饲料中添加益生菌对凡纳滨对虾抗感染和5种免疫基因表达的影响

为研究饲料中添加益生菌对凡纳滨对虾抗副溶血弧菌感染能力和非特异性免疫基因表达水平的影响,以初体质量为(6.95±1.20)g的凡纳滨对虾为研究对象,在室内养殖箱进行3周的养殖实验和2周的副溶血弧菌人工感染实验;其中,对照组每日投喂普通商品饲料,实验组每日投喂在普通商品饲料中添加地衣芽孢杆菌、枯草芽孢杆菌、地衣芽孢杆菌/枯草芽孢杆菌(1:1)配制成的3组实验饲料,实验饲料中益生菌的终浓度为10⁷ cfu/g。并采用实时荧光定量RT-PCR方法,对保护率最高的地衣芽孢杆菌/枯草芽孢杆菌实验组进行凡纳滨对虾相关免疫基因表达水平的分析。实验结果表明,在饲料中添加单一益生菌或复合益生菌均可显著提高对虾抗副溶血弧菌感染的能力(P<0.05),且复合益生菌的保护效果更佳,其相对免疫保护率为31.11%。感染副溶血弧菌后,地衣芽孢杆菌+枯草芽孢杆菌实验组凡纳滨对虾血淋巴中的先天免疫缺陷基因(innate immune deficiency gene,IMD)、对虾素3a分子(penaiedin 3a)、酚氧化酶原(prophenoloxidase,proPO)、溶菌酶(Lysozyme,LZM)和甲壳素Crustin的mRNA的相对表达量均显著上调,且分别在18～24 h达到最大值。实验结果提示:饲料中添加芽孢杆菌可有效提高凡纳滨对虾抗副溶血弧菌感染的能力,这种能力的提高可能是通过增加抗病相关基因的表达量实现的。     

饲料添加益生菌对凡纳滨对虾肠道菌群、Toll受体及溶菌酶基因表达及抗感染的影响

以初体重为(7.20±1.38)g的凡纳滨对虾(Litopenaeus vannamei)为研究对象,在室内养殖箱进行3周的养殖实验和2周的哈维氏弧菌(Vibrio harveyi)人工感染实验,其中对照组每日投喂普通商品饲料,实验组每日投喂在普通商品饲料中添加地衣芽孢杆菌(Bacillus licheniformis)1.0×10~8 CFU/m L配制成的实验饲料。目的是研究饲料中添加益生菌对凡纳滨对虾肠道菌群、Toll受体及溶菌酶基因表达量和抗哈维氏弧菌能力的影响。实验结果表明,在饲料中添加地衣芽孢杆菌可显著提高对虾抗哈维氏弧菌感染的能力,其相对免疫保护率为22.22%。与对照组相比,实验组可显著降低对虾肠道内弧菌数量(P0.05)。感染哈维氏弧菌后,实验组溶菌酶(lysozyme,LZM)m RNA的相对表达量在12 h、18 h、24 h、36 h、48 h、72 h均显著高于对照组(P0.05)。实验组感染哈维氏弧菌后在6 h、12 h、18 h、24 h、36 h、48 h、72 h、7 d的Toll受体(Toll receptor)m RNA的相对表达量均显著高于对照组(P0.05)。实验结果提示:饲料中添加地衣芽孢杆菌可有效提高对虾抗哈维氏弧菌感染的能力,这种能力的提高可能是通过降低对虾肠道内的弧菌量,并提高抗病相关基因的表达量实现的。     

饲料中益生菌对凡纳滨对虾生长、肠道菌群及部分免疫指标的影响

以初体质量为(0.11±0.00)g的凡纳滨对虾(Litopenaeus vannamei)为研究对象,在室外水泥池进行10周饲喂实验,研究饲料中添加单一益生菌和复合益生菌对凡纳滨对虾生长、消化酶、肠道和粪便菌群及部分免疫指标的影响.以基础饲料为对照组,在基础饲料中分别添加地衣芽孢杆菌(Bacillus licheniformis)、枯草芽孢杆菌(Bacillus subtilis)、虾乐333(复合益生菌)、地衣芽孢杆菌+枯草芽孢杆菌(1:1)、虾乐333+地衣芽孢杆菌+枯草芽孢杆菌(1:1:1)各1 g/kg,配制5种实验饲料.实验结果显示,复合益生菌饲喂组对虾的特定生长率显著高于对照组(P<0.05),而单一益生菌饲喂组与对照组差异不显著(P<0.05).饲料中添加益生菌能使凡纳滨对虾肠道蛋白酶和淀粉酶活力升高,其中复合益生菌饲喂组淀粉酶活力显著高于对照组(P<0.05).与对照组比,饲料中添加益生菌显著降低了肠道及粪便弧菌数(P<0.05),不同程度提高了凡纳滨对虾血清蛋白浓度、酚氧化酶活力、溶菌酶活力和总抗氧化力,其中添加复合益生菌能使血清蛋白浓度及溶菌酶活力较对照组显著提高(P<0.05).上述结果表明,与饲喂单一益生菌相比,复合益生菌对凡纳滨对虾的生长、免疫力具有更好的促进作用.     

一株芽孢杆菌PC024的鉴定及其抗WSSV感染效果的研究

为了筛选WSSV的防病益生菌株,从健康中国明对虾消化道分离纯化一株芽孢杆菌PC024,经Biolog碳源利用反应、ATB微生物自动鉴定系统、脂肪酸气相色谱分析得出该菌株与坚强芽孢杆菌的生理生化特性最为相似,该菌株为革兰氏阳性菌,有一根端极鞭毛;细胞呈球杆状,有椭圆芽孢;单个菌落呈圆形,中间略微凸起;16S rRNA序列分析表明,该菌株与坚强芽孢杆菌进化地位最接近,同源性均达到100％,综合以上4种方法的鉴定结果,该菌株被鉴定为坚强芽孢杆菌.将已鉴定的PC024菌株粘附于对虾饲料表面投喂给凡纳滨对虾20 d后,进行WSSV肌肉注射感染,测定投喂和感染后对虾血淋巴上清和肝胰腺的免疫相关酶活性,并对对虾肠道总菌数和添加菌PC024进行计数及鉴定.结果表明:添加该菌的实验组对虾相对存活率高,相对保护率达33.7％;投喂含该菌饲料的实验组对虾血清和肝胰腺的相关免疫酶活性较对照组显著提高,对虾肠道总细菌数始终显著高于对照组,并在实验组能够分离得到坚强芽孢杆菌,坚强芽孢杆菌PC024可作为WSSV的防病益生菌株应用于对虾养殖生产.     

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。     

一种复合益生菌对凡纳滨对虾抗副溶血弧菌感染能力的影响

本研究选择1株地衣芽孢杆菌(Bacillus licheniformis)BL-9、1株枯草芽孢杆菌(Bacillus subtilis)BS-12、1株金丽假交替单胞菌(Pseudoalteromonas flavipulchra)CDM8,以1∶1∶1将其组成复合益生菌,各益生菌水体添加浓度为107 CFU/ml,进行为期30 d的凡纳滨对虾(Litopenaeus vannamei)养殖实验。实验分为暂养期(7d)、益生菌处理期(15d)、副溶血弧菌(Vibrio parahaemolyticus)攻毒期(10 d)。结果显示,养殖水体中添加复合益生菌能显著增加水体和对虾肠道可培养细菌总数(P<0.05),攻毒实验结束时,实验组对虾累积存活率为(73.33±6.83)%,显著高于阳性对照组(25.33±15.43)%。对虾抗病基因热激蛋白70(Heat shock proteins 70,Hsp70)、β-1,3-葡聚糖结合蛋白-脂蛋白(Beta-1,3-glucan-binding protein-lipoprotein,βGBP-HDL)、脂多糖-β-1,3-葡聚糖结合蛋白(Lipopolysaccharide-β-1,3-glucan binding protein,LGBP)、抗菌肽Crustin在益生菌处理阶段均出现不同程度的上调,在攻毒阶段虽呈现各自不同的表达情况,但所有基因都经历了更大幅度上调。研究表明,水体中添加芽孢杆菌和假交替单胞菌组成的复合益生菌可提高凡纳滨对虾抗副溶血弧菌感染能力,对虾抗病力的提高可能与益生菌增加对虾肠道可培养细菌数量、抗病相关基因表达水平及过氧化氢酶(CAT)活性有关。     

几株肠道益生菌对凡纳滨对虾非特异免疫力和抗病力的影响

以基础饲料为对照组,在基础饲料中分别添加坚强芽孢杆菌活菌(Bacillus firmus)、坚强芽孢杆菌活菌(1.0×108 CFU/g)+美人鱼发光杆菌(Photobacterium damsela)灭活菌(1％)、坚强芽孢杆菌活菌(1.0×108 CFU/g)+溶藻弧菌(Vibrio alginolyticus)灭活菌(1％)配制3种免疫饲料.每组3个重复,对个体质量为(3.2±0.26)g的凡纳滨对虾(Litopenaeus vannamei)进行了为期30d的养殖实验.每5d取样,以血清中的酸性磷酸酶(ACP)、碱性磷酸酶(AKP)、一氧化氮合酶(NOS)、超氧化物歧化酶(SOD)和溶菌酶(UL)活性为免疫指标,探讨了肠道益生菌及其灭活菌体作为免疫制剂对凡纳滨对虾非特异性免疫水平的影响;在投喂免疫饲料后的第16天,按0.9 g/10尾剂量,直接投喂感染白斑综合征病毒(wssV)对虾病料,计算各实验组每天的累计死亡率,分析肠道益生菌及其灭活菌体作为免疫制剂对凡纳滨对虾病毒感染能力的影响.结果显示:添加益生菌的实验组对虾血清中SOD、ACP、AKP和NOS活性明显高于对照组(P＜0.05),特别是显著提高了对虾抗WSSV感染的能力.其中坚强芽孢杆菌活菌(1.0× 108 CFU/g)和美人鱼发光杆菌灭活菌(1％)实验组的抗病毒感染能力最强,感染WSSV 14 d后累计死亡率为10.71％;而对照组为64.28％.结论认为,饲料中添加肠道益生菌及其灭活菌体能提高凡纳滨对虾非特异性免疫水平和抵抗疾病的能力,有望作为新型对虾免疫增强剂应用于对虾养殖业.     

饲料中添加潜在益生菌对凡纳滨对虾肠道消化酶活性和菌群组成的影响

在基础饲料中添加不同配伍的坚强芽孢杆菌Bacillus firmus活菌（1.0×108CFU/g）、美人鱼发光杆菌Photobacterium damsela灭活菌（1%）和溶藻弧菌Vibrio alginolyticus灭活菌（1%）作为实验饲料,基础饲料为对照组。实验所用凡纳滨对虾初始质量为3.2±0.26 g,投喂15d后进行WSSV感染实验并继续观察14d。实验期间定期取样测定对虾肠道胃蛋白酶、脂肪酶和淀粉酶的活性变化,并分析可培养肠道细菌总数和优势菌数量。结果表明,实验组对虾肠道中胃蛋白酶、脂肪酶和淀粉酶活性整体水平均显著高于对照组（P<0.05）,肠道细菌数量较对照组有显著增加。其中,坚强芽孢杆菌与美人鱼发光杆菌灭活菌复合菌组具有最高的淀粉酶活性,坚强芽孢杆菌与溶藻弧菌灭活菌复合组具有最高的脂肪酶和胃蛋白酶活性,而坚强芽孢杆菌单一组具有最大的肠道细菌总数和优势菌数。本研究表明,饲料中添加潜在益生菌可以提高凡纳滨对虾肠道消化酶活性并调节肠道菌群变化,利于养分的消化吸收。其中以坚强芽孢杆菌与美人鱼发光杆菌灭活菌复合组的整体效果较佳。     

地衣芽孢杆菌对大泷六线鱼生长、肠道消化酶、血清非特异性免疫及抗病力的影响

本研究探讨饲料中添加地衣芽孢杆菌(Bacillus licheniformis)对大泷六线鱼(Hexagrammos otakii)生长、肠道消化酶、血清非特异性免疫及抗病力的影响。选取平均体重为(22.0±2.0) g的大泷六线鱼270尾,随机分成3组(对照组和2个不同菌剂水平的实验组),每组3个平行,每个平行 30尾鱼。对照组投喂仅有蛋白液包裹的基础饲料,实验组投喂含活菌量达5×107 CFU/g(0.5%)和1×108 CFU/g(1.0%)地衣芽孢杆菌的实验饲料。投喂50 d后进行致病菌杀鲑气单胞菌(Aeromonas salraonicida)攻毒感染,测定14 d内累积死亡率。结果显示,实验组饲料中地衣芽孢杆菌可显著提高大泷六线鱼的特定生长率(SGR) (P<0.05)。与对照组比较,0.5%和1.0%地衣芽孢杆菌均可提高大泷六线鱼血清中SOD、CAT及T-AOC活性(P<0.05),降低MDA含量(P<0.05);GSH-Px活性在1.0%实验组较对照组下降,而0.5%实验组较对照组提高。地衣芽孢杆菌实验组鱼血清GS、MDH和HK活性均得到提高(P<0.05),0.5%和1.0%不同剂量组的提高程度不同。地衣芽孢杆菌实验组鱼血清中AST和ALT活性较对照组均降低(P<0.05),CHE和ADA活性较对照组有提高(P<0.05)。实验组鱼肠道胰蛋白酶、淀粉酶和脂肪酶活性均有不同程度的提高,且1.0%实验组活性最强(P<0.05)。攻毒实验结果显示,1.0%地衣芽孢杆菌组大泷六线鱼14 d内累积死亡率仅有35.55% (P<0.05)。由此可见,饲料中添加地衣芽孢杆菌可促进大泷六线鱼生长,提高其肠道消化酶活性及非特异性免疫酶活性,增强其对杀鲑气单胞菌的抵抗能力。     

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。     

Isolation of a novel strain of Bacillus pumilus from penaeid shrimp that is inhibitory against marine pathogens.

A bacterium was isolated from the mid-gut of healthy black tiger shrimp, Penaeus monodon , based on a large zone of inhibition in mixed culture on solid medium. The isolate was a Gram-positive, motile spore former, with an optimum pH range for growth in tryptone soya broth containing 2% NaCl of between pH 6 and 9. The bacterium was highly salt tolerant with concentrations between 0% and 8% having no detrimental effect on growth. The isolate was identified as Bacillus pumilus based on physiological capabilities using the API50CHB and Biolog systems. Amplification and sequencing of the 16S rRNA gene followed by phylogenetic analysis confirmed its identity. The Bacillus pumilus isolate was strongly inhibitory against the marine bacterial pathogens Vibrio alginolyticus , V. mimicus and V. harveyi , and weakly inhibitory against V. parahaemolyticus in cross-streaking assays on solid medium. The organism was marginally self-inhibitory, and inhibited B. licheniformis and B. subtilis . The suitability of the B. pumilus isolate for use as a probiotic in farmed shrimp was further supported by the absence of any of the known B. cereus enterotoxin genes. Based on these in vitro results, in vivo safety and efficacy trials are underway to determine suitability of the novel strain as a commercial probiotic.     

Comparison of four antibiotics with indigenous marine Bacillus spp. in controlling pathogenic bacteria from shrimp and Artemia

Use of antibiotics for the control of bacterial diseases in shrimp culture has caused several adverse impacts to the industry. This has resulted in the search for alternative environment friendly approaches to overcome bacterial infections. This study was conducted to investigate the use of beneficial bacteria as an alternative to antibiotics. Ten pathogenic bacterial species isolated from shrimp, Penaeus monodon, and Artemia cysts were tested for susceptibility to indigenous marine Bacillus subtilis AB65, Bacillus pumilus AB58, Bacillus licheniformis AB69 and compared with oxytetracycline, chloramphenicol, gentamicin and bacitracin, which are common antibiotics used in Asian aquaculture. The Bacillus spp. were isolated from the local marine environment for bioremediation use in shrimp hatcheries and were proven to reduce total ammonium nitrogen. The pathogenic bacterial isolates were 90% susceptible to B. subtilis AB65, 70% susceptible to B. pumilus AB58 and B. licheniformis AB69 and 100% susceptible to oxytetracycline, chloramphenicol and gentamicin but only 40% to bacitracin. Two representative isolates of the vibrio group, Vibrio alginolyticus VaM11 and Vibrio parahaemolyticus VpM1, when tested for competitive exclusion by a common broth method using the marine Bacillus spp., showed decreased viable counts from 10(8) to 10(2) cfu mL(-1). The results suggest that the action of the marine bacteria appears to be significant in protecting the host shrimp against pathogenic bacteria. In addition to the alternative use of antibiotics, the selected marine bacteria had additional bioremediation properties of reducing ammonia.     

高位池养殖过程凡纳滨对虾携带WSSV情况的动态变化

为了更好地预防对虾白斑综合征(WSS)的暴发,探讨该病毒病的流行规律,笔者针对养殖过程中对虾的携带WSSV情况展开调查。调查于2010年7月-2010年11月广东省汕尾市红海湾养殖场进行,从10口凡纳滨对虾高位养殖池中随机抽取6口进行跟踪采样。收集指标包括对虾生长状况、基本环境指标、浮游微藻种群结构和对虾病毒携带量等。本文重点报道利用实时定量PCR-TaqMan探针法检测6口精养池塘对虾体内WSSV的携带量变化情况,检测结果显示:①1-3号虾池苗种携带WSSV,其波动范围在1.3×103~1.7×104copy/g之间;②对虾在养殖过程中均带毒,鳃组织中的平均病毒携带量(2.3×109copy/g)多于肌肉组织中的平均病毒携带量(3.2×108copy/g),且变化趋势一致,但没有显著性差异(P>0.05);③在整个养殖过程中对虾WSSV携带量总体呈现波动上升的趋势,期间各池出现过数次高值。前期WSSV拷贝数的波动范围在1.3×103~3.0×107copy/g之间,后期上升到1.5×106~1.2×1011copy/g,使得某些池塘养殖对虾WSS暴发。调查结果说明:1)对虾携带WSSV可以进行养殖生长;2)WSSV在对虾体内的含量是变化的,且其变化存在着一定的规律性;3)这种变化规律主要体现在带毒量随着养殖时间的进行及外界水环境中某些主要因子的变化而变化,如:养殖时间越长,带毒量越高;养殖环境中某些关键环境因子的改变,如:温差较大,不良藻相转换,天气骤变等均可引起对虾体内病毒含量较大的波动。鉴此,作者提出,构建并维持良好的浮游微藻的群落结构,注意有害藻相改变时保持养殖水体环境稳定,对环境突变前后都做好应对对虾应激的措施等可以极大程度地减少WSS暴发的可能。本研究通过对WSSV的密切跟踪,旨在更好的反映其在养殖环境下的动态变化规律,以及受各种环境因子影响的情况,从而为预防对虾WSS提供依据和参考。     

蚯蚓与蝇蛆对中国对虾生长及抗白斑综合征病毒感染的研究

以中国对虾（Fenneropenaeus chinensis）为实验材料，分别投喂配合饲料、蚯蚓、蛤蜊、蝇蛆等四种饵料，利用生长和抗病性指标综合评价蚯蚓和蝇蛆作为中国对虾饵料的可行性。生长实验前测定每个实验组的初始体长和体重，养殖40d后再次测定生长指标，之后，分别投喂不同毒饵量进行人工感染实验。方差分析表明：投喂四种饵料后，蛤蜊组生长最快，其次是蚯蚓，再次是蝇蛆，最后是配合饲料，各组之间对虾体长增长差异显著（P<0.05），体重增长差异极显著(P<0.01) 。利用线性固定模型分析不同饵料及不同病毒量感染下对中国对虾存活率的影响，其中蚯蚓组存活率最高，配合饲料组存活率最低，并且蚯蚓组存活率显著高于蛤蜊组和配合饲料组(P<0.05)，蝇蛆组显著高于配合饲料组（P<0.05）。研究结果表明：蚯蚓和蝇蛆可显著提高中国对虾的生长速度，明显提高中国对虾的抗病性，是很好的对虾饵料。     

白斑症病毒在日本对虾体内的感染增殖

用投喂患白斑症病毒病的虾组织人工感染日本对虾稚虾,每日取样,整虾冰冻切片,单克隆抗体的荧光抗体方法,原位观察病毒在虾体内的感染增殖,结果表明：感染后三天内,在感染虾的各组织器官内均未观察到明显的病毒感染的阳性细胞,每四天首先在鳃丝腔内的小量血细胞观察到病毒感染;第五天除血细胞外同时在血窦,鳃上皮组织,皮下组织内观察到,第六天进而在心脏,胃上皮组织内观察到：第七天进一步又在淋巴器官,中肠内观察到,八     

Quantitative real time PCR for the measurement of white spot syndrome virus in shrimp

Quantitative real time PCR, recently developed in molecular biology, is applied in this paper to quantify the white spot syndrome virus (WSSV) in infected shrimp tissue. The WSSV content in moribund shrimp of all species tested ( Penaeus stylirostris, P. monodon, P. vannamei ) ranged from 2.0 × 10⁴ to 9.0 × 10¹⁰ WSSV copies μg^–1 of total DNA ( n =26). In whole moribund post-larvae, 4.3 × 10⁹ WSSV copies μg^–1 of DNA were detected which is equivalent to 5.7 × 10¹⁰ WSSV copies g^–1 of post-larvae. The comparison of WSSV content between different tissues showed that muscle and hepatopancreas tissues contained 10 times less virus than gills, pleopods and haemolymph. With inocula of known virus content, bioassays by immersion challenge showed that a minimum of five logs of WSSV copies was necessary to establish disease in the challenged shrimp. In contrast, five logs of WSSV copies injected into shrimp muscle produced a LT-50 of 52 h. This real time polymerase chain reaction (PCR) technique is sensitive (four copies), specific (negative with DNA from shrimp baculoviruses and parvoviruses), dynamic (seven logs) and easy to perform (96 tests in <4 h).     

Effect of microencapsulated thyme essential oil on white spot virus-infected <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>

The present work was performed to investigate if feeding Litopenaeus vannamei with microencapsulated thyme essential oil (TEM, 1.05 g thyme essential oil per 100-g powder) adsorbed on commercial pellet feed was able to protect shrimps against white spot syndrome virus (WSSV) disease. Five treatments were tested: uninfected shrimp fed with commercial pelleted feed (T_C, negative control), WSSV-infected shrimp fed with commercial pelleted feed (T₁, positive control), WSSV-infected shrimp fed with commercial pelleted feed with 0.1% TEM (T₂), WSSV-infected shrimp fed with commercial pelleted feed with 0.5% TEM (T₃), and WSSV-infected shrimp fed with commercial pelleted feed with 1% TEM (T₄). At 72 h post infection, phenoloxidase activity of shrimps treated with 1% TEM did not show significant differences with T_C values but it was significantly higher than that of the other treatments (T₁, T₂, and T₃). Moreover, shrimps treated with T₄ presented absence of clinical signs of WSSV infection and their survival rate was significantly higher than that of T₁, T₂, and T₃ treatments. Therefore, 1% TEM seemed to protect shrimps against WSSV symptoms. Using microencapsulated thyme essential oil may help to fight against WSSV in shrimp farms.     

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。     

Pre-exposure to infectious hypodermal and haematopoietic necrosis virus or to inactivated white spot syndrome virus (WSSV) confers protection against WSSV in Penaeus vannamei (Boone) post-larvae

Larvae and post-larvae of Penaeus vannamei (Boone) were submitted to primary challenge with infectious hypodermal and haematopoietic necrosis virus (IHHNV) or formalin-inactivated white spot syndrome virus (WSSV). Survival rate and viral load were evaluated after secondary per os challenge with WSSV at post-larval stage 45 (PL45). Only shrimp treated with inactivated WSSV at PL35 or with IHHNV infection at nauplius 5, zoea 1 and PL22 were alive (4.7% and 4%, respectively) at 10 days post-infection (p.i.). Moreover, at 9 days p.i. there was 100% mortality in all remaining treatments, while there was 94% mortality in shrimp treated with inactivated WSSV at PL35 and 95% mortality in shrimp previously treated with IHHNV at N5, Z1 and PL22. Based on viral genome copy quantification by real-time PCR, surviving shrimp previously challenged with IHHNV at PL22 contained the lowest load of WSSV (0-1x10(3) copies microg-1 of DNA). In addition, surviving shrimp previously exposed to inactivated WSSV at PL35 also contained few WSSV (0-2x10(3) copies microg-1 of DNA). Consequently, pre-exposure to either IHHNV or inactivated WSSV resulted in slower WSSV replication and delayed mortality. This evidence suggests a protective role of IHHNV as an interfering virus, while protection obtained by inactivated WSSV might result from non-specific antiviral immune response.