Expression profiles of interferon gamma genes in response to immunostimulants and alloantigen in ginbuna crucian carp Carassius auratus langsdorfii

Interferon gamma (IFNγ), a Th1-derived cytokine is one of the key molecules inducing cell-mediated immune responses in mammals. The expression of 2 distinct IFNγ (ifng1 and ifng2) and IFNγrel (ifngrel) genes was examined in kidney leukocytes from clonal ginbuna crucian carp Carassius auratus langsdorfii. The expression of IFNγ and IFNγrel genes was induced in kidney leukocytes by stimulation with lipopolysaccharide or phytohemagglutinin in a dose-dependent manner. The expression levels of IFNγ and IFNγrel genes in kidney leukocytes from allo-sensitized fish cocultured with allogeneic cells were higher than those from fish cocultured with isogeneic cells. The highest expression of ifng1 was observed at day 1, whereas that of ifng2 and ifngrel was detected at day 2 after cocultivation with allogeneic cells. However, the expression pattern of ifng2 and ifngrel in kidney leukocytes from allo-sensitized fish by scale-grafting was similar to those from non-sensitized fish. These results indicate that ifng1 is a major isoform of IFNγ related to antigen-specific cell-mediated immunity (CMI), and will be a useful marker to evaluate induction of CMI in ginbuna crucian carp. In addition, IFNγ would be a crucial type-II interferon compared to IFNγrel, relevant to cell-mediated immunity in fish as in mammals.     

草鱼干扰素3蛋白多克隆抗体制备及其应用

为探讨干扰素3(Interferon 3,IFN3)在抗病毒免疫应答中的作用,以草鱼(Ctenopharyngodon idella)巨噬细胞cDNA为模板,PCR扩增IFN3成熟肽基因序列,制备草鱼IFN3蛋白的多克隆抗体,同时研究了IFN3在草鱼不同免疫组织中的蛋白表达,以及草鱼呼肠孤病毒(grass carp reovirus,GCRV)感染草鱼肾细胞系(Ctenopharyngodon idella kidney,CIK)后不同时间点的表达。结果显示,细菌表达的重组IFN3大小约为45 kD,主要以包涵体形式存在;抗体效价约为1∶3 200,制备的多克隆抗体既能识别原核表达的重组蛋白,也能识别个体水平上和细胞水平上的内源蛋白。草鱼主要免疫组织中,肝胰腺和皮肤检测到相应条带。CIK细胞感染病毒后12 h开始检测到IFN3蛋白,随感染时间的延长,IFN3蛋白表达量有所增加。蛋白水平上检测IFN3的表达,为深入研究草鱼的抗病毒免疫机制奠定了基础。     

家禽干扰素的研究进展

自1957年,Issacs等从鸡胚中发现干扰素以来,家禽干扰素的研究已取得了突破性的进展.研究表明干扰素具有抗病毒、抗肿瘤、免疫调节等功能,且干扰素基因工程产品在临床上的应用也取得了显著的成效,但是还有一些问题需要进一步的探索。本文着重就家禽IFN的产生、分类、作用机理、研究现状及目前存在的问题和应用前景做一综述。     

鱼类IRF家族在干扰素抗病毒免疫反应中的调控功能

干扰素调节因子(IRF)家族成员是一类重要的转录因子,在机体细胞感染病毒时单独或共同调控干扰素基因(IFN)的表达。IRF家族蛋白结构非常保守。其中N端DBD结构域赋予了IRF蛋白结合IFN启动子的功能,而C端IAD主要介导蛋白互作,因而不同IRF成员可能在不同的信号通路中发挥功能。哺乳动物IRF家族有9个成员,IRF1～9。鱼类IRF家族有11个成员,除了IRF1～9外,还包括硬骨鱼类特有的IRF11,以及在鸟类基因组中也存在的IRF10。哺乳类研究表明,IRF1/3/5/7/9在病毒感染的细胞中正调控IFN基因的表达,而IRF2则负调控IFN的表达。近十多年来,鱼类IRF家族的功能研究取得了重要进展,相关研究结论基本来自于体外的实验数据。本文综述了鱼类IRF家族成员的表达、亚细胞定位以及调控IFN抗病毒免疫反应的分子机制。     

Histology,immunocytochemistry and qRT‐PCR analysis of Atlantic salmon,Salmo salar L., post‐smolts following infection with infectious pancreatic necrosis virus (IPNV)

Infectious pancreatic necrosis (IPN) is a very serious viral disease in terms of its impact on production of Atlantic salmon, Salmo salar L., fry and post‐smolts. Post‐smolts of Atlantic salmon were injected with infectious pancreatic necrosis virus (IPNV) and cohabited with naive fish to produce natural infection. Cohabitant fish were sampled every 2 days, up to day 36 post‐infection (p.i.). From 90 cohabitant fish, 11 (12.2%) were positive by immunohistochemistry (IHC). The first detection of IPNV by IHC occurred on day 16 p.i. which coincided with the onset of mortality in this group. Besides the pancreas, the liver was found to be a key target organ for IPNV. For the first time, the virus was observed in the islets of Langerhans and in the kidney corpuscles of Stannius which suggests that the virus could affect the fish’s metabolism. The liver of two fish, which showed the most widespread presence of IPNV by IHC, had a pathology including focal necrosis and widespread presence of apoptotic hepatocytes, many of which did not stain for virus by IHC. Up‐regulation of cytokine gene expression was found only in the IHC‐positive (IHC+ve) fish and reflected the level of infection as determined by IHC positivity of the liver. In most fish, interferon (IFN), Mx, γIFN and γIP were up‐regulated in liver and kidney, while only IFN and Mx were up‐regulated in gill. IL1β and TNFα were not induced in any tissue. The gill showed variable levels of constitutive expression of IL1β and γIFN. The two fish with liver pathology had the highest level of IFN expression, especially relative to the level of Mx expression, in the liver compared with the other IHC+ve fish which did not have a liver pathology. The results suggest that following widespread infection of hepatocytes, the cells may over‐produce IFN, resulting in apoptosis of neighbouring cells with subsequent death from liver failure.     

4种环境源性胁迫对异育银鲫血浆生化指标的影响

以异育银鲫(Carassius auratus gibelio)幼苗[(136.70±7.98)g]为实验对象,监测4种环境源性胁迫(高pH 9.2、低溶氧2 mg·L~(-1)、高亚氮2 mg·L~(-1)和高氨氮4 mg·L~(-1))下第3、7、10、15天血浆内皮质醇(COR)、葡萄糖(GLU)、I型干扰素(IFN)、白细胞介素-2(IL-2)、超氧化物歧化酶(SOD)、免疫球蛋白M(Ig M)、补体3(C3)以及丙二醛(MDA)的变化规律。结果显示:COR经高pH、高亚氮和高氨氮胁迫时显著升高,而SOD显著降低,Ig M经高亚氮和高氨氮胁迫时亦显著升高,表明COR、SOD和Ig M可能是广源性的胁迫状态指示指标,COR和Ig M的升高以及SOD活力的降低指示异育银鲫可能处于被胁迫状态。GLU、IFN和C3只经高氨氮胁迫时显著升高,且响应幅度剧烈,表明GLU、IFN和C3可能是灵敏且狭源性的应激指示指标,高水平指示机体可能处于氨氮应激状态。IFN和MDA经高亚氮和高氨氮胁迫时,表现出不同的响应趋势:氨氮胁迫时IFN和MDA显著升高,但亚氮胁迫时则显著降低,表明IFN和MDA响应模式可能具有特异性,不适合指示综合应激状态。     

Immunomodulation and interferon gamma gene expression in sutchi cat fish,Pangasianodon hypophthalmus: effect of dietary fucoidan rich seaweed extract (FRSE) on pre and post challenge period

A 45‐days feeding trial was conducted to study the immunomodulatory effect and interferon gamma gene expression of dietary fucoidan rich seaweed extract (FRSE) from Sargassum wightii on Pangasianodon hypophthalmus fingerlings. One hundred and eighty fingerlings were distributed into six experimental groups in triplicates. Each group was stocked with 10 fish and fed to satiation with iso‐nitrogenous (34.96 ± 0.09–35.18 ± 0.03 CP%) and iso‐caloric (368.65 ± 0.86–375.09 ± 0.26 Kcal/100 g) purified diets containing either 0% FRSE (control), 1% FRSE (TF₁), 2% FRSE (TF₂), 3% FRSE (TF₃), 3% seaweed powder (TS₃) or 6% seaweed powder (TS₆) in the feed. After feeding trial the experimental fish were challenged with Aeromonas hydrophila. Immunological parameters like respiratory burst activity, lysozyme activity, phagocytic activity and total leukocyte count (TLC) were increased with the increasing level of dietary FRSE, whereas serum Albumin/Globulin (A/G) ratio and blood glucose level exhibited decreasing trend (P < 0.05). Increased TLC, blood glucose level, respiratory burst activity, serum A/G ratio, lysozyme and phagocytic activities were recorded during the post‐challenge period. Maximum expression of interferon gamma (IFN‐γ) gene was recorded in FRSE fed groups than the control group both in pre and post challenge condition. After challenged with A. hydrophila the highest survival was observed in TF₂ and TF₃ groups whereas lowest survival was observed in the control group. Hence, dietary supplementation of FRSE at an optimum level of 2% reduced the stress and improved the immune status of P. hypophthalmus fingerlings.     

病毒感染过程中青鳉irf2基因过表达的双面性

为了探究干扰素调节因子2 (interferon regulatory factor,IRF2)如何通过调控干扰素(IFN)表达影响鱼类的免疫,实验从青鳉中克隆了irf2 (Olirf2),发现该基因在青鳉各个组织中均有表达;将构建的真核表达载体pTol2/CMV-IRF2/IE1-pr转染到胖头鱥肌肉细胞系(FHM)后,发现瞬时过表达Olirf2能够显著促进鲤春病毒血症病毒(spring viremia of carp virus,SVCV)的复制,并抑制抗病毒相关基因mx1、ifn和irf3的表达。进一步通过双荧光素报告系统发现,Olirf2能够显著抑制NF-κB和ISRE的活性,说明Olirf2可能通过抑制细胞的天然免疫应答进而促进病毒的增殖。然而持续过表达Olirf2则增强了细胞的抗病毒能力,同时促进干扰素相关基因mx1、ifn和irf3的表达。因此,Olirf2基于表达的持续时间不同而具有抗病毒或者促病毒的双面效果。实验通过研究Olirf2在抗病毒信号通路中发挥的作用,为通过基因编辑或者转基因手段来构建抗病毒的鱼类提供了理论基础。     

抑制性消减杂交技术在养殖鱼类免疫基因克隆中的应用

抑制性消减杂交技术(SSH)是近年来兴起的一种分离、克隆差异基因的新技术,它结合了消减杂交和抑制PCR的优点,具有操作简便、特异性强、背景低及重复性好的优点。目前已经用SSH技术鉴定出了鱼的许多免疫相关基因,如白细胞介素、趋化因子、肿瘤坏死因子、溶菌酶、NKEF、补体、干扰素及急性期蛋白基因等,对它们的结构和功能进行了较为深入的研究。本文对SSH技术在养殖鱼类中克隆的免疫基因进行了归纳与总结,旨为全面了解鱼类的抗病免疫基因提供基础资料。     

Oral bovine serum albumin immune‐stimulating complexes improve the immune responses and resistance of large yellow croaker (Pseudosciaena crocea,Richardson, 1846) against Vibrio alginolyticus

This study aimed to investigate effects of bovine serum albumin immune‐stimulating complexes (BSA ISCOMs) on immune‐related genes expression, serum nonspecific immunity and disease resistance of large yellow croaker (Pseudosciaena crocea). Fish were fed diets containing 3.5 ml of BSA ISCOMs per kg feed (experimental group) or 3.5 ml of phosphate‐buffered saline per kg feed (control group) for 1 week. The liver, spleen, head‐kidney tissues were sampled for determining gene expression of myxovirus‐resistant protein (Mx), major histocompatibility complex class II alpha chain (MHC II α), tumour necrosis factor‐alpha (TNF‐α) and interleukin‐10 (IL‐10) 30 and 90 days after feeding. Also, blood samples were collected for determining activities of serum superoxide dismutase (SOD), interferon alpha (IFN‐α), TNF‐α and alkaline phosphatase (ALP). TNF‐α and MHCⅡα gene expression in the liver, spleen, head‐kidney, as well as IFN‐α, TNF‐α and ALP activities in the serum, of experimental fish were significantly higher 30 days after feeding; while only TNF‐α and MHC II gene expression in the head‐kidney remained upregulated 90 days after feeding. The cumulative mortality of the experimental fish was significantly lower than control. This study indicated that BSA ISCOMs improved the immune response and induced protective immunity in large yellow croaker.     

Effect of growth hormone on muscle protein synthesis in rainbow trout (Oncorhynchus mykiss) and Atlantic salmon (Salmo salar)

This paper reports on the effect of administration of mammalian growth hormone (GH) on muscle protein synthesis as measured in white muscle using the phenylalanine flooding technique. The effect of exogenous GH was compared with that of insulin and prolactin, and with endogenous GH.The rate of protein synthesis in white muscle of rainbow trout 6 h after the injection of bovine GH or bovine insulin was twice (2.6 and 2.9% d^–1) that of the control saline-injected fish (1.2% d^–1). A metabolic effect of GH, as observed with insulin, is suspected.The rates of change in body weight and body length and the fractional rate of protein synthesis in muscle of rainbow trout were enhanced by mammalian GH administration. The effect of GH on muscle RNA/protein ratios was not significant. An opposite effect of antibodies against salmon GH (Lebailet al. 1989) on growth rate and muscle protein synthesis rate was found in rainbow trout. It is suggested that the effects of exogenous and endogenous GH on capacity and efficiency of muscle protein synthesis were similar.The long-term effects of mammalian GH on presmolt Atlantic salmon was also tested. The same trends were found with ovine prolactin supplementation in Atlantic salmon but not as high as those observed with ovine GH.     

草鱼干扰素的分离纯化及某些理化和生物学特性

通过ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ阴离子交换层析、Ｓｅｐｈａｃｒｙｌ Ｓ－２００凝胶层析、高效液相层析和梯度聚丙烯酰张胶电泳等技术,对病毒诱生的草鱼血清干扰素进行了分离纯化。纯化的草鱼干扰素在ＳＤＳ－聚丙烯酰胺凝胶电泳中呈现单一组份,分子量为３８ｋＤ,等电点为５．２５,过碘酸－Ｓｃｈｉｆｆ试剂反应表现为典型的糖蛋白染色特征。理化和生物学特性研究表明,草鱼干扰素具有１０００００ｇ（２ｈ）离心不沉降、耐     

鸡干扰素生产条件的比较研究〈一〉

本试验用鸡白细胞。鸡脾细胞、鸡胚成纤维细胞以Ge－132、NDV－F、PHA、PolyI:C为诱生剂,诱生外源性IFN,将鸡胚成纤维细胞—水泡性口炎病毒作鸡IFN的检测系统,对不同种细胞、不同的细胞浓度、不同的IFN诱生剂及培养方法等诸因素对IFN产生的影响进行了比较：研究了Ge-1322、NDV－F、PHA、PolyI：C在鸡胚成纤维细胞上的最佳诱生剂量;同时也进行了不同诱生剂在不同细胞上产生的IFN的理化性状研究,结果表明：（1）在Ge－132、NDV－F、PHA、PolyI：C四种诱生剂中,诱生能力差异极显著（P＜0.01）,且以Ge－132诱生IFN的能力为最强,其他依次为NDV－F、PolyI：CPHA（2）鸡脾细胞和鸡白细胞产生的IFN效价高于鸡胚成纤维细胞;（3）鸡脾细胞和鸡白细胞悬浮培养比静置培养产生的IFN效价高近1倍;（4）雏鸡脾细胞比成鸡脾细胞产生的IFN效价高;（5）未免疫鸡鸡胚成纤维细胞比免疫鸡鸡胚成纤维细胞产生IFN效价高;（6）鸡胚成纤维细胞在IFN诱生剂的作用下,4h即可产生IFN,20－24H达到最高产量;（7）IFN诱生剂最佳诱生剂量;鸡NDV－F为128HAU／ml;Ge－132为70ug／mlPolyI：C为50ug／ml;PHA为40ug／ml     

Real‐time quantification of the immune gene expression in rainbow trout fed different forms of probiotic bacteria Lactobacillus rhamnosus

Whether it is better to use viable or non‐viable probionts in aquaculture is still a matter of debate. In this study, the molecular immunomodulation in rainbow trout Oncorhynchus mykiss induced by viable or killed forms of the probiont Lactobacillus rhamnosus JCM 1136 was investigated. Three forms of this probiont: (1) heat‐killed (HK), (2) live spray (LI) and (3) freeze‐dried (FD) were incorporated into a basal (control) diet for rainbow trout O. mykiss. The LI and FD diets are referred to as viable diets. A rearing trial, in triplicate, was conducted for 30 days, with the control and probiotic diets as treatments. The cytokine genes such as the tumour necrosis factor (TNF), transforming growth factor (TGF‐β), interferon (IFN) and immune gene Immunoglobulin (Ig) found in tissues from the kidney and spleen were assessed for their expression pattern by real‐time polymerase chain reaction. The tested immune genes were up‐regulated in the treatment groups, sometimes even in many folds like in the case of the Ig gene. The TNF gene was found to be highly (P<0.05) up‐regulated (5000‐fold) in groups fed both viable forms (LI, FD). With regard to the TGF‐β gene, the spleen of the HK and FD groups showed significant up‐regulation of 20‐ and 30‐folds respectively. The IFN gene was up‐regulated (P<0.05) in all treatments, but more in the viable diet treatments. Kidney and spleen tissues showed similar expression patterns, i.e. all of these genes were up‐regulated more with the viable diets that with the control, and in most cases, the viable diets induced a higher expression of the immune genes than the HK diet.     

Availability of culture filtrate protein-10 (CFP-10) secreted from Mycobacterium pseudoshottsii for mycobacteriosis diagnosis in ginbuna crucian carp Carrasius auratus langsdorfii

Mycobacteriosis in cultured fish is a challenge for the aquaculture industry worldwide. Treatment by chemical administration is difficult and no effective vaccine has been developed. Therefore, detection and isolation by early diagnosis are important for prevention of the spread of the disease. In mammals, interferon gamma release assays have been established for detection of tuberculosis; these tests are based on the delayed-type hypersensitivity (DTH) response against culture filtrate protein-10 (CFP-10) and the 6-kDa early secreted antigen target (ESAT-6) of Mycobacterium tuberculosis. On the other hand, little is known about the fish immune response against the ESAT-6 and CFP-10 proteins of mycobacteria, although these responses should find application in the diagnosis of mycobacteriosis in fish. In the present study, we identified ESAT-6 and CFP-10 from Mycobacterium pseudoshottsii and cloned the corresponding genes. Intraperitoneal injection of the corresponding DNA plasmid constructs in ginbuna crucian carp yielded increased expression of the fish interferon-γ1-1-encoding gene (IFN-γ1-1). In contrast, IFN-γ1-1 expression accompanied by DTH response was observed only in the CFP-10-DNA plasmid-injected fish. Furthermore, fish that had been prophylactically injected with CFP-10-DNA plasmid exhibited increased survival of M. pseudoshottsii infection. Taken together, these results suggested that CFP-10 may facilitate diagnosis of mycobacteriosis.     

Dietary Houttuynia cordata leaf extract and meal enhances the immunity and expression of immune genes in Labeo rohita (Hamilton, 1822)

A feeding trial was conducted to explore the effect of dietary Houttuynia cordata leaf extract (HCLE) and leaf meal (HCLM) on immunological responses and expression of interferon‐gamma (IFN‐γ) and tumour necrosis factor‐alpha (TNF‐α) gene in Labeo rohita fingerlings. Six isonitrogenous (350 g/kg CP) and isocaloric (17 MJ/kg DE) purified experimental diets were formulated with Houttuynia cordata leaf extract and leaf meal comprising control, C (0 g/kg HCLE and HCLM), E2.5 (2.5 g/kg HCLE), E5 (5 g/kg HCLE), E10 (10 g/kg HCLE), M10 (10 g/kg HCLM) and M20 (20 g/kg HCLM). Labeo rohita fingerlings (3.37 ± 0.23 g) were distributed in six experimental groups in triplicates following the complete random distribution. Fish were fed twice daily with respective experimental diets for a period of 60 days. A significantly (p < .05) lower lactate dehydrogenase, malate dehydrogenase, superoxide dismutase and catalase activities were registered in supplemented groups compared with control group, while respiratory burst and lysozyme activities were significantly (p < .05) higher in E10 group compared with other experimental groups. Haemoglobin, total leucocyte count, total erythrocyte count and haematocrit values were significantly (p < .05) higher in E10 group. The expression of IFN‐γ and TNF‐α in both the kidney and liver was significantly up‐regulated in leaf extract and meal supplemented groups with the highest expression in the fish of E10 group. Overall, these results suggest that the dietary supplementation of ethanolic extract of the Houttuynia cordata leaf at 10 g/kg level can enhance the immune response of L. rohita fingerlings.