弗氏柠檬酸杆菌感染诱导斑马鱼皮肤免疫相关基因的差异表达

从草鱼肠道中分离到1株细菌,通过形态学观察、生理生化特征和16S rDNA序列分析等鉴定为弗氏柠檬酸杆菌,动物试验结果表明,该菌对斑马鱼有致病性;从感染诱导的斑马鱼皮肤组织中提取总RNA,经Biotin荧光标记与拥有15 617个cDNA片段的斑马鱼基因芯片(affymetrix)杂交筛选分析,获得斑马鱼皮肤免疫相关差异表达基因88个,其中有74个上调表达基因和14个下调表达基因;进一步根据基因功能聚类(GO)分析,初步将88个差异表达基因分为8个生物学功能,其中主要参与补体激活、急性期反应、应激防御反应、细胞凋亡、抗原加工提呈、细胞迁移粘附、凝血因子和血小板激活等免疫应答过程;同时进行信号通路(pathway)分析,结果表明MAPK(hsp70、daxx、nfkbiab)、JAK/STAT(ifn1)和TGF-β(thbs1)等信号通路参与斑马鱼皮肤抗弗氏柠檬酸杆菌感染免疫应答。采用基因芯片方法筛选与鱼类皮肤免疫相关的功能基因,为将来以斑马鱼为模型研究鱼类皮肤局部免疫应答的分子机制提供科学依据。     

中国对虾不同凝集素基因应答WSSV侵染的表达差异

本研究探讨了7种细胞凝集素基因在中国对虾中应答白斑综合征病毒(WSSV)感染上的生物学功能差异.结果显示,LT1 (DQ167572.1)、LT2(EU834289.1)、LT3 (EU834292.1)和LT5(EU834290.1)是肝胰腺组织特异、受WSSV感染诱导表达的基因,其他3种基因主要在肌肉、肠和腮中表达,与WSSV感染诱导无明显相关；在应答WSSV感染不同时间段的表达特征上,7种凝集素基因各不相同；在各组织中的表达量上,方差分析表明7种凝集素基因除在肝胰腺中的表达上存在显著差异外,其他组织中不存在显著差异.这些结果显示这7个凝集素基因在应答WSSV侵染的表达特征上存在较大差异,推测其在中国对虾体内免疫防御功能上的作用也各不相同.     

中华鳖MHCⅡα基因cDNA的克隆及组织表达分析

为了探究中华鳖(Pelodiscus sinensis)的主要组织相容性复合体(major histocompatibility complex, MHC)的结构和功能,本研究利用RACE技术成功克隆获得中华鳖MHCⅡα基因的cDNA全长序列,其长度为1296 bp,开放阅读框(ORF)为807 bp,共编码268个氨基酸,分为信号肽序列、Ⅱ类组织相容性抗原α结构域、IGc1结构域及跨膜结构域4个功能结构域。通过NJ法构建的系统进化树分析显示,中华鳖与西部锦龟(Chrysemys picta bellii)亲缘较近,而与哺乳类、鸟类及鱼类亲缘关系较远。荧光定量PCR结果显示,MHCⅡα mRNA在中华鳖8个组织中均有表达,在脾、心、肝及肠组织中表达水平较高,在肌肉组织中表达量最低。感染嗜水气单胞菌(Aeromonas hydrophila) 12 h后,中华鳖MHCⅡα mRNA在肝和肠组织中的表达显著上调;感染1 d时,在脾组织中的表达显著上调;感染1 d及5 d时,在肾组织中的表达显著上调。研究表明,中华鳖MHCⅡα基因参与了中华鳖免疫反应。     

半滑舌鳎甘露糖结合凝集素相关丝氨酸蛋白酶1基因的克隆及表达分析

甘露糖结合凝集素相关丝氨酸蛋白酶1(Mannan-binding lectin associated serine protease 1,MASP1)是补体凝集素途径中起重要作用的激活蛋白。本研究以半滑舌鳎(Cynoglossus semilaevis)为研究对象,应用RACE技术和实时荧光定量qRT-PCR技术对半滑舌鳎MASP1基因(Cynoglossus semilaevis MASP1,CsMASP1)进行了克隆和表达模式分析。结果显示,CsMASP1基因c DNA序列全长为2507 bp,5′非编码区长82 bp,3′非编码区长142 bp,开放阅读框长2283 bp,共编码760个氨基酸;CsMASP1基因包含13个外显子和12个内含子,与多数已知鱼类的MASP1基因结构一致;SMART分析显示,CsMASP1包含6个结构域,与哺乳动物、鸟类、其他鱼类的结构域一致;同源比对发现,CsMASP1和鱼类的相似度较高,与金目鲈(Latescalcarifer)的相似性最高,为76%。CsMASP1基因在11种健康组织(血液、脑、鳃、性腺、心脏、头肾、肠、肝、皮肤、脾和后肾)中均有表达,其中,在肝、脾和头肾中表达量较高;鳗弧菌(Vibro anguillarum)感染后,CsMASP1基因在6种免疫组织(血液、鳃、头肾、肠、肝和脾)中呈现不同的表达模式,在6种免疫组织中呈现明显的上调表达,肝的表达峰值出现在感染后24 h;脾和鳃的表达峰值出现在感染后6 h;肠、头肾和血液的表达峰值均出现在感染后12h;随着病原菌感染时间增加,基因表达量逐渐降低并恢复至正常水平。研究表明,CsMASP1基因参与了半滑舌鳎的免疫应答过程,本结果为半滑舌鳎的免疫机理研究奠定了基础。     

嗜水气单胞菌感染翘嘴鳜头肾转录组分析

嗜水气单胞菌(Aeromonas hydrophila)是严重危害翘嘴鳜(Siniperca chuatsi)养殖生产的主要病原之一，为揭示嗜水气单胞菌感染翘嘴鳜后宿主基因表达水平的变化，筛选免疫相关基因，解析翘嘴鳜应答病原细菌感染的分子机制，本研究以病原嗜水气单胞菌感染翘嘴鳜，于感染24 h后，采集感染组与对照组翘嘴鳜头肾组织，采用Illumina Hiseq 2000进行了RNA-Seq分析，原始数据拼接后组装共获得53 040个单基因(unigene)。基因差异表达分析结果显示，感染组和未感染组翘嘴鳜存在526个差异表达基因，包括254个上调基因和272个下调基因，其中，免疫相关的显著上调的差异基因主要有炎症和免疫原性细胞因子白介素、补体系统、MHCⅠ型抗原提呈、溶菌酶、丝氨酸蛋白酶抑制因子、泛素蛋白连接酶等。GO富集分析发现，差异基因主要涉及免疫应答反应和炎症反应等，经KEGG富集分析显示，89个通路富集显著，免疫相关的代谢通路主要有内吞作用和吞噬体等。此外，实时荧光定量PCR验证结果表明，所选取7个差异表达免疫相关基因与RNA-seq结果具有相似的表达趋势。本研究为揭示翘嘴鳜对病原微生物感染的防御分子机制奠定了理论基础。     

高盐胁迫下大弹涂鱼MHC Iα基因对病毒拟似物poly(I:C)的免疫反应

主要组织相容性复合体(major histocompatibility complex,MHC)是一类编码细胞表面糖蛋白的基因,在所有硬骨鱼的适应性免疫系统中起着至关重要的作用,而关于MHC基因的研究一直是鱼类分子免疫学和鱼类抗病辅助育种的研究热点之一。本研究首次分析了大弹涂鱼(Boleophthalmus pectinirostris)MHCIα基因的cDNA序列特征,构建了系统发生树,评估了大弹涂鱼MHC Iα基因mRNA在健康个体不同组织中的表达差异,研究了注射病毒拟似物poly(I:C)后MHC Iα基因在机体主要免疫器官肝和脾的表达情况。结果显示,大弹涂鱼MHC Iα基因具有由1101个碱基组成的开放阅读框(ORF),共编码366个氨基酸残基,具有3个蛋白激酶C-磷酸化位点、1个酪蛋白激酶Ⅱ磷酸化位点和1个N-糖基化位点。系统发育分析显示与大弹涂鱼MHCIα基因亲缘关系最密切的是河川沙塘鳢(Odontobutis potamophila)。RT-PCR分析显示,MHCIα基因mRNA在不同组织中均有表达,其中肾和脾组织中表达量最高,鳃和肠组织中表达次之。大弹涂鱼在腹腔注射poly(I:C)后,肝和脾组织中mRNA表达量明显上升,在12 h时, MHC Iα基因mRNA表达量在肝和脾中均达到峰值。本研究结果表明, MHC Iα基因参与了大弹涂鱼在高盐胁迫下的免疫应答。     

尼罗罗非鱼补体C9基因的克隆和组织表达分析

为了探究补体C9在尼罗罗非鱼(Oreochromis niloticus)免疫中发挥的作用,本研究克隆并分析了尼罗罗非鱼补体C9基因(OnC9)。结果显示:OnC9的cDNA序列全长2 502 bp,包含1 761 bp的开放阅读框(ORF),编码586个氨基酸。氨基酸序列同源性分析表明,OnC9与牙鲆(Paralichthys olivaceus)补体C9氨基酸相似性最高,达73.0%,与其他鱼类补体C9的相似性介于49.3%~71.4%之间。实时荧光定量PCR检测结果表明,OnC9基因在所检测的各个组织或器官中表达水平有明显差异,在肝脏中表达量最高,其次是肠道、后肾、皮肤、肌肉、鳃,在脑、脾脏、心脏、头肾、胸腺和血液中表达量最低。在无乳链球菌(Streptococcus agalactiae)感染鱼体后,肝脏、后肾等组织中OnC9表达量表现为在感染12 h、48 h(或36 h)、120 h先后出现三个峰值的波动表达的规律。说明OnC9在无乳链球菌感染尼罗罗非鱼后的免疫应答中发挥作用。     

稀有鮈鲫MHCⅡβ基因克隆及鲁氏耶尔森菌感染后的表达分析

为探究稀有鲫MHCⅡβ基因的分子特征及其表达特点，采用PCR扩增技术获得了稀有鲫MHCⅡβ cDNA序列810 bp，包括开放阅读框(ORF)759 bp，编码252个氨基酸。生物信息分析表明，MHCⅡβ氨基酸序列存在4个保守的半胱氨酸残基和GXXGXXXGXXXXXXG结构，与其他亲缘鱼类的一致性为51.78%～80.56%，其编码的蛋白质分子包括1个信号肽、1个MHCⅡβ (β-1)结构域、1个IGc1 (β-2)结构域和1个跨膜螺旋区域。实时荧光定量PCR (qRT-PCR)结果显示，MHCⅡβ在脾脏表达量最高，头肾、鳃、皮肤表达量较高。人工感染鲁氏耶尔森菌后，6 h时在头肾表达呈显著上调，肝脏中在12 h开始出现极显著上调，皮肤中在24 h和48 h表达极显著，鳃中在6～24 h表达极显著，脾脏中则是在6 h出现极显著下调，于96 h接近对照组表达水平。初步研究表明，MHCⅡβ在鱼类抵御细菌感染的免疫反应中发挥着重要作用，为进一步揭示稀有鲫MHC家族的功能提供了参考依据。     

牙鲆精氨酸酶Ⅱ基因的克隆以及免疫应答表达分析

本研究克隆得到牙鲆(Paralichthys olivaceus)精氨酸酶Ⅱ基因(ArginaseⅡ,Arg-Ⅱ)全长cDNA序列,并检测了Arg-Ⅱ在牙鲆免疫组织和细菌感染过程中的时空表达模式。结果显示,Arg-Ⅱ基因cDNA全长1882 bp,包含1050 bp开放阅读框,编码349个氨基酸(预测分子量为38.02 kDa,等电点为6.42)。Arg-Ⅱ基因编码的氨基酸序列具有典型的精氨酸酶结构特征,推测与哺乳动物中的功能类似。系统进化分析显示,鱼类Arg-Ⅱ基因合成一簇,其中,Arg-Ⅱ基因与鲈鱼(Lates calcarifer)相关度最高(93%)。实时荧光定量结果显示,Arg-Ⅱ基因在健康牙鲆的肝、脾和鳃等免疫组织中有较高表达。进一步研究发现,经迟缓爱德华氏菌(Edwardsiella tarda)感染的牙鲆成鱼中,主要免疫组织中Arg-ⅡmRNA的表达量在细菌感染后6~12 h显著上调,随后表达量恢复正常。离体培养的牙鲆巨噬细胞经迟缓爱德华氏菌感染后,Arg-Ⅱ基因也呈显著上调模式。因此,本研究结果表明,Arg-Ⅱ基因参与牙鲆响应迟缓爱德华氏菌感染的免疫过程,揭示Arg-Ⅱ基因可能在牙鲆抗病免疫中发挥重要作用。     

长鳍吻(鳍)免疫因子基因cDNA部分序列克隆及其组织表达差异

根据鲤科鱼类同源序列设计并合成了长鳍吻(鳍)(Rhinogobio ventralis)应激因子HSP70、抗体IgM、抑炎性因子IL-10和促炎性因子IL-1b基因特异引物,以长鳍吻(鳍)皮肤组织总RNA为模板,RT-PCR 扩增获得长度分别为412、463、520和217 bp的上述4种免疫因子基因cDNA部分序列.同时通过RT-PCR比较患小瓜虫病长鳍吻1715和对照组长鳍吻(鳍)皮肤和肠道组织中各免疫因子的表达差异,结果显示:在皮肤组织中,IgM和IL-10在感染组鱼体中表达量显著高于对照鱼(P<0.05), HSP 70和 IL-1b的表达则没有差异;在肠道组织中,IgM、IL-10和 IL-1b在感染组鱼体中表达量显著增高,HSP 70的表达量没有差异.本研究首次对长鳍吻(鳍)的免疫因子进行了分析,指出免疫因子IgM和IL-10在鱼体中免疫应答反应中较为灵敏,为养殖过程中长鳍吻(鳍)应激监测方面奠定了理论基础.     

Role of major histocompatibility complex II antigen-presentation pathway genes in orange-spotted grouper infected with Cryptocaryon irritans

Cryptocaryon irritans, a pathogen model for fish mucosal immunity, causes skin mucosal and systematic humoral immune response. Where and how MHC II antigen presentation occurs in fish infected with C. irritans remain unknown. In this study, the full-length cDNA of the grouper cysteine protease CTSS was cloned. The expression distributions of six genes (CTSB, CTSL, CTSS, GILT, MHC IIA and MHC IIB) involved in MHC II antigen presentation pathway were tested. These genes were highly expressed in systematic immune tissues and skin and gill mucosal-associated immune tissues. All six genes were upregulated in skin at most time points. Five genes expected CTSS was upregulated in spleen at most time points. CTSB, CTSL and MHC IIA were upregulated in the gill and head kidney at some time points. These results indicate that the presentation of MHC II antigen intensively occurred in local infected skin and gill. Spleen, not head kidney, had the most extensive systematic antigen presentation. In skin, six genes most likely peaked at day 2, earlier than in spleen (5–7 days), marking an earlier skin antibody peak than any recorded in serum previously. This significant and earlier mucosal antigen presentation indicates that specific immune response occurs in local mucosal tissues.     

Skin‐injured Zebrafish,Danio rerio,are more Susceptible to Vibrio anguillarum Infection

Vibrio anguillarum, which is part of normal microflora on fish, is the causative agent of vibriosis in aquaculture. It is speculated that V. anguillarum does not affect the host in most situations, but can cause a severe disease once the host is compromised. In the study reported herein, skin‐injured and intestine‐injured zebrafish, Danio rerio, were established as a model to mimic the natural infection caused by V. anguillarum when fish suffered an injury to a mucosal surface. Our results showed the lethal dose to 50% of the population (LD₅₀) of skin‐injured zebrafish was 6.8 × 10³ colony‐forming unit (CFU)/mL, which was much lower than intestine‐injured zebrafish (1.9 × 10⁶ CFU/mL) or non‐injured zebrafish (5.5 × 10⁶ CFU/mL). With the quantitative polymerase chain reaction and immunohistochemical analysis, we found that V. anguillarum proliferated rapidly in the skin and muscle after the bacteria entered into the host via the skin injury. The bacteria were subsequently transported to the immune organs and then caused a systemic infection in the fish. However, mortality of skin‐injured zebrafish significantly decreased if the fish were allowed to heal. These results indicate that minimizing injury to the mucosal surfaces of fish, especially the skin, will reduce infections caused by V. anguillarum.     

1,3‐1‐6 ß‐glucans enhance tissue regeneration in zebrafish (Danio rerio): Potential advantages for aquaculture applications

High aquacultural rearing density and handling of fish may frequently result in skin or gills wounds, thereby facilitating the onset of secondary infections. The capacity of the zebrafish to regenerate tissues, as well as fins and other organs, makes it an ideal animal model for studying the mechanisms of tissue regeneration. Since macrophages are involved in tissue regeneration, a diet including ß‐glucans might positively affect the process through activation of macrophages and other immune pathways. Consequently, the aim of this study was to investigate the effects of the oral administration of 1,3‐1,6 β‐glucans on the regeneration process of the caudal fin after its amputation in zebrafish. One hundred and twenty zebrafish were randomly distributed into four groups with three replicates each: an untreated non‐amputated group (CNA) and an untreated amputated group (CA) fed a control diet; two treated and amputated groups (MI and MII) fed for 14 days the same diet with the addition of two differently extracted 1,3‐1,6 ß‐glucans (MacroGard^® and Experimental MacroGard^®, Biorigin^©). ß‐glucans were added to allowed the administration of 12.5 mg/kg of fish body weight (0.35 g/kg of feed). Results showed that 1,3‐1,6 ß‐glucans decreased fish mortality rate and enhanced both daily and cumulative regenerated fin area, independent of the specific ß‐glucan extraction method used. Based on the mechanisms similarities of the innate immune system and tissue regeneration among different teleost species, these results may likely be extended to species of interest for the aquaculture sector.     

Identification of a localized mucosal immune response in rainbow trout, Oncorhynchus mykiss (Walbaum), following immunization with a protein-hapten antigen

The ability of rainbow trout, Oncorhynchus mykiss (RBT), to produce a localized mucosal immune response was investigated following intraperitoneal (i.p.) or peranal (p.a.) immunization with a protein-hapten carrier, fluorescein isothiocyanate conjugated to keyhole limpet haemocyanin (FITC/KLH). Antibody levels in serum, mucus, tissue culture supernatant from blood and spleen leucocytes, and excised skin, intestine and gill tissues were determined by ELISA. Significantly, elevated antigen-specific antibodies were elicited in both serum and mucus of fish immunized i.p. Mucosal antibody responses, in general, paralleled serum responses over time. Leucocytes isolated from spleen and blood of i.p. immunized fish at week 10 produced significantly elevated antibody levels against FITC when cultured in vitro. Excised skin, intestine and gill tissues from these fish also exhibited significantly elevated antibody responses indicating localized production in the mucosa from tissue-specific B cells. A localized mucosal immune response was elicited only after i.p. and not p.a. immunization, suggesting that systemically stimulated B cells migrate to mucosal tissues where they produce antibodies locally.     

Cutaneous antibodies in excised skin from channel catfish, Ictalurus punctatus Rafinesque, immune to Ichthyophthirius multifiliis

This study determined whether cutaneous antibodies were present in the excised skin of channel catfish, Ictalurus punctatus Rafinesque, immune to Ichthyophthirius multifiliis Fouquet (Ich). Theronts were immobilized on or near the excised skin from immune fish. The survival of immobilized theronts was significantly reduced after exposure for 8 h to the culture of excised skin from immune fish. Culture fluids from excised skin of immune fish immobilized theronts with a peak in the immobilization titre at 24 h post-exposure. Immobility of theronts in the culture fluid from immune skin was removed after immunoabsorption with theronts. Indirect immunofluorescent staining of theronts treated with culture fluid from excised skin of immune fish revealed strong and uniform fluorescence on the cilia and cell surface of theronts. Western blot analysis of the culture fluid from immune fish revealed a 70-kDa band which corresponded to the molecular weight of catfish immunoglobulin heavy chain. The results of this study show that cutaneous antibodies to Ich theronts were present in and released from the excised skin from fish immune to Ich. Immobilization and killing of the theronts are two characteristics of the antibody response that appear to prevent the successful invasion of theronts into excised skin.     

鱼类粘膜免疫研究进展

Fish immunology has achieved great progress in recent years. While before 1990s, most researches focused on the fish systematic immunity, and the mucosal immunity of fish had not been given enough attention. Indeed, it has been shown that fish mucosal immunity plays an important role in disease defense. Fish mucosal immunity research has made some exciting progress in this decade. This review will focus on such progress: Constitution of mucosal-associated tissues and distribution of different immune cells, including T/B lymphocytes, granules, monocytes, macrophages, goblet cells, etc, in these sites have been well described with the development of some monoclonal antibody to these cells and associated techniques. Non-specific immune response mechanism of mucosal tissues reported these years, such as secretion of non-specific anti-bacteria and anti-fungi substances in mucus, the respiratory burst, enzyme activity of immune cells and so on, is believed important for fish disease defense. The specific immunity of mucosal tissues also attracts much interest and makes great achievement in antigen presenting, MHC genes, antibody producing and antibody secreting cells, comparison of serum and mucus immunoglobulin, relationships of immune response between different mucosal immune tissues. Whether mucosal immune system is independent of systematic immune system is another interesting question and causes great concern. In recent years, some evidences from phyletic evolution and ontogenesis show that mucosal immunity is prior to systematic immunity in evolution. Dynamics of antibody producing of mucosal tissues and serum in immersion or oral vaccines immunized fish also shows immune response can be elicited in mucosal tissues independent of systematic immune system. Some researchers also begin to pay attention to factors involved in mucosal immune regulations, for instance, neuromodulators and cytokines. The level of these factors changes in fish immune response process but the mechanisms of regulation still remain unknown. Prospect of the promising future of fish mucosal immunity has also been discussed in this review.     

Current knowledge of nocardiosis in teleost fish

Nocardia sp. is the causative agent of nocardiosis, a lethal granulomatous disease of the skin, muscle, and various inner tissues affecting various teleost and shellfish. Four species of Nocardia have been isolated from diseased fish and shellfish, namely Nocardia asteroides, Nocardia seriolae, Nocardia salmonicida and Nocardia crassostreae. Therefore, in fish aquaculture, nocardiosis has caused severe economic losses, especially in the Asian region. Considerable research has been performed, since the first report of identified Nocardia sp. in fish, to characterize Nocardia sp. and identify rapid detection techniques, immune response against infection and prophylactic approaches. In this review, the current state of knowledge about nocardiosis in fish has been presented, including the pathogenesis, diagnosis, host immune response and vaccine development.