红螯光壳螯虾(Cherax quadricarinatus)酚氧化酶原基因的克隆与表达分析

为深入了解红螯光壳螯虾酚氧化酶原(proPO)基因的非特异性免疫机制,利用RACE技术从红螯光壳螯虾血细胞中克隆到酚氧化酶原基因cqproPO,cqproPO基因cDNA全长为2 962 bp,开放阅读框为1 998 bp,编码665个氨基酸,其结构中含有两个铜离子结合位点,预测分子量为75.86 ku;同源性比对结果显示,红螯光壳螯虾CqproPO与克氏原螯虾酚氧化酶原的同源性最高为79％,其次是淡水螯虾74％、挪威龙虾69％、美国龙虾67％等;进化分析发现CqproPO与克氏原鳌虾、淡水螯虾、挪威龙虾、美国龙虾等的酚氧化酶原亲缘关系最近;Realtime-PCR实验结果表明,CqproPO在血细胞中表达水平最高,其次是肠、触角腺、鳃等;在肝胰腺中有适量表达;WSSV感染后红螯光壳螯虾CqproPO mRNA在血细胞、肝胰腺和鳃组织中具有不同的时空表达趋势,但感染组和免疫后感染组mRNA表达量分别在感染后12h和24 h达到最大值,且在3种组织中2个感染组的CqproPO表达量为对照组的1.3 ～2.55倍,显著高于对照组(P＜0.05),之后cqproPO基因的转录水平明显下降.免疫后再受病毒感染的虾,CqproPO mRNA的表达量在3种组织中总体高于感染组,感染7d后的免疫保护率达到51.86％,表明免疫增强剂可使机体的抗病毒能力增强,对防御WSSV感染具有一定的免疫保护作用.     

二种对虾白斑综合征组织与细胞病理研究

对自然发病、投喂感染和注射感染的中国对虾、凡纳滨对虾白斑综合征进行组织与细胞病理研究，结果发现：中国对虾与凡纳滨对虾的病理变化相似，但不同感染方式的患病对虾有差异。临床病理：相同点表现为游动无力，反应迟钝，胃中无食，体色变暗，肌肉浑浊，肝胰腺肿大；不同点为自然发病和投喂感染的对虾临床病理变化比注射人工感染明显，病程长。显微病理：相同点表现为细胞核肿大，细胞变性、坏死等；不同点为自然发病和投喂感染的病虾胃、肝胰腺的病理变化比注射感染严重，坏死细胞数量多，组织大面积坏死溶解；注射感染病虾的肌肉组织显微病理变化比自然发病、投喂感染严重。超微病理：相同点表现为细胞肿大、变性、坏死、溶解，线粒体、内质网等细胞器形态变异，肿大，膜溶解或整体崩解；细胞核肿大或固缩或溶解．部分细胞核中可见WSSV；不同点为注射感染的患病对虾肌肉组织的超微病理变化比自然发病、投喂感染严重，肌细胞核中观察到WSSV的几率较高；投喂感染、自然发病对虾的胃上皮细胞、肝胰腺细胞的超微病理变化比注射感染严重。另外，不同类型的细胞对WSSV的易感性不相同，血细胞为最常见的被病毒感染的靶细胞，血细胞通过细胞变形或形成伪足，贴附于其它组织细胞膜上。     

罗氏沼虾肌肉白浊病的病原和组织病理

对患肌肉白浊病的罗氏沼虾进行细菌分离、寄生虫检查、组织病理观察以及虾池的水质分析和疾病的流行病学调查,排除了细菌、寄生虫以及水质环境因素造成该病的可能性,并发现病症多发生在体长0 8～3cm的幼虾中,而体长3cm以上的虾病症明显减轻。病虾的显微和亚显微组织病理观察发现,病虾腹部白浊的肌肉组织肌纤维肌浆内存在嗜碱性包涵体,肝胰腺、血细胞、心脏和鳃组织细胞胞浆内也有嗜碱性包涵体出现,包涵体内有大量以晶格状排列、无囊膜的二十面体球状病毒颗粒,直径为(23.6±3.8)nm(n=40)。在包涵体外的胞浆内,尤其在白浊肌肉组织肌纤维肌浆内也分布有大量病毒。心脏和鳃组织出现不同程度的变性,肌肉组织、肝胰腺、血细胞除变性外还出现坏死;肌肉组织病变尤为严重,肌肉白浊症状是肌肉组织变性、坏死的结果。将病虾白浊肌肉组织以口服方式感染健康虾,健康虾出现与自然发病虾一样的症状,发病率和死亡率亦与自然感染病虾相近,表明上述二十面体球状病毒是罗氏沼虾肌肉白浊病的病原。     

对虾发光病病原菌的致病性及病理学观察

本文报道了对虾发光病病原菌的致病性、发病症状和病理变化。结果表明:哈维氏弧菌对虾类有极强的致病性,并引起组织和器官不同程度的病变。病理变化表现为:肌纤维颗粒变性,纵裂,坏死:肝胰腺空泡变性,坏死;淋巴器官坏死等。注射感染的发病虾,肝胰腺出现血细胞浸润,说明对虾对细菌感染有一定的防御能力。     

中国对虾溞状幼体的细小病毒(Parvovius)类疾病初检

通过对病变的中国对虾溞状幼体组织学切片观察发现,病虾的肠粘膜、肌肉、神经索、心脏等组织器官普遍坏死。明显的特征是,角质层皮下组织的细胞核内有巨大嗜伊红染料的包涵体。故推论此病是由细小病毒感染所致。     

中国对虾涵状幼体的细小病毒（Parvovius）类疾病初检

通过对病变的中国对虾溞状幼体组织学切片观察发现，病虾的肠粘膜，肌肉，神经索，心脏等组织器官普遍坏死，明显的特征是，角质层皮下组织的细胞核内有巨大嗜伊红染料的包涵体，故推论此病是由细小病毒感染所致。     

大连地区中国对虾暴发性流行病病理学研究

本文报道１９９４年－１９９５年大连地区由杆状病毒引起的自然发病和人工感染实验中国对虾组织病理学研究。结果表明，自然发病虾和人工感染的虾组织病理相同，在胃、皮下、类淋巴、造血组织、鳃、中肠、后肠、肝胰腺、心脏等组织器官的上皮细胞、干细胞和结缔组织细胞中都可观察到显著胀大，呈空泡状或被苏木精浓染的细胞核。胃是病理变化出现最早且最严重的器官。在电镜下，胃、甲壳下上皮细胞核中可见大量长２７０～３５０ｎｍ，宽１００～１２０ｎｍ的杆状病毒颗粒，细胞核、线粒体出现胀大，嵴断裂、消失情况。干细胞、上皮细胞和疏松结缔组织细胞为靶细胞。     

南美白对虾（Litopenaeus vannamei）幼虾和杜拉对虾（Farfantepenaeus duorarum）稚虾对六种白斑综合病病毒（WSSV）地理隔离株的免疫比较

白斑综合病病毒(WSSV)是一种对对虾养殖危害性最大的病原体之一。它广泛分布在大多数养殖对虾的亚洲国家以及墨西哥湾、美国。我们采用南美白对虾(Litopenaeus vannamei)幼虾和杜拉对虾(Farfantepenuaeus duorarum)稚虾对六种WSSV地理隔离株的病毒性进行免疫比较。这6组WSSV地理隔离株分别取自中国、印度、泰国、美国德克萨斯州、南卡来罗纳州的对虾以及美国国家动物园养殖的淡水龙螯虾。为了进行免疫性试验研究，我们把病毒感染组织接种到南美白对虾幼虾和杜拉对虾稚虾中。通过组织检查结果证实发生WSSV感染。在这次免疫性试验中，被六组WSSV地理隔离株感染的南美白对虾幼虾的死亡率为100％。其中，德克萨斯州的病毒比其它的病毒厉害，最快引起实验对象死亡，而淡水龙虾的WSSV病毒致病性最慢。与之形成显著对比，杜拉对虾稚虾的死亡率仅为35％--60％，并且对不同的病毒其死亡率也有所不同。值得注意的是，杜拉对虾对德克萨斯州病毒感染也是最为严重，而对淡水龙虾病毒的感染最轻。这个实验结果表明：不同地区的WSSV病毒对实验对象的致病性略有不同，不同品种的对虾及同一种对虾在其生长的不同阶段对病毒的易感性也有所不同。     

南美白对虾的病害防(二)

3.黄头病【病因】该病病原是黄头病病毒,呈杆状,有囊膜,病毒粒子(50～95)纳米×(130～380)纳米,属单链RNA病毒。【症状】病虾早期游动迟缓,头胸甲呈黄色或发白,膨大,鳃变成淡黄色到棕色,肝胰腺变成淡黄色。主要感染鳃组织、淋巴器官、血细胞和结缔组织等。该病毒毒力较强,对虾被感染后3～5天内可全军覆灭。患病虾组织多处坏死。胞核缩小或破裂,核周围细胞质内有嗜碱性的球形包涵体。染毒组织的血窦中有一种独特的数目不等的球形细胞。其胞质嗜碱性、球形胞核居中。【流行及危害】该病首先在印度斑节对虾的精养塘中发现,后在东亚地区的其它…     

一种虹彩病毒感染大菱鲆的病理学研究

对我国虹彩病毒感染的大菱鲆Scophthalmus maximus进行的组织病理和超微病理学研究发现,该病典型的病理学特点是在病鱼的脾脏、肾脏、肠、肝脏、鳃、心脏和皮肤等器官组织内出现嗜碱性的肿大细胞。病毒感染导致患病大菱鲆多个器官组织发生了不同程度的病理变化,其中以脾脏组织的病理变化最为显著,表现为造血组织的严重坏死。此外,肾脏造血组织发生坏死、肠固有膜和黏膜下层出血和水肿、肝细胞水样变性、心肌局灶性坏死以及皮肤真皮层出血并伴有水肿和炎性渗出也是该病常见的组织病理学变化。超微病理研究表明,肿大细胞内有虹彩病毒粒子存在。病毒分布于受感染细胞的胞质、组织间隙以及血管腔内。受感染细胞出现线粒体和内质网等细胞器肿胀、崩解等细胞病理变化。研究认为,病毒感染造成皮下组织血管损伤出血,是虹彩病毒感染的大菱鲆发生＂红体病＂的原因所在。虹彩病毒感染所致的机体严重贫血是患病大菱鲆死亡的主要原因,而主要器官组织的病变使得病鱼器官功能衰竭则可加速鱼的死亡。     

A synthetic antibacterial peptide from Mytilus galloprovincialis reduces mortality due to white spot syndrome virus in palaemonid shrimp

White spot syndrome virus (WSSV) isolated from Penaeus monodon was found to be highly infective for the western Mediterranean shrimp, Palaemon sp. Using polymerase chain reaction (PCR), it was demonstrated that such shrimp are not naturally carriers of WSSV. Following challenge with virus, mortality reached 100% 3.5-4 days after injection at 22 degrees C. Incubation of infected shrimp at 10 degrees C totally suppressed the mortality which rapidly developed when shrimp were returned to 18 or 22 degrees C. Preincubation of WSSV with mature synthetic mytilin significantly reduced shrimp mortality with a 50% efficient dose of about 5 microM. Survival of shrimp was not due to the development of an active mechanism of defence as re-injection of WSSV produced the same mortality pattern. Mortality was probably due to WSSV replication as dot blot failed to detect viral DNA in the injection sample but was positive 1 day post-injection. Protection by mytilin was by interaction at the virus level, preventing replication as no WSSV nucleic acid was detected by PCR even after 7 days in shrimp injected with WSSV preincubated with 10 or 50 microM mytilin.     

Experimental host range and histopathology of white spot syndrome virus (WSSV) infection in shrimp, prawns, crabs and lobsters from India

Experimental studies were conducted by injecting or feeding white spot syndrome virus (WSSV) derived from infected shrimp, Penaeus monodon (Fabricius), collected from the south-east coast of India, to five species of shrimp, two species of freshwater prawns, four species of crabs and three species of lobsters. All species examined were susceptible to the virus. Experimental infections in the shrimp had the same clinical symptoms and histopathological characteristics as in naturally infected P. monodon . A cumulative mortality of 100% was observed within 5–7 days in shrimp injected with WSSV and 7–9 days in shrimp fed with infected tissue. Two species of mud crab, Scylla sp., survived the infection for 30 days without any clinical symptoms. All three species of lobsters, Panulirus sp., and the freshwater prawn, Macrobrachium rosenbergii (De Man), survived the infection for 70 days without clinical symptoms. However, bioassay and histology using healthy P. monodon revealed that crabs, prawns and lobsters may act as asymptomatic carriers/reservoir hosts of WSSV. This is the first report to suggest the carrier/reservoir capacity of these hosts through histological and bioassay evidences. Ultrastructural details of the virus in experimentally infected shrimp, P. vannamei , (Boone), were also studied.     

Production of polyclonal antiserum against recombinant VP28 protein and its application for the detection of white spot syndrome virus in crustaceans

The VP28 gene of white spot syndrome virus (WSSV) was cloned into pRSET B expression vector. The VP28 protein was expressed as a protein with a 6-histidine taq in Escherichia coli GJ1158 with NaCl induction. Antiserum was raised against this recombinant-VP28 protein in rabbits and it recognized VP28 protein in naturally and experimentally WSSV-infected shrimp, marine crabs, freshwater prawns and freshwater crabs. The antiserum did not recognize any of the other known WSSV structural proteins. Various organs such as eyestalks, head muscle, gill tissue, heart tissue, haemolymph, tail tissue and appendages were found to be good materials for detection of WSSV using the antiserum and detection of WSSV was successful in experimentally infected Penaeus monodon and P. indicus at 12 and 24 h post-infection (p.i.), respectively. The antiserum was capable of detecting WSSV in 5 ng of total haemolymph protein from WSSV-infected shrimp.     

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。     

High efficacy of white spot syndrome virus replication in tissues of freshwater rice‐field crab,Paratelphusa hydrodomous (Herbst)

An attempt was made to determine the replication efficiency of white spot syndrome virus (WSSV) of shrimp in different organs of freshwater rice‐field crab, Paratelphusa hydrodomous (Herbst), using bioassay, PCR, RT‐PCR, ELISA, Western blot and real‐time PCR analyses, and also to use this crab instead of penaeid shrimp for the large‐scale production of WSSV. This crab was found to be highly susceptible to WSSV by intramuscular injection. PCR and Western blot analyses confirmed the systemic WSSV infection in freshwater crab. The RT‐PCR analysis revealed the expression of VP28 gene in different organs of infected crab. The indirect ELISA was used to quantify the VP28 protein in different organs of crab. It was found that there was a high concentration of VP28 protein in gill tissue, muscle, haemolymph and heart tissue. The copy number of WSSV in different organs of infected crab was quantified by real‐time PCR, and the results revealed a steady increase in copy number in different organs of infected crab during the course of infection. The viral inoculum prepared from different organs of infected crab caused significant mortality in tiger prawn, Penaeus monodon (Fabricius). The results revealed that this crab can be used as an alternate host for WSSV replication and production.     

Response of crayfish, Procambarus clarkii, haemocytes infected by white spot syndrome virus

White spot syndrome virus (WSSV) is a serious pathogen of aquatic crustaceans. Little is known about its transmission in vivo and the immune reaction of its hosts. In this study, the circulating haemocytes of crayfish, Procambarus clarkii, infected by WSSV, and primary haemocyte cultures inoculated with WSSV, were collected and observed by transmission electron microscopy and light microscopy following in situ hybridization. In ultra-thin sections of infected haemocytes, the enveloped virions were seen to be phagocytosed in the cytoplasm and no viral particles were observed in the nuclei. In situ hybridization with WSSV-specific probes also demonstrated that there were no specific positive signals present in the haemocytes. Conversely, strong specific positive signals showed that WSSV replicated in the nuclei of gill cells. As a control, the lymphoid organ of shrimp, Penaeus monodon, infected by WSSV was examined by in situ hybridization which showed that WSSV did not replicate within the tubules of the lymphoid organ. In contrast to previous studies, it is concluded that neither shrimp nor crayfish haemocytes support WSSV replication.     

Transmission of white spot syndrome virus (WSSV) from Dendronereis spp. (Peters) (Nereididae) to penaeid shrimp

Dendronereis spp. (Peters) (Nereididae) is a common polychaete in shrimp ponds built on intertidal land and is natural food for shrimp in traditionally managed ponds in Indonesia. White spot syndrome virus (WSSV), an important viral pathogen of the shrimp, can replicate in this polychaete (Desrina et al. 2013); therefore, it is a potential propagative vector for virus transmission. The major aim of this study was to determine whether WSSV can be transmitted from naturally infected Dendronereis spp. to specific pathogen‐free (SPF) Pacific white shrimp Litopenaeus vannamei (Boone) through feeding. WSSV was detected in naturally infected Dendronereis spp. and Penaeus monodon Fabricius from a traditional shrimp pond, and the positive animals were used in the current experiment. WSSV‐infected Dendronereis spp. and P. monodon in a pond had a point prevalence of 90% and 80%, respectively, as measured by PCR. WSSV was detected in the head, gills, blood and mid‐body of Dendronereis spp. WSSV from naturally infected Dendronereis spp was transmitted to SPF L. vannamei and subsequently from this shrimp to new naïve‐SPF L. vannamei to cause transient infection. Our findings support the contention that Dendronereis spp, upon feeding, can be a source of WSSV infection of shrimp in ponds.     

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。     

Effect of VP28 DNA vaccine on white spot syndrome virus in <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>

White spot syndrome virus (WSSV) occurs worldwide and causes high mortality and considerable economic damage to the shrimp farming industry. Considering the global environmental, the economic and sociological importance of shrimp farming, and the constraints of high intensity cultivation, development of novel control measures against the outbreak of WSSV become inevitable. In this study, we have explored the protective efficacy of DNA vaccination and tissue distribution of the recombinant plasmid in immunized Litopenaeus vannamei. The VP28 gene was cloned in the eukaryotic expression vector pVAX1, and the construct vector was named as lpv28. The protective effect of lpv28 against WSSV was evaluated in L. vannamei by injecting lpv28 construct and later challenging with WSSV. Expression of these proteins from the recombinant plasmids was confirmed in vitro by RT-PCR and Western blot analysis. The result of vaccination trials showed that a survival rate in shrimp vaccinated with lpv28 was 52.5% at most compared to control groups (100% mortality). The immunological parameters analyzed in the vaccinated and control groups showed that the vaccinated groups owned a high level of lysozyme, alkaline phosphatase, and total superoxide dismutase when compared to the control group. Furthermore, protein expression analysis indicated that VP28 can be detected in gill, muscle and head soft tissue of the shrimps in the immunized group after 14th day injection. Thus, the result indicated that DNA vaccination strategy has a potential utility against WSSV.     

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。