双齿围沙蚕与赤子爱胜蚓对凡纳滨对虾生长和免疫指标的影响

以鲜活饵料双齿围沙蚕（Perinereis aibuhitensis）、赤子爱胜蚓（Eisenia foetida）和人工配合饵料投喂凡纳滨对虾（Litopenaeus vannamei），并测定各种饵料对其生长和免疫指标的影响。结果表明，沙蚕和蚯蚓单独投喂或与人工配合饵料配合投喂都可显著提高凡纳滨对虾的生长速率，但用沙蚕单独投喂的对虾成活率较低。蚯蚓与人工配合饵料配合投喂可显著提高对虾血清中抗菌活力、溶菌活力、酚氧化酶活力和血细胞数量；蚯蚓单独投喂可显著提高血清中抗菌活力、酚氧化酶活力和血细胞数量，对血清中溶菌活力作用不明显。沙蚕与人工配合饵料配合投喂可显著提高对虾血清中抗菌和溶菌活力．但对血清中酚氧化酶活力和血细胞数量变化作用不明显；单独投喂沙蚕对所测各项免疫指标影响均不显著。研究结果证实：利用蚯蚓和沙蚕可显著提高对虾的生长速率，但蚯蚓的饵料效果优于沙蚕。[中国水产科学，2006，13（4）：561—565]     

中国对虾血红细胞吞噬功能的研究

本研究建立了检测对虾血细胞吞噬的功能吖啶橙检测法，该检测同时直观地中国对虾血细胞对金黄色葡萄球菌的吞噬和杀伤情况；一项试验可同时获得吞噬和杀伤百分率，吞噬和杀伤指数四个指标。用本法研究四种免疫增强剂对对，血细胞吞功能的作用，结果表明，均呆提高血细胞对金葡菌的吞噬活力以及血淋巴中的溶菌活力和酚氧化酶活力，但经统计学分析，结合攻毒试验的结果，浙农Ｉ号和湖州Ｉ号的免疫效果优于湖州Ⅱ号和湖州Ⅲ号。     

感染白斑综合病毒（WSSV）对虾相关免疫因子的研究

129尾中国对虾（Penaeus chinensis)分别捕自未暴发白斑综合症（WSSV病毒所致）虾池、WSSV暴发虾池以及曾暴发WSSV虾池。用斑点杂交和组织病理学方法确定各尾对虾的染毒（WSSV）程度。用96孔酶标板法测量相应个体血淋巴上清液的抗菌活力（Ua)、溶菌活力（UL）、酚氧化酶（PO）活性以及过氧化酶（POD）相对活性；用硝酸纤维膜斑点法测定其碱性磷酸酶（ALP）相对活性；用血凝法测定其凝集效价（HAT）。通过对以上免疫指标进行统计分析，结果表明，WSSV感染与对血淋巴PO活性以及ALP相对活性变化有紧密联系；不同虾池各免疫因子差异显著，发病虾池虾样各免疫指标平均值均低于其他虾池；曾发病虾池的虾样PO活性较强；WSSV与HPV感染无统计学意义上的相关性；未发病虾池与曾发病虾池实验对虾的Ua与UL相关性极显著，发病虾池实验对虾Ua与UL呈负相关；发病虾池对虾PO与ALP活性相关性显著。不同性别中国对虾血淋巴上清液的免疫因子活性没有显著差异。     

中国对虾血细胞吞噬功能的研究↑（*）

本研究建立了检测中国对虾血细胞吞噬功能的橙检测法。该检测法可同时直观地观察中国对虾血细胞对金黄色葡萄球菌的吞噬和杀伤情况：一项试验可同时获得吞噬和杀伤百分率，吞噬和杀伤指数四个指标。用本法研究四种免疫增强剂对时虾血细胞吞噬功能的作用，结果表明，他们均可提高血细胞对金葡菌的吞噬活力以及血淋巴中的溶菌活力和酚氧化酶活力，但经统计学分析，结合攻毒试验的结果，浙农Ⅰ号和湖州Ⅰ号的免疫效果优于湖州Ⅱ号和湖州Ⅲ号。     

氨氮对凡纳对虾免疫指标的影响

以凡纳对虾(Litopenaeus vannamei)为研究对象,研究氨氮对其免疫指标的影响。实验氨氮质量浓度梯度设置为0.05(对照)、0.5、1.0、1.5、2.0、2.5 mg／L,各氨氮梯度用4g／L的氯化铵溶液来调节。将暂养在自然海水(对照)中的凡纳对虾分别放入各实验梯度中,对虾体长为(8.5±0.5)cm。结果表明,氨氮对凡纳对虾血细胞数量、血清中的酚氧化酶活力、溶菌和抗菌活力的影响显著(F>F0.05),且随着氨氮质量浓度的升高,血细胞数量和溶菌、抗菌活力降低,酚氧化酶活力升高;在0-24 h实验时间内,各处理组(对照组除外)对虾血细胞数量和溶菌、抗菌活力呈下降趋势,酚氧化酶活力呈上升趋势,24 h后稳定在较低水平上。实验说明,随着氨氮水平升高,凡纳对虾免疫力明显下降,对病原菌的易感性提高,因此在养殖过程中,环境氨氮变化幅度不应超过0.5 mg／L,或长时间维持在较高氨氮水平(>0.5 mg／L)。     

泥蚶血清及血细胞免疫功能研究

初步研究了泥蚶(Tegillarca granosa)血细胞离体条件下的吞噬作用以及血清因子对其的影响,并测定经大肠杆菌注射以后,泥蚶血清中抗菌、溶菌、酚氧化酶(PO)、超氧化物歧化酶(SOD)活力的变化。结果表明,泥蚶血清能促进血细胞对大肠杆菌的吞噬,但不能识别鸡红细胞。大肠杆菌激发后泥蚶血清中抗菌活力在1h、15h和30h时实验组均显著高于对照组。溶菌活力实验组均高于对照组,但在15h差异不显著。PO活力实验组均低于对照组,在30h时差异极显著。SOD活力在1h时显著低于对照组,而在30h时显著高于对照组。     

凡纳滨对虾C-型凝集素基因原核表达与免疫作用的研究

本文利用原核表达系统对凡纳滨对虾C-型凝集素基因的开放阅读框进行了重组表达,并通过纯化分离获得重组目的蛋白,采用血细胞悬浮培养的方法,研究了重组C-型凝集素对凡纳滨对虾血细胞免疫作用的影响。实验梯度设置为：对照组和重组C-型凝集素蛋白（1.0 mg/mL）,分别在0、3、6、9、12、24h取培养的血细胞和培养液进行免疫相关指标的测定。结果表明：重组C-型凝集素经SDS-PAGE分析显示在39KD处有显著诱导条带,与预测的分子量大小基本一致,Western blot分析可以与anti-His抗体特异性结合,从而证明C-型凝集素重组蛋白在大肠杆菌中成功表达。重组C-型凝集素对大肠杆菌具有明显的凝集作用,对凡纳滨对虾血细胞数量、存活率和吞噬率影响显著（P<0.05）;培养基酚氧化酶活力在培养3-12h内呈峰值变化,于9h时达到最大值（P<0.05）。研究表明,C-型凝集素不仅具有凝集作用,而且还能激活酚氧化酶原系统,具有重要的免疫防御作用。     

泥蚶血清及血细胞免疫功能研究

初步研究了泥蚶（Ｔｅｇｉｌｌａｒｃａｇｒａｎｏｓａ）血细胞离体条件下的吞噬作用以及血清因子对其的影响,并测定经大肠杆菌注射以后,泥蚶血清中抗菌、溶菌、酚氧化酶（ＰＯ）、超氧化物歧化酶（ＳＯＤ）活力的变化。结果表明,泥蚶血清能促进血细胞对大肠杆菌的吞噬,但不能识别鸡红细胞。大肠杆菌激发后泥蚶血清中抗菌活力在１ｈ、１５ｈ和３０ｈ时实验组均显著高于对照组。溶菌活力实验组均高于对照组,但在１５ｈ差异不显著。ＰＯ活力实验组均低于对照组,在３０ｈ时差异极显著。ＳＯＤ活力在１ｈ时显著低于对照组,而在３０ｈ时显著高于对照组。     

虫草菌粉对日本沼虾免疫功能的影响

在饵料中添加0.5%的虫草菌粉,以口服形式对日本沼虾进行免疫。日本沼虾血淋巴吞噬活性,血清抗菌、溶菌活力及酚氧化酶活力均显著提高。经嗜水气单胞菌攻毒后,试验组的免疫保护率也明显提高。因此,投喂虫草菌粉,能够明显地增强日本沼虾的免疫抵抗能力。     

细菌糖蛋白对螯虾免疫因子的影响

实验检测了气味黄杆菌的胞外糖蛋白对克氏原螯虾（Ｐｒｏｃａｍｂａｒｕｓ ｃｌａｒｋｉｉ）的几咱非特异性免疫因子－凝集素、酚氧化酶、溶菌、抗菌和超氧化物歧化酶等活力的影响。注射１０ｍｇ糖蛋白后,实验组敖蛳主血清的免疫因子活力均高于对照组,凝集素活力在第１～４天最高;酚氧化酶在第１～５天维持较高活力,最大活力在第２天,溶菌、抗菌活力在第１～６天维持较高水平,分别在第３天、第２天达到最大。与对照组相比,实     

病原感染条件下中国对虾二倍体和三倍体血液学变化

以热休克诱导获得中国对虾三倍体，通过口服或注射的方式对二倍体和三倍体中国对虾进行了白斑综合征病毒(WSSV)和鳗弧菌感染实验，研究了病原感染前后二倍体和三倍体中国对虾血液学的变化并首次报道了三倍体中国对虾血细胞的组成。实验结果表明：三倍体和二倍体中国对虾均对WSSV敏感，三倍体未显示出明显的抗性。WSSV感染4d后，对虾外周循环系统的血细胞数量明显减少，三倍体和二倍体对虾血细胞数量均降为对照组的10％左右。对虾血细胞组成发生变化，大颗粒细胞明显减少，小颗粒细胞相应增加，而透明细胞所占比例基本不变；大颗粒细胞和小颗粒细胞内颗粒物质均有所减少，出现空泡或细胞外突起等异常结构。注射鳗弧菌感染后两种对虾的血细胞数量均有所减少，血细胞数量的减少与弧菌的注射量呈正相关关系：三倍体中国对虾血细胞组成与二倍体相似，但其密度仅为二倍体的40％左右：本研究结果为三倍体对虾抗逆特性的研究提供了一定依据和参考。     

中华绒螯蟹感染“颤抖病”病毒后血清免疫因子的变化

对中华绒螯蟹进行颤抖病病毒悬液和PBS(对照)注射感染,研究其血清免疫因子抗菌活性(Ua)、溶菌活性(UL)、凝集活性(HA)、酚氧化酶(PO)、过氧化酶(POD)、超氧化物歧化酶(S0D)、酸性磷酸酶(ACP)、碱性磷酸酶(AKP)活性的变化,并对实验的结果进行统计学分析。结果表明,在中华绒螯蟹人工感染颤抖病病毒后,蟹体内的免疫因子发生了明显的变化,血清中Ua、UL活性先是降低,然后上升至高于正常值水平。P0活性先上升然后下降至低于正常值水平,POD、HA、SOD活性逐渐升高至显著水平;ACP及AKP活性则显著下降。     

Passive immunity in tiger shrimp (Penaeus monodon) fed with monoclonal antibody to white spot syndrome virus

A study was conducted on the stability of monoclonal antibody (MAb) in the hepatopancreas and hemolymph of Penaeus monodon and its effect on protection against white spot syndrome virus (WSSV) upon challenge. MAb C-5 raised against WSSV was purified and coated onto a commercial shrimp feed at dosages of 5, 10 and 15 mg/kg feed. The feed was fed to P. monodon and stability of the MAb in hepatopancreas and hemolymph was determined by immunodot and Western blot. Immunodot results indicated the presence of MAb for 2 h post-feeding in hepatopancreas and hemolymph which was dose-dependent. MAb was also detected in hemolymph by Western blot up to 1 h post-feeding. Shrimp fed with MAb were challenged with WSSV by oral and injection methods. In shrimp fed with 15 mg antibody/kg feed (0.45 μg MAb/g shrimp/day) WSSV infection significantly delayed both in oral and injection challenges with a survival of 65 and 70 % (p < 0.05), respectively, during 15 days post-challenge. MAb was stable in shrimp for passive immunization against WSSV and could be a potential tool for prophylaxis against the virus.     

复方中草药对青虾肌肉和肝胰脏中几种非特异性免疫因子的影响

以青虾(Macrobrachium nipponense)为试验对象,研究了复方中草药对其肌肉和肝胰脏中几种非特异性免疫因子的影响。结果显示:投喂添加复方中草药的饲料60 d后,与对照组相比,青虾肌肉和肝胰脏中溶菌活性、抗菌活性、酚氧化酶、过氧化物酶活性变化不明显(P>0.05)。肌肉中的血凝活性敏感度低以至无法测出,肝胰脏中的血凝活性试验饲料组显著高于对照组(P<0.05)。肌肉和肝胰脏中的酸性磷酸酶、碱性磷酸酶、超氧化物歧化酶活性显著高于对照组(P<0.05),说明复方中草药对增强青虾免疫活性具有一定的作用。     

Immune Responses and Resistance against Vibrio parahaemolyticus Induced by Probiotic Bacterium Arthrobacter XE-7 in Pacific White Shrimp, Litopenaeus vannamei

A 63-d feeding experiment was conducted to determine the effects of dietary supplementation of probiotic bacterium Arthrobacter XE-7 on immune responses and resistance against Vibrio parahaemolyticus in the Pacific white shrimp, Litopenaeus vannamei . The probiotic bacteria were administered orally at four different doses of 0, 10⁶, 10⁸, and 10¹⁰ colony-forming unit (CFU)/g feed for shrimp. On Day 50, the shrimp were challenged with Vibrio parahaemolyticus by bath. On Days 7, 21, 49, and 63, six shrimp per tank were sampled to take intestine and hemolymph. With increasing dietary supplementation of probiotic bacteria, shrimp mortality decreased from 63.16% (the control) to 55.9% (10⁶ CFU/g feed), 51.75% (10⁸ CFU/g feed), and 51.78% (10¹⁰ CFU/g feed), respectively. Vibrio counts in intestine of shrimp fed probiotic bacterium Arthrobacter XE-7 was generally lower than that in the control shrimp ( P  < 0.05). The probiotic bacteria generally increased the immune parameters in shrimp, that is, total hemocyte counts, percentage phagocytosis, respiratory burst activity, and serum phenoloxidase activity. The results showed that probiotic bacterium Arthrobacter XE-7 can be used as probiotic in shrimp feed.     

The feasibility of using primary shrimp hemocyte culture to screen herbal immunostimulants

An analysis of the immune activity of herbal extract in vivo and in vitro assays was performed to explore the feasibility of using primary shrimp hemocyte culture to screen herbal immunostimulants. In vitro experiment was conducted by adding herbal extract into the medium of primary cultured hemocytes and incubating for 24h. In vivo experiment was carried out by feeding shrimp with diets incorporated with herbal extract for 21 days. Challenge test can be used to directly reflect comprehensive immune capacity of organisms. Four immune indexes, total hemocyte counts (THC and lysozyme, phenoloxidase (PO), and phagocytic activity were examined both in vitro and in vivo. The results showed that effects of herbal extract on THC or lysozyme activity were different between in vitro and in vivo. On the contrary, effects of herbal extract on PO activity and phagocytic activity in vitro and in vivo showed a strong correlation. Herbal extract, which enhanced PO activity and phagocytic activity in vitro, had ultimate immune-promoting function in vivo. Therefore, it was feasible to use primary hemocyte culture to screen herbal immunostimulants by determining the effects of herbal immunostimulants on PO and phagocytic activities.     

Hemagglutinating activity and electrophoretic pattern of hemolymph serum proteins of Penaeus monodon and Macrobrachium rosenbergii to white spot syndrome virus injections

The giant fresh water prawn Macrobrachium rosenbergii is known to be highly tolerant to white spot syndrome virus (WSSV) infections when compared to the widely cultured marine tiger shrimp Penaeus monodon. At present, the exact mechanism of tolerance by M. rosenbergii to WSSV is not known. In this study, we attempt to study the effect of WSSV injections on the hemagglutination activity of the hemolymph serum of both P. monodon and M. rosenbergii and look for changes if any, on their hemolymph serum protein electrophoretic patterns. Our results show that M. rosenbergii had significantly (p < 0.05) higher hemagglutinating activity against mouse erythrocytes when compared to P. monodon. As the infection progressed to 48 h there was a further increase (p < 0.05) in the hemagglutination activity in M. rosenbergii, while it decreased in P. monodon. 12% SDS-PAGE analysis of the hemolymph serum of M. rosenbergii infected with WSSV did not show any new protein bands, whereas few bands with decreased intensity was observed in moribund P. monodon where the hemagglutinating activity was also observed to be decreased. The results indicate that hemolymph hemagglutinin levels are modulated in crustaceans as a response to viral infections.