regarding immunological parameters such as phagocytic activity, lysozyme, respiratory burst activity, antioxidant enzymes, complement system as well as immune-related gene expression, in the aim to foster research efforts on effective strategies for the successful use of probiotics in the tilapia farming industry.
相似文献17.
ABSTRACT: Stress and immune responses were studied in Nile tilapia Oreochromis niloticus under both conditions of self-feeding and scheduled feeding. Self-feeding fish had a significantly lower cortisol level in their blood plasma than that of scheduled feeding fish. Furthermore, skin color was found to be paler in the self-feeding fish compared with that of the scheduled feeding fish. Thus, the self-feeding fish had a less stressful physiological status than the other experimental fish. Immunological analyses revealed that the self-feeding fish significantly exceeded the scheduled feeding fish in the phagocytic activity of their macrophages, antibody production, and the number of blood-circulating lymphocytes. The higher immune responses of self-feeding fish seem to be attained through a less stressful environment. This self-feeding behavior could be exploited to create reduced stress for disease control in fish farming. 相似文献
18.
Marco Paredes Katerina Gonzalez Jaime Figueroa Enrique Montiel-Eulefi 《Fish physiology and biochemistry》2013,39(5):1215-1221
The in vitro and in vivo effect of prolactin (PRL) on kidney macrophages from Atlantic salmon (Salmo salar) was investigated under the assumption that PRL stimulates immune innate response in mammals. Kidney macrophages were treated two ways: first, cultured in RPMI 1640 medium containing 10, 25, 50 and 100 ng/mL of PRL and second, isolated from a fish with a PRL-injected dose of 100 ng/Kg. Reduced nitro blue tetrazolium (formazan) was used to produce intracellular superoxide anion. Phagocytic activity of PRL was determined in treated cells by optical microscopy observation of phagocytized Congo red-stained yeast. Kidney lysozyme activity was measured in PRL-injected fish. In vitro and in vivo macrophages treated with PRL presented an enhanced superoxide anion production, elevated phagocytic index and increased phagocytic activity. Treated fish showed higher levels of lysozyme activity in the head kidney compared to the control. These results indicate that PRL-stimulated innate immune response in Atlantic salmon and future studies will allow us to assess the possibility of using PRL as an immunostimulant in the Chilean salmon industry. 相似文献
19.
海豚链球菌感染对不同品系罗非鱼血液生化指标和肝脏HSP70 mRNA表达的影响 总被引：4，自引：3，他引：1
以吉富罗非鱼、新吉富罗非鱼、埃及尼罗罗非鱼和红罗非鱼为研究对象,饲养100d后,进行海豚链球菌(2.95×108CFU/mL)感染试验,分析攻毒前后各品系罗非鱼的血液生化指标和肝脏HSP70 mRNA表达量的变化规律。另从各桶中取20尾鱼进行同样的攻毒试验,统计攻毒后各时间点的累积死亡率。结果表明,感染海豚链球菌96h后,吉富罗非鱼和新吉富罗非鱼对病原较为敏感,累积死亡率分别达到36.67%和38.33%;埃及尼罗罗非鱼对病原敏感性较差,试验期间未见死亡。吉富罗非鱼、新吉富罗非鱼和红罗非鱼血清皮质醇和葡萄糖水平以及肝脏HSP70 mRNA的表达量在攻毒后明显提高,血清谷丙转氨酶、谷草转氨酶与溶菌酶活力也呈上升趋势,碱性磷酸酶活力与甘油三酯和胆固醇水平低于攻毒前。埃及尼罗罗非鱼可以利用糖原和脂类产生的能量,提高了HSPS与一些特定免疫蛋白(溶菌酶、球蛋白等)的合成,增强了鱼体的非特异性免疫力。罗非鱼选育过程中,需要将抗病力与生长性能进行有效的结合,在注重生长速度的同时也要增强其抗应激能力,从而为罗非鱼产业的可持续发展提供保证。 相似文献
20.
Pasnik DJ Evans JJ Panangala VS Klesius PH Shelby RA Shoemaker CA 《Journal of fish diseases》2005,28(4):205-212
Streptococcus agalactiae is a major bacterial pathogen that is the cause of serious economic losses in many species of freshwater, marine and estuarine fish worldwide. A highly efficacious S. agalactiae vaccine was developed using extracellular products (ECP) and formalin-killed whole cells of S. agalactiae. The vaccine efficacy following storage of S. agalactiae ECP and formalin-killed S. agalactiae cells at 4 degrees C for 1 year was determined. The stored ECP containing S. agalactiae formalin-killed cells failed to prevent morbidity and mortality among the vaccinated fish, and the relative percentage survival was 29. Serum antibody responses of the stored ECP and freshly prepared ECP against soluble whole cell extract of S. agalactiae indicated that significantly less antibody was produced in fish immunized with stored ECP and S. agalactiae cells than in those fish immunized with freshly prepared ECP and S. agalactiae cells at day 31 post-vaccination. Silver staining of sodium dodecyl sulphate-polyacrylamide gels and immunostaining of Western blots with tilapia antiserum to S. agalactiae revealed that predominant 54 and 55 kDa bands were present in the freshly prepared ECP fraction. The 55 kDa band was absent from the stored ECP and new bands below 54 kDa appeared on the Western blot. The results of this study on S. agalactiae ECP provide evidence for a correlation between protection and antibody production to ECP and for the importance of the 55 kDa ECP antigen for vaccine efficacy. 相似文献