海湾扇贝养殖过程中的流行病学调查研究

2001~2002年,对莱州湾养殖海湾扇贝进行了较系统地流行病学调查研究.以海湾扇贝养殖集中海区莱州市金城镇扇贝养殖场为定点调查和样品采集点.调查内容主要包括海湾扇贝各养殖期的死亡率和可疑病原生物检测,同时对pH,溶解氧,温度,盐度等理化环境因子进行连续监测.调查结果显示:海湾扇贝在0.5~1.3cm的幼贝阶段死亡率较高,达50%;随后死亡率降低,1.3~2.2cm的幼贝阶段死亡率约为20%,以后各阶段死亡率平稳,均在7%以下,累积死亡率约为60%.对环境理化因子的调查显示,pH,溶解氧、盐度的变化幅度不大,与扇贝死亡率无明显相关性;温度变化与死亡率有一定的相关性,即低温(20℃以下)可能是养殖海湾扇贝幼贝重要的环境胁迫因子,而高温(27℃左右)对海湾扇贝成贝生长无不良影响.通过对可疑致病性生物因子的调查研究及病理学观察表明,海湾扇贝在养殖过程中可感染多种微生物,其中原核微生物类立克次体(RLO)在养殖过程各生长阶段均可检出,即0.5,1.5,3.0,3.6和5.0cm的海湾扇贝RLO的感染率分别为15%,35%,80%,50%和20%;RLO的感染强度分别为1.0,1.5,16.0,11.5和5.0.在病原生物检查过程中,除RLO以外还发现有类支原体原核生物(MLO)寄生.结合感染强度和组织病理学的相关性分析,初步认为类立克次体(RLO)可能是海湾扇贝在海上吊养的幼贝阶段发病死亡的致病因子.     

栉孔扇贝体内寄生的病毒的分离纯化及其形态学观察

栉孔扇贝(Chlamys farreri)是我国传统海水养殖贝种,养殖范围主要分布于黄海、渤海沿岸的山东、河北、辽宁等地,但是,近年来因病害发生,造成了巨大的经济损失.扇贝大规模死亡流行病学调查研究已进行多年,流行病学调查、病理学、病原感染实验及病原分离纯化等研究初步认为类立克次体是扇贝(包括栉孔扇贝和海湾扇贝Argopecten irradians)的病原生物[1～4].在用泛影葡胺密度梯度离心纯化栉孔扇贝类立克次体时发现一种具囊膜的病毒粒子,本文首次报道栉孔扇贝体内该球形病毒纯化的形态学特征.     

栉孔扇贝大规模死亡的病原研究新进展

对导致我国北方海区栉孔扇贝在养成期大规模死亡的病原研究已成为国内养殖界各方关注的焦点之一。其中对致病原因的探查工作至今至少涉及到原核类微生物 (如立克次体 ,衣原体等 )、病毒、寄生原生动物、环境因子 (如饵料、水温等 )以及微生物等几方面［１～４，６～９］。在国家重大基础研究规划项目 (９７３)的资助下 ,我们自２０００年１月起对导致栉孔扇贝大规模死亡的各种可能诱因分别从流行病学、环境、组织病理学以及病原学角度开展了深入、全面的调查与检测 ［２］。在此基础上 ,本课题组新近又开展和完成了对栉孔扇贝各类可疑病原的…     

热带海洋珍珠贝类立克次体(RLO)病研究 Ⅷ.养殖大珠母贝贝苗养成期群体RLO病流行病学研究

大珠母贝人工养殖过程中,幼贝死亡是一个严重的问题。1993年11月至1995年5月,采用现场调查、实验室研究和数理分析相结合的方法,在海南省临高县新盈珍珠贝养殖场自然海区吊养的A,B,C3批养殖贝和陵水新村珍珠贝养殖场养殖池吊养的D批养殖贝的发病和死亡情况进行了流行病学凋查.结果显示,大珠母贝幼贝群体的死亡是以暴发性方式发生,幼贝群体的大批量死亡高峰一般发生在4～6月龄期,8个月以后随着龄期的延长,死亡率显著降低.幼贝群体死亡率与贝体平均体长的关系是4cm以下的贝体死亡率较高,1～3cm阶段处于死亡高峰期内,5cm以上的贝体死亡率显著降低.大珠母贝幼贝群体的死亡率与类立克次体感染(即RLO平均感染严重度指数SI)之间存在显著的相关性,在RLO感染的高峰之后或当中部相应伴随贝群体死亡率的高峰,随着RLO感染的降低,贝群体的死亡率也明显降低。在4批养殖贝群体中,仅在A批养殖贝群体样本中发现有少量纤毛虫寄生(感染率为87.5%,感染强度为3.56个/10倍物镜).这几批幼贝群体的死亡与海水温度、盐度之间均无相关性。     

热带海洋珍珠贝类立克次体(RLO)病研究 Ⅶ 大珠母贝育苗期幼虫群体RLO病流行病学研究

对流行病学分析显示大珠母贝幼虫群体的死亡是以暴发性方式发生,从胚胎发育前期至变态期,幼虫存在两个死亡高峰,分别位于壳顶期之前或壳顶期和壳顶后期.大珠母贝幼虫群体的死亡率与RLO感染之间存在显著的相关性,在RLO感染的高峰之后均相应伴随幼虫群体死亡率的高峰,随着RLO感染率和感染强度的降低,幼虫群体死亡率也明显降低.在TEM下未发现未受精卵母细胞、受精卵和胚胎早期幼虫感染RLO,最先出现RLO包涵体阳性感染的时期为24h尚未摄食的D形期幼虫,故认为RLO可能不是通过垂直传播,而是通过外界接触感染的方式感染宿主,贝体感染至发病死亡存在潜伏期.     

栉孔扇贝大规模死亡的原因探讨

于１９９８年７－９月,在发生贝类大规模死亡的山东工岛县和烟台四十里湾养殖区,选取濒死的栉孔扇贝,通过组织病理分析和电镜观察,发现大量寄生原核生物－－衣原体样原核生物和类立克次氏体。病理学研究表明,寄生原核生物造成栉孔扇贝组织细胞严重病变,可能是引起栉孔扇贝大规模死亡的重要原因。     

山东沿岸夏季栉孔扇贝大规模死亡原因分析

在对夏季高温期山东沿海栉孔扇贝大规模死亡现状调查的基础上,从山东沿岸栉孔扇贝养殖业发展的历史,生态,环境和种质等方面分析了栉孔扇贝大规模死亡的原因,认为大规模死亡的根本原因是长期订养殖导致养殖环境的老化或恶化及栉孔扇贝抗逆能力下降,而在夏季高温期,海区饵料明显不足,栉孔扇贝本身消耗增大,养殖水体自身污染加剧及病原体大量滋生更为直接的原因。     

温度和盐度对华贵栉孔扇贝免疫相关酶的联合效应

为研究温度和盐度对华贵栉孔扇贝免疫相关酶活力的联合效应,采用中心复合设计(CCD)和响应曲面分析法(RSM)进行试验,在实验室条件下研究了温度(19~31℃),盐度(22~38)对华贵栉孔扇贝酸性磷酸酶(ACP)、碱性磷酸酶(AKP)、谷胱甘肽还原酶(GR)三种免疫相关酶活性的联合效应,旨在考察温度与盐度对ACP,AKP和GR活性的影响及华贵栉孔扇贝免疫水平较高时的最佳温度和盐度组合,为华贵栉孔扇贝人工养殖所需条件提供理论基础。结果表明:温度的一次效应和二次效应对ACP,AKP和GR活力影响显著(P0.05);盐度的一次效应和二次效应对ACP,AKP和GR的活力影响显著(P0.05);温度和盐度的交互作用对ACP,AKP和GR活力的影响显著(P0.05)。采用响应曲面法,建立了温度和盐度对华贵栉孔扇贝ACP,AKP和GR活性影响的模型方程,该模型方程决定系数分别为0.986 6,0.981 3和0.957 6(P0.01),预测系数分别为0.934 5,0.888 0和0.738 4,表明该模型可以用于预测华贵栉孔扇贝ACP,AKP和GR活力变化。通过模型优化和验证实验,得到在温度24.30℃、盐度30.55时,ACP和AKP活力达到最小值,分别为170.43,181.74U/mg,而GR活力最大为0.34U/mg,满意度达0.95。     

海洋岛海域水温异常波动与养殖栉孔扇贝死亡的关系

本文对海洋岛海域水温异常波动,与该海域养殖栉孔扇贝大批死亡的关系,进行了研究。首先,总结了几年来海洋岛海域养殖栉孔扇贝死亡的特征。其次,结合水温观测数据分析了水温异常波动与栉孔扇贝大批死亡的关系。得出的结论是:大幅度、跳跃型水温异常波动是导致扇贝死亡主要原因。最后,通过生物试验证实了上述观点。     

4种养殖扇贝的群体遗传多样性及特异性AFLP标记研究

应用AFLP技术对我国4种主要养殖扇贝进行了遗传分析.虾夷扇贝、海湾扇贝、华贵栉孔扇贝、栉孔扇贝4种养殖扇贝群体中,种内遗传相似度依次分别为0.841 5,0.786 3,0.719 0和0.673 1,香侬氏指数分别为43.52,58.87,80.16和92.83,栉孔扇贝和华贵栉孔扇贝的遗传多样性水平明显高于海湾扇贝和虾夷扇贝;AFLP标记共享位点分布表明,在4种扇贝中栉孔扇贝与华贵栉孔扇贝之间的共享位点最多,同时4种扇贝的种间遗传相似度也以栉孔扇贝和华贵栉孔扇贝之间为最高(0.769 5),海湾扇贝和虾夷扇贝之间为最低(0.662 6),揭示所研究的几种扇贝中栉孔扇贝和华贵栉孔扇贝的遗传关系最近(遗传距离为0.262 1);找到了21个种内特异性AFLP标记和种间共享标记,可用于种质鉴定和分子辅助分类.     

Studies on rickettsia-like organism (RLO) disease of tropical marine pearl oyster-Epidemiological investigation of RLO disease in juvenile populations of maricultured Pinctada maxima

The death which occurred in juvenile population was a severe problem in the course ofPinctada maxima artificial culture. With the methods of field investigation, histological study and statistic analyses, the epidemiological study was carried out on the disease and death in juvenile populations of Pinctada maxima in the Xinying Pearl Oyster Mariculture Farm of Lingao County (for the A, B and C batches of cultured juveniles hanged in the sea) and the Xincun Pearl Oyster Mariculture Farm of Lingshui County (for the D batch of cultured juvenile hanged in the pond), Hainan Province from November 1993 to April 1995.The results show that the deaths which occurred in juvenile populations of Pinctada maxima presented an outbreak pattern. The peak of mortality rates, in general, occurred in 4-to 6-month old pearl oyster juveniles, and the mortality rates gradully declined with the extention of pearl oyster age after cul-turing 8 months. The correlation between the mortality rates of juvenile populations and mean     

Studies on rickettsia-like organism(RLO)disease of tropical marine pearl oyster--Epidemiologicai investigation of RLO disease in juvenile populations of maricultured Pinctada maxima

The death which occurred in juvenile population was a severe problem in the course ofPinctada maxima artificial culture. With the methods of field investigation, histological study andstatistic analyses, the epidemiological study was carried out on the disease and death in juvenile popula-tions of Pinctada maxima in the Xinying Pearl Oyster Mariculture Farm of Lingao County (for the A,B and C batches of cultured juveniles hanged in the sea) and the Xincun Pearl Oyster Mariculture Farmof Lingshui County (for the D batch of cultured juvenile hanged in the pond), Hainan Province fromNovember 1993 to April 1995. The results show that the deaths which occurred in juvenile populations of Pinctada maxima pre-sented an outbreak pattern. The peak of mortality rates, in general, occurred in 4-to 6-month old pearloyster juveniles, and the mortality rates gradully declined with the extention of pearl oyster age after cul-turing 8 months. The correlation between the mortality rates of juvenile populations and mean bodylengths of juvenile populations show that the mortality rate become higher under 4 cm of mean bodylengths of juvenile population and become obvious declined over 5 cm of mean body lengths. The peak ofmortality rate occurred in 1 ～3 cm of mean body lengths. The results of histological observation showedthat rickettsia-like organism inclusions were common, histological widespread infective agent among ev-ery batches (A, B, C and D) of cultured juvenile populations. The mean severity indices (SI) of rick-ettsia-like organism (RLO) infection were positively correlated with mortality rates of juvenile popula-tions. After or within every peaks of RLO infection were all accompanied with the peaks of mortalityrates of host populations and the mortality rates declined with decreased RLO infection. So the evidencesof histological observation and epidemiology in this study indicated that rickettsia-like organism (RLO)may be as an important pathogenic organism of disease and death of cultured Pinctada maxima. In addi-tion, a few ciliates were discovered only in part of batch A of cultured juvenile population. The mortalityrates in juvenile populations were not correlated with the weekly mean temperature and salinity of seawater.     

Rickettsia-like organism infection associated with mass mortalities of blood clam, Tegillarca granosa, in the Yueqing Bay in China

A series of mass mortalities of the cultured blood clam, Tegillarca granosa, occurred in the Yueqing Bay of China from 2005 to 2009. An obligate intracellular prokaryote, designated as rickettsia-like organism (RLO), was frequently found in the moribund or dead blood clam sample during ultrastructural examination. These organisms were usually round, ellipsoid or occasionally dumbbell-shaped, ranged from approximately 0.28 to 0.71 μm in size and had a trilaminar cell wall. Two reproductive modes of organisms, transverse binary fission and budding, were observed. The organisms were able to form eosinophilic inclusions. Most inclusions were found within epithelial and connective tissues of the mantle, gills and digestive tube. The biological and morphological characteristics indicate that these organisms may belong to the family Rickettsiaceae. RLOs exhibited significant pathogenicity. Cytopathological examinations revealed extensive necrosis and destruction in the infected cell. The degree of tissue destruction was positively related to the number of RLO inclusions in the tissues, and the cytopathological effects were positively related to the number of intracellular RLO. RLOs and their inclusions were discovered throughout different disease areas and in different time periods. The infection intensity of the RLOs was positively correlated with the mortality rate of clams. Therefore, RLO infection might be associated with mass mortalities of cultured blood clams in the Yueqing Bay.     

Studies on rickettsia-like organism (RLO) disease of tropical marine pearl oyster——Epidemiological investigation of RLO disease in larvae populations of maricultured Pinctada maxima

The onset and sPread of diseases in dricultured Anrnals are resulted frOm the mutule ef-fect of pathogen, hOSt and environrnnt. Epidemology takes the diseased AnInal poPulationsas the study object, rnakes observation and exPeriment on the field as the primp method ofstUdy and stuha the mutule effec Of PathOgen, hOS and environrnen by inVeSigating the na-ttire distribution of diseases and ePidendc mechedsm. To date, succeSSful exarnple of epidend-obo study on lerne shellfish deseases is…     

热带海洋珍珠贝类立克次体病研究 Ⅳ.组织细胞病理学研究

类立克次体（RLO）对大珠母贝和合浦珠母贝具有强烈的致病性,引起基本一致的病理变化．在急性坏死破坏期内,RLO导致外套膜、鳃、消化管、肝胰腺、生殖腺腺管及全身血管内皮系统等多器官组织的变性坏死,使器官组织结构的完整性遭到破坏,破坏程度与RLO包涵体的数目密切相关,而细胞的破坏与细胞内RLO的生长发育及繁殖到大量数目密切相关．鉴于此,将RLO引起的珍珠贝病称为类立克次体病（RLO病）．RLO病呈急性变质性炎症和慢性增生性炎症病理,在前者细胞呈现崩解性坏死、溶解性坏死和退变性坏死;在后者存在实质细胞增生和纤维母细胞增生为纤维细胞并形成纤维化．根据炎症病理的发展过程,RLO病可分为急性坏死破坏期和慢性增生修复期．     

中国北方沿海6个栉孔扇贝群体的AFLP分析

利用4对AFLP引物首次对我国北方沿海主要的6个栉孔扇贝养殖区的自然采苗群体进行了遗传多样性分析,共扩增得到191条清晰带谱,其中175条具有多态性。对各群体进行遗传多样性分析,结果表明各群体均具有较高的遗传多样性水平,香农式多样性指数在0.347 4～0.391 7之间。AMOVA结果表明遗传变异主要来源于群体内,占总遗传变异的87.22%。个体聚类结果表明,烟台、长岛、大连3个群体的个体聚在一起,可看做1个群体;而荣成群体的个体单独聚在一起,具有独立的遗传结构;胶南和日照的一部分个体聚在一起,其他大部分各群体单独聚在一起;推测是海洋环境和人工养殖影响的共同结果。