克氏原螯虾酚氧化酶部分生化特性的研究

以克氏原螯虾(Procambarus clarkii)酚氧化酶(PO)为材料,采用L-DOPA为反应底物,分光光度计测定不同温度和pH条件下PO活力,研究克氏原螯虾PO的特性,为其免疫学研究及优化PO准确测定方法提供依据。结果显示PO活力最高时的温度为35℃,低于和高于35℃活力逐渐下降,45℃以上活力迅速下降;0℃仍具有最高活力的24%。在pH4.0～9.5内PO均具有活力,鳃组织最佳pH5.5,血清最佳pH6.5。因此测定克氏原螯虾PO活力的最佳参数为温度35℃,pH5.5(鳃)或6.5(血清)。以上结果说明冰浴终止反应法测定克氏原螯虾PO活力会影响结果的准确性,要慎用。     

秦皇岛地区克氏原螯虾源嗜水气单胞菌分离及生物学特征分析

为鉴定引起克氏原螯虾死亡的病原菌及分析其生物学特征,从秦皇岛地区病死的疑似细菌感染的克氏原螯虾的肝脏和胰腺分离出1株病原菌,暂定名为ProcQHD。经细菌分离鉴定和16S rRNA基因序列分析,该菌株被鉴定为嗜水气单胞菌。通过人工感染动物试验、PCR法及K-B药敏纸片对其致病性、毒力基因、耐药基因及耐药性进行了研究。结果表明,ProcQHD株的LD₅₀为1.0×10⁵ CFU/mL,携带ser、act、exu、lip和Luxs 5种毒力基因;ProcQHD株对替米考星、头孢曲松、环丙沙星、土霉素、诺氟沙星等12种药物高度敏感且携带Tet(A)、Tet(C)、Tet(E)、qepA、gyrB、aac4、aadB、Ant-Ia、Sul-1、LAP-1、SHV 11种耐药基因。分析发现ProcQHD株的某些耐药表型与耐药基因之间有一定相关性。     

克氏原螯虾掘洞行为研究

克氏原螯虾(Procam barus clarkii)掘洞时间多在夜间,可持续掘洞6~8 h,一夜挖掘深度成虾可达40 cm,幼虾可达25 cm。成虾的洞穴深度大部分在50~80 cm之间,少部分可以达到80~150 cm;幼虾洞穴的深度在10~25 cm之间;体长1.2 cm的稚虾已经具备掘洞的能力,洞穴深度在10~20 cm之间。洞穴分为简单洞穴和复杂洞穴两种:85%的洞穴是简单的,即只有一条穴道,位于水面上、下10 cm之间;15%较复杂,即有2条以上的穴道,位于水面以上20 cm处。洞穴的密度为2.8~5.6个/m2。每个洞穴中一般有1~2只虾,但冬季也常发现一个洞中有3~5只虾。克氏原螯虾在繁殖季节掘洞强度增大,在寒冷的冬季,掘洞强度微弱。     

白斑综合征病毒感染克氏原螯虾后的PCR检测及组织病理学研究

采用投喂感染白斑综合征病毒(White Spot Syndrome Virus,WSSV)对虾肌肉的方式,对养殖克氏原螯虾(Procambarus clarkii)进行人工感染,以确定WSSV对养殖克氏原螯虾的易感性。结果发现,投喂病虾感染组螯虾的死亡率达到90%,而对照组未出现死亡。采用PCR对试验组螯虾的肌肉进行WSSV检测,发现投喂感染组的阳性检出率为100%,对照组的阳性检出率均为0。PCR检测结果发现,濒死螯虾的肝胰腺、中肠、肌肉、鳃、性腺、心脏六种组织的PCR结果均为WSSV阳性,而对照组的各组织检测结果均为阴性。组织切片的光镜观察也证实,濒死螯虾的肝胰腺、中肠、肌肉、鳃、性腺、心脏及血淋巴等组织均发生了不同程度的病变。     

常规PCR与巢式PCR法快速检测克氏原螯虾白斑综合征病毒(WSSV)

对传统对虾白斑综合征病毒(WSSV)的PCR检测方法中的病毒模板DNA的提取方法加以改进,建立了一种快速检测克氏原螯虾(Procambarus clarkii)WSSV的PCR方法。本方法将克氏原螯虾肝胰腺组织匀浆液冻融3次进行差速离心后,在病毒沉淀中加入50μL蛋白酶K溶液,室温消化5 min,沸水中煮10 min后,10000 r/min高速离心5 min即可取上清液用于PCR扩增,结果显示:与传统的病毒模板DNA提取方法相比,本方法的PCR结果特异性强,扩增效率高,准确率高,可应用于快速大规模检测克氏原鳌虾中的WSSV。     

泗洪地区克氏原螯虾病原维氏气单胞菌鉴定及致病性分析

为了探究江苏省泗洪县多家养殖场克氏原螯虾(Procambarus clarkii)出现暴发性死亡的病因,本研究以濒死的克氏原螯虾进行病原检测分析,从濒死虾肝胰腺中分离到优势生长菌(菌株编号CPA2020),经对分离菌进行致病性、形态与生理生化特征及gyrB基因同源性与系统发育分析,结果表明引起克氏原螯虾暴发性死亡的病原为维氏气单胞菌(Aeromonas veronii),人工感染实验结果表明分离菌CPA2020对克氏原螯虾具有较强的致病性,LD₅₀为6.31×10⁵ CFU/mL。组织病理学观察发现克氏原螯虾的肝胰腺与肠道组织出现严重的病理损伤,肝细胞裂解,空泡化严重,部分肝细胞坏死,肠壁结缔组织萎缩,皱嵴减少,肠上皮细胞坏死,与自然患病虾的病理变化相同。毒力基因检测表明分离菌CPA2020携带lip、hly、flgN、ompA、flgM、flaH、aha和flgA 8种毒力基因。此外分离菌CPA2020可分泌卵磷脂酶、明胶酶和溶血素。耐药谱测定结果显示,该菌对链霉素、万古霉素、环丙沙星等12种药物耐药,对头孢唑林、克拉霉素等9种药物中介,对...     

白斑综合征病毒(WSSV)在市售克氏原螯虾中携带情况调查

近年来白斑综合征成为制约克氏原螯虾养殖的重要病害。对市场销售的克氏原螯虾抽样调查,采用PCR仪检测白斑综合征病毒(WSSV)的携带情况,结果为:湖泊养殖的克氏原螯虾不携带WSSV,池塘单养的克氏原螯虾及江河野生克氏原螯虾携带WSSV。     

克氏原螯虾繁殖生物学研究

对克氏原螯虾(Procambarus clarkii)繁殖生物学进行了研究。结果显示:克氏原螯虾的性成熟年龄为1年左右;雌虾最小体长为6.4 cm,最小体重为10 g;雄虾最小体长为7.1 cm,最小体重为20 g。5~9月为交配期,其中以6~8月为交配高峰期。克氏原螯虾交配后大约为30 d左右产卵。繁殖期为7~10月,高峰期为8~9月。10月底以后抱卵的虾由于水温逐步降低,一直延续到第2年春季才孵化。克氏原螯虾的繁殖行为与掘洞行为密切相关,繁殖期的掘洞数量较非繁殖期明显增多。7~10月,亲虾均栖息在洞内繁殖,洞穴深度为50~80 cm。卵巢为一次产卵类型。雌雄比例为1∶1。克氏原螯虾的个体绝对繁殖力的变动范围为172~1158粒,平均为517粒。个体相对繁殖力(F/W)的变动范围为2~41粒/g,平均为21粒。个体相对繁殖力(F/L)的变动范围为47~80粒/cm,平均为63粒。     

养殖克氏原螯虾体内白斑综合征病毒的绝对定量分析

近年来克氏原螯虾的养殖受到WSSV的威胁,病毒在宿主组织中的绝对定量对于了解病毒的致病性具有重要意义,但克氏原螯虾组织中WSSV的绝对定量分布还有待研究。实验调查了湖北省5个主养区克氏原螯虾WSSV的感染率,结果表明80%以上克氏原螯虾都携带有WSSV。采用WSSV-VP28蛋白特异性抗体对克氏原螯虾提取蛋白进行Western Blot检测,在WSSV-PCR阳性样品中可检测到VP28特异性条带,在WSSV-PCR阴性样品中没有检测到相应条带。采用实验室建立的WSSV绝对定量PCR方法,对携带病毒的克氏原螯虾6个组织(鳃、胃、肠、血淋巴细胞、肝胰腺和心脏)进行检测。结果表明,在鳃、胃和肠可检测到较多病毒量(约108拷贝/mg),其次是血淋巴细胞(107拷贝/mg)、肝胰腺(106拷贝/mg),在心脏中病毒的含量最低(103拷贝/mg),表明病毒的复制存在组织特异性。结果显示WSSV主要存在于消化系统中,预示着克氏原螯虾可能主要在摄食过程中感染WSSV;不同地区克氏原螯虾组织病毒携带量表现出一定差异,预示着WSSV感染可能受到环境因素的影响。     

复方中草药对克氏原螯虾生长和脱壳的影响

研究了饲料中添加复方中草药(质量分数为1%,2%和4%)对克氏原螯虾(Procambarus clarkii)生长和脱壳的影响。结果显示,添加复方中草药后,虾的脱壳周期缩短,脱壳死亡率和非脱壳死亡率均明显降低(P<0.05),但各中草药添加组间差异不显著(P>0.05)。三个剂量的复方中草药添加组均能不同程度地提高虾的脱壳数、成功脱壳数、硬壳虾重和软壳虾重,与未添加组相比差异显著(P<0.05),其中2%组增加最为显著,分别比对照组高出71.1%、103.2%、20.3%、10.0%。回归分析表明,克氏原螯虾的特定生长率达到最大值时的中草药添加质量分数为2.74%。试验表明克氏螯虾饲料中复方中草药水平以2%左右为宜。     

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.     

Oral vaccination trials with crayfish, Procambarus clarkii, to induce resistance to the white spot syndrome virus

The potential of oral vaccination against white spot syndrome virus (WSSV) in crayfish Procambarus clarkii was investigated. The protective effect of binary ethylenimine (BEI)-inactivated WSSV was tested by oral vaccination, followed by an oral challenge with WSSV. The crayfish fed with feed pellets coated with BEI-inactivated WSSV showed a resistance to WSSV on the seventh day post vaccination (dpv). The relative percentage survival values were 60%, 70% and 75% for the vaccinated once, twice and thrice with inactivated WSSV. Following an intramuscular injection experiment, no mortality was recorded in the inactivated WSSV group and the negative control at 17 days post challenge. The cumulative mortalities in the heated WSSV group and WSSV group were 100%. Shrimp that survived the WSSV challenge on the seventh day after cessation of oral vaccination were positive for the presence of WSSV by a polymerase chain reaction assay specific for WSSV. This result indicated that inactivated WSSV could protect crayfish against WSSV by oral delivery.     

克氏原螯虾自噬相关基因PcAtg2的克隆及其在白斑综合征病毒胁迫下的表达分析

为了解自噬相关基因Atg2在克氏原螯虾(Procambarus clarkia)先天免疫中的作用,本研究克隆了克氏原螯虾Atg2 (PcAtg2)基因全长序列。生物信息学分析显示,PcAtg2蛋白编码序列全长为9 966 bp,推测其编码2 189个氨基酸。组织定量表达分布显示,PcAtg2在克氏原螯虾的各个组织中均有表达,其中在肝胰腺中表达最高,在眼柄中表达最低。在白斑综合征病毒(WSSV)感染实验中,PcAtg2基因表达量在不同组织中均呈现显著上调趋势。RNA干扰(RNAi)实验显示,PcAtg2基因沉默后,WSSV在克氏原螯虾体内的增殖明显被抑制,同时,自噬相关基因的表达量上调。透射电镜分析结果显示,在WSSV感染后,PcAtg2基因沉默组中克氏原螯虾肝胰腺组织中的自噬小体多于对照组。本研究结果可为了解克氏原螯虾应对WSSV胁迫下的调控机制提供理论参考。     

Increased resistance to white spot syndrome virus in Procambarus clarkii by injection of envelope protein VP28 expressed using recombinant baculovirus

In order to investigate whether protein structure has an effect on protective effect of envelope protein of WSSV, VP28 protein was expressed both in Escherichia coli (pET-VP28) and insect (BmN) cells (BmNPV-VP28). The baculovirus (BmNPV) expression system was used to obtain correctly folded VP28 protein. Procambarus clarkii crayfish were intramuscularly injected with lysates of cells infected with recombinant pET-VP28 and BmNPV-VP28, respectively, and then challenged by intramuscular injection of WSSV to assess the duration of protection. The crayfish injected with BmNPV-VP28 showed generally lower mortality rates when compared to crayfish injected with pET-VP28, resulting in relative percent survivals of 92% and 39%, respectively, when compared to the control groups injected with empty vectors BmNPV and pET-30a. These results showed that VP28 protein produced in BmN cells gave much better protection than VP28 protein produced in E. coli.     

Effect of low water temperature on viral replication of white spot syndrome virus in Procambarus clarkii

This study evaluated the effect of low water temperature (10 ± 1 °C) on viral infection and replication of white spot syndrome virus (WSSV) in crayfish, Procambarus clarkii, under standardized conditions. Crayfish were (i) maintained at 24 ± 1 °C before challenge and 10 ± 1 °C afterwards, or (ii) maintained at 10 ± 1 °C before challenge and 24 ± 1 °C afterwards. No mortality was observed when crayfish were held at 10 ± 1 °C after challenge, but mortality reached 100% when they were transferred to 24 ± 1 °C. Competitive PCR showed that viral levels at 10 ± 1 °C rose from 10⁶ to 10⁸ copies/mg of gill tissues, while at 24 ± 1 °C levels increased from 10⁶ to 10¹⁰ copies/mg of gill tissues during the same time interval. These results showed that a low water temperature of 10 ± 1 °C could reduce viral replication when compared to 24 ± 1 °C but could not prevent it.     

温度、pH对克氏原螯虾血清酚氧化酶活力及稳定性的影响

以克氏原螯虾(Procam barus clarkii)为材料,研究温度、pH对其血清酚氧化酶活力及稳定性的影响。实验以L-dopa为反应底物,在试验设计的温度范围(20,30,40,50,60,70℃)内,测得酚氧化酶活力的最适温度为20℃,随温度升高酶活力迅速下降,该酶在20~40℃范围内表现出较高的热稳定性,而30℃时最稳定;该酶的最适pH 7.0,在pH 6.0和7.0的缓冲系统中表现出较强的稳定性。     

60Co辐照对对虾白斑综合征病毒(WSSV)的灭活效果

以超低温保存的感染了白斑综合征病毒(WSSV)的中国对虾制备的病毒粗提液为毒种，注射感染凡纳对虾并收集濒死虾，DNA斑点杂交检测每尾凡纳对虾WSSV感染状况。取DNA斑点杂交呈强阳性的30尾对虾，平均分为3组，病毒粗提液也平均分成3组，3组材料分别通过^60Co辐照，辐照时间分别为12、24和36h，辐照剂量为0．8KGy／h。辐照后的材料经PCR检测证实^60Co辐照不能完全破坏WSSV的DNA组成。以辐照后的感染白斑综合征病毒的对虾个体和白斑综合征病毒粗提液为感染毒种，人工感染健康凡纳对虾，验证^60Co辐照对病毒感染力的破坏作用，证实^60Co辐照可显著降低WSSV病毒粗提液的感染力，^60Co辐照可适当降低WSSV感染对虾的感染力。