Molecular cloning and expression analysis of a CXC chemokine gene from large yellow croaker Pseudosciaena crocea

In the present study, we report the cloning of a CXCL12 chemokine gene homologue from the large yellow croaker Pseudosciaena crocea (LycCXCL12). The complete cDNA of LycCXCL12 is 678 nucleotides (nt) encoding a protein of 97 amino acids (aa), with a putative molecular weight of 11.1 kDa. The deduced LycCXCL12 contains a 22-aa signal peptide and a 75-aa mature polypeptide, which possesses the typical arrangement of four cysteines as found in other known CXC chemokines. It shares 57-68% and 32-36% aa sequence identities to known CXCL12 chemokines in fish species and other vertebrates, respectively. The LycCXCL12 gene was constitutively expressed in all tissues examined although at different levels. Upon induction with poly(I:C) or inactivated trivalent bacterial vaccine, LycCXCL12 gene expression was significantly up-regulated in gills, liver, kidney, spleen and blood at 24 h after stimulation. Time course analysis using real-time PCR showed that LycCXCL12 gene expression reached peak level in spleen and kidney at 12 h or in gills at 24 h post-induction by poly(I:C), while its expression increased to the highest level in kidney at 24h or in gills and spleen at 48 h post-induction by bacterial vaccine, indicating that LycCXCL12 gene expression was differentially regulated by poly(I:C) and bacterial vaccine.     

Characterization of tilapia (Oreochromis niloticus) viperin expression,and inhibition of bacterial growth and modulation of immune-related gene expression by electrotransfer of viperin DNA into zebrafish muscle

Viperin is an anti-viral protein, induced by viral infection. In this study, we examined whether over-expression of viperin in fish muscle could inhibit bacterial growth. We first obtained the cDNA sequence of tilapia viperin, through RT-PCR-mediated cloning and sequencing. The cDNA sequence was similar to those of several fish viperins in GenBank, and it was predicted to encode the conserved domain of radical S-adenosylmethionine superfamily proteins. Phylogenetic analysis revealed that tilapia viperin was most closely related to viperin of Sciaenops ocellatus, Coreoperca kawamebari, and C. whiteheadi. Expression of tilapia viperin was significantly up-regulated in the kidney, liver, spleen, and gills upon challenge with lipopolysaccharide (LPS) and poly(I:C) in a time- and dose-dependent manner. Injection of Vibrio vulnificus (204) and Streptococcus agalactiae (SA47) bacteria into tilapia resulted in significant induction of viperin expression in the whole body, kidney, liver, and spleen. Electrotransfer of a viperin-expressing plasmid into zebrafish muscles decreased bacterial numbers and altered expression of immune-related genes. These data indicate that such altered expression may account for the improvement in bacterial clearance following electroporation of viperin, suggesting that fish viperin has antiviral and antibacterial activities.     

How Ebola virus counters the interferon system

Zoonotic transmission of Ebola virus (EBOV) to humans causes a severe haemorrhagic fever in afflicted individuals with high case-fatality rates. Neither vaccines nor therapeutics are at present available to combat EBOV infection, making the virus a potential threat to public health. To devise antiviral strategies, it is important to understand which components of the immune system could be effective against EBOV infection. The interferon (IFN) system constitutes a key innate defence against viral infections and prevents development of lethal disease in mice infected with EBOV strains not adapted to this host. Recent research revealed that expression of the host cell IFN-inducible transmembrane proteins 1-3 (IFITM1-3) and tetherin is induced by IFN and restricts EBOV infection, at least in cell culture model systems. IFITMs, tetherin and other effector molecules of the IFN system could thus pose a potent barrier against EBOV spread in humans. However, EBOV interferes with signalling events required for human cells to express these proteins. Here, we will review the strategies employed by EBOV to fight the IFN system, and we will discuss how IFITM proteins and tetherin inhibit EBOV infection.     

干扰素诱导跨膜蛋白1对猪源冠状病毒吸附宿主细胞的影响

为探索干扰素诱导跨膜蛋白1（interferon-inducible transmembrane protein 1,IFITM1）对猪源冠状病毒复制的影响,本研究选用猪传染性胃肠炎病毒（Transmissible gastroenteritis virus,TGEV）高致病毒株及其宿主细胞PK15作为研究对象。正常PK15细胞接种TGEV后,实时荧光定量PCR检测感染后不同时间点PK15细胞中一些干扰素刺激基因（interferon-stimulated genes,ISGs）的mRNA表达水平;利用慢病毒表达系统构建稳定表达和稳定干扰IFITM1表达的PK15细胞系。将一段靶向干扰猪源IFITM1的shRNA序列及猪源IFITM1全长,分别插入pLKO.1-EGFP-Puro载体及pLVML-Myc-MCS-IRES-Puro载体中,分别构建出pLKO.1-IFITM1shRNA-EGFP-Puro及pLVML-Myc-IFITM1-IRES-Puro重组质粒。将重组质粒与慢病毒包装质粒共转染293FT细胞后获得带有目的基因的重组慢病毒,慢病毒侵染PK15细胞后用嘌呤霉素进行筛选,获得稳定表达及稳定干扰IFITM1表达的PK15细胞系,分别命名为PK15-IFITM1及PK15-IFITM1^-/-,并分别用实时荧光定量PCR、间接免疫荧光试验（IFA）及Western blotting检测IFITM1干扰效率及表达情况;TGEV接种PK15-IFITM1^-/-和PK15-IFITM1,实时荧光定量PCR测定细胞中TGEV的拷贝数。结果显示,PK15细胞接种TGEV后的48 h内,一些ISGs的mRNA水平均有所上升;PK15-IFITM1^-/-细胞系的干扰效率为70%,PK15-IFITM1细胞系表达成功;在PK15-IFITM1^-/-细胞系中,IFITM1的mRNA水平显著下调,促进了TGEV的复制。反之,在PK15-IFITM1细胞系中,TGEV的复制受到了抑制。但IFITM1的表达或缺失却不影响TGEV对PK15的吸附作用。总之,IFITM1对TGEV有显著的抗病毒作用,IFITM1不影响TGEV对PK15细胞的早期吸附,这为后续IFITM1抗冠状病毒机制的研究奠定了基础。     

Prevalence of Flavobacterium psychrophilum bacterial cells in farmed rainbow trout: Characterization of metallothionein A and interleukin1-β genes as markers overexpressed in spleen and kidney of diseased fish

The aim of the present study was to assess the prevalence of the flavobacteria within farmed trout and to quantify their bacterial burden. A total of 61 fish were sampled from seven farms, and were distributed in two groups: (1) visibly diseased fish suffering from the rainbow trout fry syndrome or the bacterial cold water disease caused by the bacteria Flavobacterium psychrophilum and (2) normally appearing fish. F. psychrophilum cells were titered by qPCR, targeting a specific area of the 16S rRNA gene in skin, muscle, gills, liver, spleen and kidney from all fish. The pathogen was detected in these organs whatever the health status, with titers ranging from 10⁴ to 6 × 10⁷ bacteria/g of tissue in normally appearing fish, thus showing they were bacterial carriers. Two organs allowed differentiation between diseased and normally appearing fish: spleen and kidney, with titers ranging from 10⁶ to 10⁷ bacteria/g of tissue in normally appearing fish vs 10¹¹ to 10¹² bacteria/g of tissue in diseased fish. No relationship was found between immunoglobulin M-like titer in plasma and health status. Gene expression analysis in fish organs revealed two genes that were markers of the bacterial infection: mt-a and il-1β genes encoding the metallothionein A and the interleukin1-β, respectively. These genes were both over-expressed in gills, liver, spleen and kidney of diseased fish. Four genes encoding immunity markers were down-regulated in spleen (a key organ implicated in immunity) of diseased fish: tgf-β, cd8-α, mhc2-β and igt, suggesting a weakened immune system in diseased fish.     

大黄鱼营养需求研究进展

文章综合论述了近年来大黄鱼营养研究的进展。主要包括:饲料中蛋白质和氨基酸、脂肪和脂肪酸、维生素和矿物元素等的适宜含量;不同蛋白源替代鱼粉以及原料消化率;非营养性添加剂;大黄鱼配合饲料发展现状和研究展望。     

Atypical presentation of mycobacteriosis in a collection of frogfish (Antennarius striatus).

Severe systemic mycobacteriosis without typical granuloma formation was diagnosed in a group of six mature, captive, striated frogfish Antennarius striatus approximately 5 mo after fish originating from Brazil were purchased by Mote Marine Laboratory Aquarium. Beginning 1 mo after spawning, over a period of 9 mo, individuals began to show a variety of signs including egg retention, ocular opacity, poor buoyancy control, ascites with coelomic distension, skin lesions, and anorexia. Two fish died, and four were euthanatized. At necropsy, raised pigmented skin nodules; pale pink gills; and pale yellow or tan, fatty livers were noted. A systemic fungal infection was diagnosed histopathologically in one female, and the remaining fish had severe, systemic, histiocytic inflammation and necrosis. Although large numbers of acid-fast bacterial rods were identified in each fish, no bacteria were cultured aerobically from skin, kidney, spleen, liver, or brain. Mycobacterium marinum was cultured from the liver of the last fish that was euthanatized after it became moribund and failed to respond to symptomatic treatment.     

发酵豆粕替代鱼粉对大黄鱼幼鱼生长性能、体成分、血清生化指标及肝脏组织形态的影响

本试验旨在研究发酵豆粕替代鱼粉对大黄鱼(Larimichthys crocea)幼鱼生长性能、体成分、血清生化指标及肝脏组织形态的影响,探索发酵豆粕替代大黄鱼幼鱼饲料中鱼粉的适宜比例。以鱼粉、小麦蛋白粉为主要蛋白质源,鱼油、大豆油和大豆卵磷脂为主要脂肪源,配制含40%鱼粉的基础饲料。以发酵豆粕替代基础饲料中0(R0组,作为对照组)、15%(R15组)、30%(R30组)、45%(R45组)、60%(R60组)、75%(R75组)的鱼粉,并在除对照组饲料外的各饲料中添加适量晶体氨基酸(赖氨酸和蛋氨酸),配制6种等氮(蛋白质水平为45%)等脂(脂肪水平为10%)的试验饲料。养殖试验在海水网箱(1.5 m×1.5 m×2.0 m)中进行,每种试验饲料投喂3个网箱,每个网箱放养60尾初始体重为(10.49±0.03)g大黄鱼幼鱼,养殖时间持续56 d。结果表明:各组大黄鱼幼鱼的存活率无显著差异(P0.05),但随着发酵豆粕替代鱼粉比例的升高有下降趋势;与R0组相比,R15、R30、R45组的特定生长率(SGR)、增重率(WGR)、饲料系数(FCR)无显著变化(P0.05),进一步提高鱼粉替代比例(R60、R75组),SGR、WGR显著降低(P0.05),FCR显著升高(P0.05);各组全鱼粗蛋白质、粗脂肪、水分含量无显著差异(P0.05),随着发酵豆粕替代鱼粉比例的升高,全鱼粗灰分含量有上升的趋势;各组血清生化指标没有显著差异(P0.05),但随着发酵豆粕替代鱼粉比例的升高,血清中总胆固醇含量有下降趋势,谷丙转氨酶活力有升高趋势;通过肝脏组织学观察发现,发酵豆粕替代鱼粉比例超过30%后会造成肝脏细胞的损伤,替代比例越高损伤越严重。综合各项测定指标,本研究认为:发酵豆粕替代饲料(含40%鱼粉)中30%的鱼粉较为适宜,过高的替代比例会造成大黄鱼幼鱼肝脏组织病变,导致生长速度、存活率下降。     

Genetic characteristics and polymorphisms in the chicken interferon-induced transmembrane protein (IFITM3) gene

Veterinary Research Communications - The interferon-induced transmembrane protein 3 (IFITM3) gene is classified as a small interferon-stimulated gene and is associated with a broad spectrum of...     

Identification and characterization of Japanese flounder, Paralichthys olivaceus interferon-stimulated gene 15 (Jf-ISG15)

Previously, using cDNA microarray analysis, we demonstrated that an EST clone of Japanese flounder (Paralichthys olivaceus) with homology to mammalian interferon-stimulated gene 15 (ISG15) was strongly induced by treatment with DNA vaccine encoding the glycoprotein gene of Hirame rhabdovirus (HIRRV). In this study, we conducted molecular cloning and expression analysis of the Japanese flounder ISG15 (Jf-ISG15). Jf-ISG15 encoded two exons. The first exon was non-coding, while the second exon encoded a protein of 158 amino acids. The coded protein has two tandem ubiquitin-like domains with a carboxyl-terminus conjugation motif “LRLRGG”. Phylogenetic analysis revealed an evolutionary relationship among Jf-ISG15, mammalian and fish ISG15 orthologues. The interferon-stimulated response element (ISRE) sites were conserved among DNA sequences of Jf-ISG15 and mammalian ISG15 promoter regions. An RT-PCR analysis of healthy tissues showed that Jf-ISG15 mRNA was notably strongly expressed in gills, PBLs and spleen. Expression of Jf-ISG15 was strongly induced by poly-I:C treatment in head-kidney cells, peripheral blood leukocytes (PBLs) and spleen cells, and by HIRRV infection in kidney of juvenile fish suggesting that Jf-ISG15 plays a role in fish antiviral response.     

芦花鸡IFITM3基因克隆及生物信息学分析

本研究旨在对芦花鸡的干扰素诱导的跨膜蛋白-3（interferon-induced transmembrane protein 3，IFITM3）基因进行克隆及生物信息学分析。根据GenBank中鸡IFITM3基因序列（登录号：NM_001350061.1）设计特异性引物，利用RT-PCR技术对芦花鸡IFITM3基因进行克隆、测序和生物信息学分析。结果表明，芦花鸡IFITM3基因片段大小为414 bp，与GenBank中发表的鸡IFITM3基因序列（登录号：NM_001350061.1）同源性为99.9%；编码137个氨基酸。IFITM3基因理论分子质量为14.95 ku，理论等电点（pI）为6.89，不稳定系数为33.98，脂肪系数为103.14，平均疏水指数为0.300，存在3个明显的疏水区，无信号肽，存在2个跨膜区，分别位于第46-68、101-123位氨基酸处；二级结构预测显示，IFITN3蛋白主要以无规则卷曲为主，存在多个α-螺旋和β-折叠区域。本研究成功克隆了芦花鸡IFITM3基因，为进一步研究IFITM3蛋白功能及阐明其抗病毒分子机制奠定了理论基础。     

Pathological effects of Arcobacter cryaerophilus infection in rainbow trout (Oncorhynchus mykiss Walbaum)

Arcobacter cryaerophilus was isolated from naturally infected rainbow trout (Oncorhynchus mykiss Walbaum), and its pathogenicity was tested by intramuscular injection into 40 healthy 1-year-old rainbow trout at 16 degrees C. The lethal dosage of 50% end point (LD50) for A. cryaerophilus was calculated 2.25 x 10(4) viable cells. Experimental infection caused deaths with gross clinical abnormalities such as degenerated opercula and gills, liver damage, haemorrhagic kidney and serous fluid in swollen intestines. The counts of A. cryaerophilus in kidney, liver and gills of experimentally infected fish ranged from 1.59 x 10(10) colony forming units (cfu)/g to 7.41 x 10(12) cfu/g. The means of erythrocyte (RBC) count, haematocrit level, serum cholesterol, triglyceride, albumin and total protein concentrations in the blood of the experimentally infected rainbow trout group were significantly lower than in the healthy fish. Leukocyte (WBC) counts of the experimentally infected rainbow trout were significantly higher than those of healthy fish. The present work shows that the selected blood characteristics may be good indicators of response to infections in rainbow trout.     

Amoeba-like infections in cultured marine fishes: systemic infection in pompano Trachinotus falcatus L. from Singapore and gill disease associated with Paramoeba sp. in sea bream Sparus aurata L. from Greece

Two cases of Amoeba-like infections in cultured warmwater marine fish are described, an unusual systemic infection in pompano Trachinotus falcatus L. from Singapore and a gill infection in Mediterranean sea bream Sparus aurata L. All pompano showed marked systemic infection of Amoeba-like parasites in gills, kidney, intestine, pancreas and spleen. The most severe lesions were in the gills and renal tissue with minimal tissue reaction in other organs.     

Quantitation of immune response gene expression and cellular localisation of interleukin-1beta mRNA in Atlantic salmon, Salmo salar L., affected by amoebic gill disease (AGD)

The characterisation of selected immune response genes during amoebic gill disease (AGD) in Atlantic salmon, Salmo salar L., was performed using semi-quantitative RT-PCR, quantitative real-time RT-PCR (qRT-PCR), and in situ hybridisation (ISH). The immune response genes of interest were interleukin-1beta (IL-1beta), inducible nitric oxide synthase (iNOS), serum amyloid A (SAA), and serum amyloid P-like pentraxin (SAP). Atlantic salmon were inoculated with the ectoparasite Neoparamoeba sp., the causative agent of AGD, and gill, liver and anterior kidney tissue sampled at 0, 7 and 14 d post-inoculation (p.i.). Semi-quantitative RT-PCR was performed on the tissue samples to identify up/down-regulated mRNA expression relative to uninfected control fish and normalised to the housekeeping gene, beta-actin. Interleukin-1beta (IL-1beta) was the only immune response gene of those investigated whose mRNA was differentially regulated in any of the tissues and was found to be up-regulated in the gills by semi-quantitative RT-PCR. Increased gill IL-1beta mRNA expression was then accurately quantitated and confirmed using probe-based qRT-PCR. The cellular localisation of the IL-1beta mRNA expression in the gills of uninfected and infected fish was then determined by ISH using an IL-1beta-specific biotinylated cRNA probe. Expression of IL-1beta mRNA was localised to filament and lamellar epithelium pavement cells in gills of uninfected and infected Atlantic salmon. These data implicate the involvement of IL-1beta at the site of infection, the gills, of Atlantic salmon during AGD. This work supports previous studies that suggest IL-1beta is important in the regulation of the fish immune response to parasitic infection but additionally shows the cellular localisation of fish IL-1beta mRNA expression during infection.     

共轭亚油酸对大黄鱼生长和免疫指标的影响

本试验旨在研究共轭亚油酸(CLA)对大黄鱼(Pseudosciaena crocea,R)生长和免疫指标的影响.选用初体重为(146.25±10.62) g的大黄鱼1 600尾,随机分为4组,每组2个重复,每个重复200尾,分别饲喂在基础日粮中添加了0%、1%、2%和4%共轭亚油酸的试验日粮,试验共进行10周.结果表明,与未添加共轭亚油酸组相比,各共轭亚油酸添加组的饵料系数(FE)、特定生长率(SGR)、肥满度(CF)、肝体比(HSI)和脏体比(VSI)未产生显著影响(P>0.05).添加共轭亚油酸能够显著提高血清中溶菌酶、免疫球蛋白M(IgM)、血清超氧化物歧化酶(SOD)和肝脏SOD活性(P<0.05),并且随共轭亚油酸含量的升高,活性也升高.血清补体(C3、C4)活性2%和4%共轭亚油酸组显著提高(P<0.05),且以2%组活性最高,并显著高于1%和4%组(P<0.05);肌肉SOD 2%和4%共轭亚油酸组显著提高(P<0.05),且以4%组活性最高,但与1%和2%组差异不显著(P>0.05).由此得出,日粮中添加1%～4%CLA不能提高大黄鱼的生长性能,但可显著提高大黄鱼的机体免疫能力.[动物营养学报,2009,21(2):205-211][中文全文见<动物营养学报>网站(www.ChinaJAN.com)中文版2009年21卷2期]