自然养殖水体投喂CpG ODN和表面展示VP28的解脂耶罗维亚酵母对凡纳滨对虾(Litopenaeus vannamei)抗WSSV的免疫保护作用

用添加CpG寡聚核苷酸(CpG ODN)和表面展示VP28的解脂耶罗维亚酵母(VP28-yl)的饵料投喂凡纳滨对虾,进行田间中试实验.投喂30 d后进行WSSV感染实验,评估其对凡纳滨对虾的免疫保护作用.投喂实验结束后,CpG ODN投喂组对虾的相对增重率达到(65.8±7.8)% (P＜0.05),这暗示CpG ODN可能具有促生长作用.WSSV攻毒后,CpG ODN和VP28-yl投喂组对虾中WSSV拷贝数与对照组相比均显著降低(P＜0.05),相对免疫保护率分别可达到 26.7%和 36.7%.在投喂结束和WSSV刺激后,CpG ODN组对虾中的呼吸爆发水平均显著升高(P＜0.05).而在VP28-yl投喂组,WSSV引起的细胞凋亡则显著受到抑制(P＜0.05).此外,WSSV刺激后,STAT基因在CpG ODN组和VP28-yl组对虾中的表达水平均显著上调(P＜0.05),分别在第5天和第3天达到最大值,而对照组中则显著下调.研究结果表明,CpG ODN和VP28-yl增强了凡纳滨对虾抗病毒免疫力,对养殖对虾病毒性疫病的防控具有显著作用,可以作为免疫增强剂添加在饵料中,具有在养殖生产中推广使用的前景.     

转基因vp28蓝藻口服疫苗半数有效量测定及其对斑马鱼的安全评价

蓝藻是对虾幼苗的天然饵料,使用转vp28基因蓝藻(Synechococcus sp. PCC 7942)直接投喂凡纳滨对虾(Litopenaeus vanname)幼苗来防治白斑综合征病毒(WSSV),可起到“药食同源”的作用。转基因蓝藻作为对抗WSSV的口服疫苗,目前面临环境释放的安全性问题。斑马鱼是最常见的模式生物之一,本研究将转vp28基因蓝藻投喂凡纳滨对虾幼苗,验证其剂量梯度抗WSSV的影响,并测定其半数有效剂量(ED50)。以该剂量为依据投喂斑马鱼后,通过测定斑马鱼体内的酶学指标、进行组织切片观察以及养殖水体中氮磷等的变化,来探究转vp28基因蓝藻对水生生物、水体环境造成的影响。研究结果表明,随着转基因蓝藻口服疫苗剂量的增加,凡纳滨对虾幼苗抗WSSV的能力也随之增强,测得ED50为0.027 g。分别使用野生型和转基因型蓝藻配合饲料投喂斑马鱼15天,投喂结束后其生长状态良好、游动正常；投喂前后身体颜色无明显差异,不同组间体长增长的差异不显著(p > 0.05)。转基因组投喂的过氧化氢酶活力稍低于空白组和野生型组,过氧化物酶活力随着投喂天数的增长而呈现降低趋势；水质监测发现总氮和总磷无较大差异,投喂蓝藻的两组氨氮均要低于空白对照组,氨氮含量的提升对斑马鱼的抗氧化能力存在一定影响；组织切片发现斑马鱼各组的肝脏、心脏组织细胞无明显差异。研究表明转vp28基因蓝藻可有效增强凡纳滨对虾抗WSSV能力,且抗病能力随着口服疫苗剂量的升高而增强；投喂斑马鱼后,斑马鱼无明显的毒理效应，且对水质影响较小；转vp28基因蓝藻作为亚单位疫苗投放对水生生物的影响较小,可为后续产业规模应用提供基础。     

饲料中添加芽孢杆菌PC465对凡纳滨对虾生长和STAT基因表达的影响

将坚强芽孢杆菌PC465浓缩菌液直接与饲料原料混匀后制成含106、108和1010CFU芽孢杆菌/g干物质的颗粒饲料,或者经冷冻干燥制成冻干粉,与饲料原料混匀制成含107CFU芽孢杆菌/g饲料的颗粒饲料,设连续投喂组、间隔“4+3”投喂组、间隔“1+1”投喂组,研究投喂剂量和投喂频率对对虾生长和类淋巴STAT基因表达的影响。研究发现,饲料中添加不同剂量的坚强芽孢杆菌PC465均能显著提高凡纳滨对虾的生长率和STAT基因表达(P<0.05),而且跟添加量有一定关系;实验采取不同投喂频率投喂凡纳滨对虾,都能显著提高对虾的生长率和对虾淋巴器官中STAT基因的表达水平(P<0.05),其中以连续投喂组的效果最明显。研究结果对对虾健康养殖有一定的参考价值。     

养殖水体中添加蜡样芽孢杆菌PC465 对凡纳滨对虾抗病力的影响

将蜡样芽胞杆菌(Bacillus cereus)PC465添加到凡纳滨对虾(Litopenaeus vannamei)养殖水体中至终浓度分别为10~4、10~5和10~6 CFU/mL,以无益生菌添加的养殖组为对照组,定期检测实验对虾肠道和养殖水体中的细菌总数、弧菌总数以及水体中氨氮含量和亚硝酸氮含量。实验结果表明,水体中添加益生菌能降低凡纳滨对虾肠道内细菌数量,且与对照组相比差异显著(P0.05);高浓度的益生菌处理组可以明显降低养殖水体内弧菌数量(P0.05),但是益生菌并没有显著影响水体中的氨氮含量和亚硝酸氮含量。养殖4周后进行WSSV投喂感染实验,感染实验表明,实验组H组(益生菌浓度为10~6 CFU/mL)和M组(益生菌浓度为10~5 CFU/mL)凡纳滨对虾的累计死亡率分别为63.9%和74.6%,显著低于对照组100%的累计死亡率(P0.05)。感染实验期间采用荧光定量PCR方法测定了凡纳滨对虾3种免疫相关基因的表达情况,统计数据显示,益生菌处理组的脂多糖-β-1,3-葡聚糖结合蛋白(lipopolysaccharide-β-1,3-glucan-binding protein,LGBP)、β-1,3-葡聚糖结合蛋白-脂蛋白(beta-1,3-glucan-binding protein-lipoprotein,βGBP-HDL)、热激蛋白70(heat shock proteins,Hsp70)mRNA的表达量在WSSV感染后呈显著上调趋势。实验结果提示,水体中添加蜡样芽孢杆菌PC465可以提高凡纳滨对虾抗WSSV感染能力,其作用机制可能是降低对虾肠道和水体中的细菌和弧菌数量,或调节免疫相关基因的表达水平。     

毒死蜱胁迫下WSSV对凡纳滨对虾的致病性

为了评价养殖水环境中毒死蜱对凡纳滨对虾生存的危害性,开展了毒死蜱胁迫下白斑综合征病毒(WSSV)对凡纳滨对虾致死实验,分析了毒死蜱胁迫下凡纳滨对虾鳃组织WSSV含量和肌肉组织乙酰胆碱酯酶活性变化。通过急性毒性实验测定了毒死蜱对凡纳滨对虾的半致死浓度(LC₅₀),随着暴露时间的延长,LC₅₀值显著下降,存在着浓度-反应的正向关系,96 h LC₅₀为0.758 μg/L(0.521～0.987 μg/L)。在此基础上,确定了毒死蜱胁迫实验浓度为0.2 μg/L,此浓度下药浴4 d后对凡纳滨对虾注射WSSV,结果显示:毒死蜱胁迫下注射WSSV组的对虾死亡率(83.33?Ee4.7%)极显著高于乙醇-WSSV组(40.00?Ee0.9%);对虾鳃组织WSSV荧光定量PCR检测结果显示:感染72 h后,毒死蜱-WSSV组WSSV含量约是乙醇-WSSV组的4倍;感染96 h后,毒死蜱-WSSV组WSSV含量显著增加,约是72 h毒死蜱-WSSV组的4.9倍,是96 h乙醇-WSSV组的5.9倍。毒死蜱胁迫下,对虾肌肉组织乙酰胆碱酯酶(AchE)活性低于对照组20%左右。由此可见,毒死蜱胁迫下,WSSV增殖速率加快,导致对虾死亡率升高。     

2株消化道优势菌对凡纳滨对虾免疫酶活性和抗白斑综合征病毒感染力的影响

以凡纳滨对虾为研究对象,在基础饲料中分别添加从健康对虾消化道中分离纯化的优势菌菌株——美人鱼发光杆菌PC463和坚强芽孢杆菌PC465(菌含量≥1011CFU/g)的活菌和破碎菌各1 g/kg,观察其对凡纳滨对虾血淋巴免疫酶活性和抗WSSV感染保护率的影响。经过20 d养殖实验后发现,与对照组相比,饲料中添加坚强芽孢杆菌活菌的免疫组和添加美人鱼发光杆菌灭活菌的免疫实验,其凡纳滨对虾血淋巴中酸性磷酸酶(ACP)、碱性磷酸酶(AKP)、超氧化物歧化酶(SOD)和过氧化氢酶(CAT)活性在不同程度上有所提高,并显著高于对照组(P<0.05)。WSSV感染后饲料中添加坚强芽孢杆菌活菌的免疫组存活率(53%±12%)和添加美人鱼发光杆菌灭活菌的免疫组存活率(49%±15%)显著高于对照组(P<0.05)。结果表明:饲料中添加坚强芽孢杆菌活菌和美人鱼发光杆菌灭活菌可以在一定程度上提高凡纳滨对虾免疫酶活性和抗WSSV感染能力,上述有防病作用的益生菌株以饲料添加剂的方式应用于对虾养殖生产,可望成为对虾白斑病生物防治的有效途径之一。     

复方中草药对凡纳滨对虾生长、消化酶和免疫因子活性及抗WSSV的影响

研究复方中草药(葛根、黄连、金银花、板蓝根、黄芪、甘草、柴胡、当归、山楂、陈皮和茯苓)对凡纳滨对虾生长、消化酶和免疫因子活性及抗WSSV的影响,为对虾健康养殖提供科学依据。分别在饲料中添加0%(对照组)、0.4%、0.8%、1.2%、1.6%和2.0%该复方中草药,连续投喂凡纳滨对虾[体质量(0.026±0.007)g]28 d(每组3个重复,每个重复160尾),每隔1周随机采样10尾/桶测量体长、体质量,并取肝胰腺用于生长指标(WGR、SGR、FR和SR)、相关酶(淀粉酶、蛋白酶、脂肪酶、SOD、GPT和GOT)及MDA含量的测定。研究结果表明,复方中草药对凡纳滨对虾的生长均有提高,其中0.8%和1.2%添加组效果最佳,差异显著;且饵料系数显著下降。对虾肝胰腺消化酶(淀粉酶、蛋白酶、脂肪酶)和免疫相关因子(SOD、GPT和GOT)随养殖时间的延长,高酶活性逐渐由高添加量组向低添加量组移动,其中以0.8%和1.2%添加组效果最佳,差异显著;与对照组相比,0.4%,0.8%和1.2%添加组MDA含量差异显著下降。对照组和0.4%添加组对虾经投喂感染WSSV后全部死亡,但0.4%添加组对虾第1尾死亡时间、50%死亡时间以及100%死亡时间分别比对照组延后8 h、16 h和8 h;0.8%添加组成活率最高(33.3%),与其他组差异显著。复方中草药能促进对虾生长、消化,提高免疫和抗WSSV能力,其中以添加0.8%和1.2%效果最佳。     

养殖密度、饵料种类和丰度对日本囊对虾(Marsupenaeus japonicus)争胜行为的影响

本研究设置10、25、50尾/m2 3个养殖密度,人工饵料、鲜菲律宾蛤仔肉两种饵料,不喂(N)、少喂(L)、饱喂(F) 3个处理,分别于投喂前1 h、投喂时、投喂后1 h测定日本囊对虾(Marsupenaeus japonicus)的争斗次数、平均优势指数等指标,分析养殖密度、饵料种类及丰度对日本囊对虾争胜行为的影响。结果显示,随着养殖密度的增加,对虾的争胜行为逐渐增强,投喂时与投喂前、投喂后对虾的争胜行为差异显著(P<0.05);投喂时对虾的争斗次数最多;饵料种类和丰度显著影响日本囊对虾的争胜行为,投喂菲律宾蛤仔肉的争胜行为强于投喂人工饵料(P<0.05),少喂与不喂、饱喂处理间差异显著(P<0.05)。研究表明,养殖密度、饵料种类及丰度均显著影响日本囊对虾的争胜行为。     

不同饲料能量源及其水平对凡纳滨对虾生长、抗氧化能力及蛋白质利用的影响

为探究不同饲料能量源及其水平对凡纳滨对虾(Litopenaeusvannamei)生长性能的影响,以饲料蛋白质含量为37%,蛋白能量比为20.23 mg/kJ的饲料作为对照组饲料(C),在此基础上分别通过提高饲料碳水化合物、脂肪含量调节饲料能量水平,制作饲料蛋白能量比分别为19.51 mg/kJ (中碳水化合物组, MC)、18.85 mg/kJ (高碳水化合物组, HC)、19.45 mg/kJ (中脂肪组, ML)和18.54 mg/kJ (高脂肪组, HL)的4组实验饲料,在淡水养殖条件下投喂初始体重为(0.6±0.02)g的凡纳滨对虾幼虾56d。结果表明,与对照组相比,提升饲料脂肪水平能显著提高凡纳滨对虾的生长性能和蛋白质沉积率(P<0.05),HL组凡纳滨对虾获得最大特定生长率及蛋白质沉积率;增加饲料碳水化合物水平对凡纳滨对虾的生长性能及蛋白质沉积率无显著影响(P>0.05)。与对照组相比, ML组、MC组、HC组对虾肌肉粗蛋白含量显著提高(P<0.05),HL组对虾肌肉粗蛋白含量较对照组有所提高,但差异不显著(P>0.05),对虾肌肉总脂肪含量则随着...     

复合中草药制剂对凡纳滨对虾生长和免疫指标的影响

研究一种复方中草药制剂(黄芪∶板蓝根∶金银花∶生石膏=1∶1∶1∶1)对凡纳滨对虾生长和免疫的影响。生长与免疫试验设7个处理,试验虾饲喂在基础饲料中分别添加0.0%、0.5%、0.9%、1.3%、1.7%、2.1%、2.5%的饲料,凡纳滨对虾的初始体重为0.10 g,养殖周期56 d。结果显示:随着复方中草药水平增加,凡纳滨对虾的增重率和特定生长率随之增加,但仅最高组(2.5%组)与对照组有显著差异(P<0.05),其它各组与对照组均无显著差异;饲料系数和成活率各组与对照组均无显著差异,用二次回归曲线得到复方中草药水平为2.07%时增重率最高;复方中草药对凡纳滨对虾血清中超氧化物歧化酶、碱性磷酸酶、酚氧化物酶和溶菌酶有显著影响,随着复方中草药水平的增加,这4种酶的活力出现先显著增加后下降的趋势,酶活力最高的组是2.1%组;而对酸性磷酸酶和过氧化物酶活力没有显著影响。攻毒试验设3个处理组,不添加中草药的对照组、间隔投喂组和连续投喂添加了2.1%中草药制剂组,养殖试验结束后采用注射和投喂病毒两种方法攻毒(1.4×108cfu/mL弧菌病毒)。结果表明:注射和投喂后连续投喂组的免疫保护率分别为50%和79.19%,比对照组(试验Ⅰ组)高27.1%和17.2%。因此,在本实验条件下连续投喂添加2.1%复方中草药制剂的饲料,可以促进凡纳滨对虾的生长并提高其免疫力。     

Potential role of LvDscam in specific immune response of Litopenaeus vannamei against white spot syndrome virus by oral delivery of VP28 using Bacillus subtilis

Envelope protein VP28 has been suggested as a candidate vaccine component to evoke a better protection against white spot syndrome virus (WSSV). We have reported that Bacillus subtilis spores harbouring VP28 (rVP28‐bs) can specifically protect shrimp against WSSV. However, the mechanism that supports the production of unique molecules induced by rVP28‐bs to trigger specific immunity is originally unknown. It has recently been suggested that Dscam (Down syndrome cell adhesion molecule) plays an essential role in the alternative adaptive immunity of invertebrates. In this study, we compared the diversity of Litopenaeus vannamei Dscam (LvDscam) variable regions by different antigens immunization. A total of 13, 15 and 11 expressed alternative sequences were identified for N‐terminal Ig2, N‐terminal Ig3 and the entire Ig7 domain, respectively. More than half of the unique variants (16 out of 22) were found in the Ig2/Ig3 domains. Further analysis of the interaction between VP28 and unique Ig2/Ig3 variants was confirmed by both yeast two‐hybrid and GST pull‐down approach. We also found that the percentage of haemocytes phagocytosing WSSV was significantly higher (P < 0.001) in the shrimp injected with control‐siRNA (43.8 ± 2.2) than those with Dscam‐siRNA (11.3 ± 5.4) in the rVP28‐bs groups. With Dscam‐siRNA injection, survivorship significantly decreased (P < 0.001) in the rVP28‐bs group after WSSV challenge. Our data suggested that LvDscam‐mediated pathway may be involved in the specific immune response of shrimp against WSSV induced by rVP28‐bs.     

转vp28蓝藻口服剂对凡纳滨对虾抗白斑综合征病毒能力及免疫反应的影响

为探讨转vp28蓝藻(Anabaena sp.PCC7120)口服剂对凡纳滨对虾抗白斑综合征病毒能力及其相应的免疫反应,本研究将此口服剂免疫幼虾7 d,再分别通过投喂攻毒和浸泡攻毒,测定其存活率及相应的免疫指标。投喂攻毒和浸泡攻毒的实验组存活率分别为78.8%和83.19%,表明该口服剂能显著增强对虾抗白斑综合征病毒的能力。蓝藻口服剂免疫对虾的酶活性检测结果显示,超氧化物歧化酶(SOD)、酚氧化酶(PO)、过氧化氢酶(CAT)和碱性磷酸酶(AKP)活性在免疫后2 h均有上升趋势,且在48或96 h达到最高值,这表明该口服剂能引起对虾体内酶活性变化。投喂攻毒的对虾酶活性检测结果显示,实验组攻毒后的对虾肝胰腺SOD活性分别比阳性对照组、野生型组、空载体组显著提高42.10%、32.26%和16.04%,且攻毒后的肌肉SOD活性分别比阴性对照组、阳性对照组、野生型组和空载体组略微提高17.70%、11.50%、15.00%以及10.00%。实验组攻毒后的对虾肝胰腺PO、CAT和AKP活性比阳性对照组分别提高12.17%、88.80%和240.07%,比野生型组分别提高21.49%、30.90%和100%;酸性磷酸酶(ACP)活性比阴性对照组略微提高,而在肌肉中各组ACP活性无显著性差异。同时浸泡攻毒组结果与投喂攻毒组具有类似的趋势。浸泡攻毒的实验组CAT和AKP活性显著高于其余处理组,且CAT活性比投喂攻毒更为显著。浸泡攻毒的实验组肝胰腺PO活性显著高于阳性对照组、野生型组和空载体组,而各组肌肉ACP活性无显著性差异。研究表明,转vp28蓝藻口服剂能够增强凡纳滨对虾抗病能力并延缓对虾死亡。转vp28蓝藻PCC7120本身可作为幼虾饵料直接投喂,无需提取纯化,有望大规模应用于对虾养殖产业。     

Protection of Fenneropenaeus chinensis (Osbeck, 1765) against the white spot syndrome virus using specific chicken egg yolk immunoglobulins by oral delivery

Two kinds of specific chicken egg yolk immunoglobulins (IgYs), IgY‐WSSV and IgY‐VP28, were, respectively, raised against the 2 mM binary ethylenimine (BEI)‐inactivated white spot syndrome virus (WSSV) and a principal envelope protein VP28. The activity of purified specific IgYs was stable under the conditions of 20–70 °C, pH 3.0–10.0 and 0–700 g L^?1 sucrose solution. In the neutralization assay, these high‐affinity IgY antibodies can specifically bind with the virus particles to protect shrimp (Fenneropenaeus chinensis) against WSSV infection. After oral delivery for 20 days, the IgY‐WSSV exerted a higher protection effect (RPS: 71.5%) than IgY‐VP28 (RPS: 63.7%). Moreover, an increase in RPS (79.2%) was found on addition of IgY‐WSSV:VP28 (0.1% IgY‐VP28 plus 0.2% IgY‐WSSV). This may indicate that neutralization of WSSV refers to the multiple‐hit model. By time‐course study of the levels of the specific IgYs in vivo, the data showed that the titre was enhanced to a relatively high level (P/N=8.35±0.45) at 3 days post administration, declined slightly (P/N=7.13±1.01) at 7 days post administration and then remained stable for further investigation. The stable antibody level potentially contributes towards blocking a large number of WSSV particles from entering and infecting on the major tissues at the early and late stages after challenge in shrimp.     

Immune responses of whiteleg shrimp,Litopenaeus vannamei (Boone, 1931), to bacterially expressed dsRNA specific to VP28 gene of white spot syndrome virus

In this study, dsRNA specific to VP28 gene of white spot syndrome virus (WSSV) of shrimp was synthesized in Escherichia coli in large scale and studied the immune response of shrimp to dsRNA‐VP28. The haematological parameters such as clotting time and total haemocytes counts, and immunological parameters such as prophenoloxidase (proPO), superoxide dismutase (SOD), superoxide anion (SOA) and malondialdehyde content, as well as the mRNA expression of ten immune‐related genes were examined to estimate the effect of dsRNA‐VP28 on the innate immunity of Litopenaeus vannamei. The activities of proPO, SOA and SOD significantly increased in haemocyte after dsRNA‐VP28 treatment, whereas MDA content did not change significantly. Among the ten immune‐related genes examined, only the mRNA expression of proPO, cMnSOD, haemocyanin, crustin, BGBP, lipopolysaccharides (LPs), lectin and lysozyme in haemocytes, gill and hepatopancreas of L. vannamei, was significantly upregulated at 12 h after dsRNA‐VP28 treatment, while no significant expression changes were observed in Toll receptor and tumour receptor genes. The increase of proPO and SOD activities, and SOA level and mRNA expression level of proPO, cMnSOD, haemocyanin, crustin, BGBP, LPs, lectin and lysozyme after dsRNA‐VP28 stimulation indicate that these immune‐related genes were involved in dsRNA‐VP28‐induced innate immunity in shrimp.     

Kinetic analysis of internalization of white spot syndrome virus by haemocyte subpopulations of penaeid shrimp,Litopenaeus vannamei (Boone), and the outcome for virus and cell

Little is known about the innate antiviral defence of shrimp haemocytes. In this context, the haemocytes of penaeid shrimp Litopenaeus vannamei (Boone) were separated by iodixanol density gradient centrifugation into five subpopulations (sub): sub 1 (hyalinocytes), sub 2 and 3 (prohyalinocytes), sub 4 (semigranulocytes) and sub 5 (granulocytes) and exposed to beads, white spot syndrome virus (WSSV) and ultraviolet (UV)‐killed WSSV. In a first experiment, the uptake of beads, white spot syndrome virus (WSSV) and UV‐killed WSSV by these different haemocyte subpopulations was investigated using confocal microscopy. Only haemocytes of sub 1, 4 and 5 were internalizing beads, WSSV and UV‐killed WSSV. Beads were engulfed by a much larger percentage of cells (91.2 in sub 1; 84.1 in sub 4 and 58.1 in sub 5) compared to WSSV (9.6 in sub 1; 10.5 in sub 4 and 7.9 in sub 5) and UV‐killed WSSV (12.9 in sub 1; 13.3 in sub 4; and 11.8 in sub 5). In a second experiment, it was shown that upon internalization, WSS virions lost their envelope most probably by fusion with the cellular membrane of the endosome (starting between 30 and 60 min post‐inoculation) and that afterwards the capsid started to become disintegrated (from 360 min post‐inoculation). Expression of new viral proteins was not observed. Incubation of haemocyte subpopulations with WSSV but not with UV‐killed WSSV and polystyrene beads resulted in a significant drop in haemocyte viability. To find the underlying mechanism, a third experiment was performed in which haemocyte subpopulations were exposed to a short WSSV DNA fragment (VP19) and CpG ODNs. These small DNA fragments induced cell death. In conclusion, WSSV is efficiently internalized by hyalinocytes, semigranulocytes and granulocytes, after which the virus loses its envelope; as soon as the capsids start to disintegrate, cell death is activated, which in part may be explained by the exposure of viral DNA to cellular‐sensing molecules.     

Protection of crayfish, Cambarus clarkii, from white spot syndrome virus by polyclonal antibodies against a viral envelope fusion protein

White spot syndrome virus (WSSV) is a large double-stranded DNA virus, causing considerable mortality in penaeid shrimp and other crustaceans. WSSV produces five major structural proteins, including two major envelope proteins, VP28 and VP19. To produce VP28 and VP19 as a single protein for antibody production, DNA sequences encoding both open reading frames were fused together and cloned into pET-22b(+) expression vector. The fusion protein, VP(19+28), was expressed in Escherichia coli, purified using Ni2+ His affinity chromatography and injected into a rabbit. Antiserum collected from the immunized rabbit was tested in vivo for ability to protect crayfish, Cambarus clarkii, from disease caused by WSSV. Fifteen days after challenge with WSSV, treatment with VP(19+28) antiserum gave 100% protection against disease in the ambient temperature range of 15-22 degrees C and 65% protection at a constant temperature of 26 degrees C. These results demonstrated VP(19+28) antiserum is effective in protection of crayfish from WSSV and confirmed that VP19 and VP28 play an important role in WSSV host infection. Targeting both VP19 and VP28 may be effective for the design of both immunotherapeutic medicines and reagents to detect WSSV.     

饲料中添加复合芽孢杆菌对凡纳滨对虾抗病毒感染能力及抗病基因表达的影响

自健康凡纳滨对虾(Litopenaeus vannamei)分离到枯草芽孢杆菌(Bacillus subtilis)、地衣芽孢杆菌(B. licheniformis)和短小芽孢杆菌(B. pumilus),将上述芽孢杆菌以单一和3株复合的方式包裹在基础饲料表面,制成益生菌饲料;每日投喂对虾,3周后进行白斑综合征病毒(WSSV)人工感染。统计实验组和对照组的累积死亡率,测定对虾鳃组织内WSSV拷贝数,分析对虾肠道组织含半胱氨酸的天冬氨酸蛋白酶基因(Caspase)和硫氧还原蛋白基因(Trx)的相对表达量。结果显示,感染实验结束时,A组(枯草芽孢杆菌)、B组(地衣芽孢杆菌)、C组(短小芽孢杆菌)和D组(枯草芽孢杆菌+地衣芽孢杆菌+短小芽孢杆菌复合益生菌)的对虾累积死亡率分别为(73.3±7.0)%、(63.3±5.5)%、(75.0±7.9)%和(50.0±5.3)%,显著低于对照组(PBS组)(100%);在整个感染阶段,各实验组的病毒拷贝数呈先上升后下降的趋势,但对照组呈现一直上升趋势,且显著高于实验组。抗病基因表达结果显示,WSSV感染后,各组对虾肠道Caspase相对表达量随感染时间的延长呈先上调再下调的趋势,且在18 h各组对虾肠道Caspase表达量达到最大值;益生菌摄取和WSSV感染都能刺激Trx的表达,益生菌的刺激相对平缓,且各实验组对虾肠道Trx相对表达量在WSSV感染后的18 h时陡升到最大值,极显著高于对照组,且以D组的激活能力最强。研究证实,枯草芽孢杆菌、地衣芽孢杆菌和短小芽孢杆菌均可提高对虾抗WSSV感染能力,复合芽孢杆菌抗病毒能力最突出。对虾抗病力的提高可能与芽孢杆菌减缓了病毒在靶组织的增殖速率、提高了Caspase和Trx基因表达水平相关。     

Effect of hot water extracts of brown seaweeds Sargassum spp. on growth and resistance to white spot syndrome virus in shrimp Penaeus monodon postlarvae

An experiment was conducted to evaluate the effect of a hot water extract of brown seaweeds Sargassum duplicatum and Sargassum wightii on the growth and white spot syndrome virus (WSSV) resistance in shrimp Penaeus monodon postlarvae (PL). Artemia nauplii (instar II) were enriched with both seaweed extracts at various concentrations (250, 500 and 750 mg L^?1) and fed to the respective P. monodon (PL15–35) group for 20 days. A control group was also maintained without seaweed extract supplementation. The weight gain of the experimental groups was significantly higher (0.274–0.323 g) than the control group (0.261 g). Similarly, the specific growth rate was also significantly higher (16.27–17.06%) in the experimental groups than in the control group (16.03%). After 20 days of the feeding experiment, the shrimp PL were challenged with WSSV for 21 days. During the challenge test, the control shrimp displayed 100% mortality within 8 days. In contrast, the mortality percentage of the highest concentration (750 mg L^?1) of seaweed extract enriched Artemia nauplii fed shrimp was 54–79%. Comparatively, low mortality was observed in S. wightii extract‐enriched Artemia nauplii fed shrimp. The polymerase chain reaction analysis indicated the concentration‐dependent infection of WSSV in P. monodon PL.     

Effects of Dietary Administration of Probiotic Halomonas sp. B12 on the Intestinal Microflora,Immunological Parameters,and Midgut Histological Structure of Shrimp,Fenneropenaeus chinensis

The experiment was conducted to investigate the effects of oral administration of probiotic Halomonas sp. B12 (previously isolated from the intestine of Fenneropenaeus chinensis) on the intestinal microflora, immunological parameters, and midgut histological structure of F. chinensis. Shrimp (initial weight: 4.00 ± 0.10 g) were fed diets containing Halomonas sp. B12 at 0 (control), 3.68 × 10⁷ (T1), and 7.18 × 10¹⁰ (T2) colony‐forming units per gram for 6 wk, respectively. The results showed that the total bacterial counts significantly increased (P < 0.05) with supplementation of dietary probiotic B12. However, Vibrio spp. counts significantly decreased (P < 0.05) in the intestine of shrimp with increasing dietary probiotic B12. Hemocyte counts in the shrimp fed the diets supplemented with probiotic B12 were significantly higher (P < 0.05) than the control group. Both phenoloxidase (PO) activity in plasma and hemocyte lysate supernatant were higher in the shrimp fed diets supplemented with probiotic B12 compared with the control group. No significant difference was found in PO activity in plasma between the T1 and the control (P > 0.05). PO activity in plasma was higher in T2 than that in T1. Cumulative shrimp mortality after 10‐d white spot syndrome virus (WSSV) challenge test significantly decreased with increasing dietary probiotic B12 (P < 0.05). No significant difference was found between T1 and control in cumulative shrimp mortality after challenge test (P > 0.05). The oral administration of probiotic B12 to F. chinensis was beneficial to protect the integrity of shrimp intestinal mucosal layer. In summary, even though the low dose (T1) had some effects on bacterial counts and immunological parameters, only the high dose (T2) significantly increased the resistance of the shrimp to WSSV.