栉孔扇贝大量死亡的流行病学调查研究

2001~2003年,对养殖的栉孔扇贝进行了较系统地流行病学调查研究.以栉孔扇贝养殖集中海区长岛县亚美扇贝养殖场为定点调查和样品采集点.调查内容主要包括栉孔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、盐度等环境理化因子进行连续监测.调查结果显示:栉孔扇贝在养殖初期幼贝阶段死亡率较低,随后死亡率逐渐升高,至8月24日月死亡率达最高点71%.9月份以后死亡率开始下降.至10月份累积死亡率达90%以上.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;温度变化与死亡率似有一定的相关性,即高温(月平均23℃以上)可能是养殖栉孔扇贝重要的环境胁迫因子.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,栉孔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,其感染率和感染强度与扇贝死亡率呈现一致的趋势.在病原病理检测中,发现RLO的大量感染与组织细胞严重病理损伤密切相关.在病原生物检测过程中,除RLO以外还发现有类枝原体原核生物(MLO)和类病毒(VLP)寄生.RLO的人工感染实验结果说明RLO是栉孔扇贝重要的病原菌.结合感染率和死亡率、感染强度和组织病理学的相关性以及感染实验的结果分析,认为类立克次体(RLO)可能是栉孔扇贝养殖过程中大量死亡的主要致病因子.     

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。     

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.     

热带海洋珍珠贝类立克次体病研究──大珠母贝病原类立克次体包涵体的组织学及超微结构研究

于1993年11月和1994年11月在海南省临高县新盈珍珠贝养殖场采集患病或濒死的大珠母贝幼贝,采用组织切片和扫描电镜技术对其病原类立克次体（Rickettsia-likeorganism,RLO）的包涵体进行组织学和超微结构研究,以期找出RLO包涵体在宿主细胞内形态发生的内在规律。结果表明,RLO包涵体寄生在宿主多处内脏组织,但RLO具有明显的嗜细胞特异性,感染的靶细胞为上皮（或表皮）、结缔组织和血管内皮细胞;包涵体存在不同的发育时期,可分为颗粒前期和颗粒期,后者又可分为颗粒Ⅰ—Ⅲ期;包涵体是由两种颗粒外包一界膜而形成,其中小而纤细的颗粒可能为游离的RLO,大而圆形的颗粒可能是吞噬溶酶体内RLO繁殖形成的微克隆。     

赤潮对浙江近海养殖业危害的初步调查

本文报导了浙江北部海域赤潮发生时对海湾扇贝、皱纹盘鲍幼体和养殖幼贝毒害的观察结果,证明赤潮毒素是赤潮杀死养殖生物的重要原因。文章还根据已有文献综合讨论了该水域有利于发生赤潮的环境条件。     

墨西哥湾扇贝稚贝对盐度的耐受力

用壳高15～21mm的墨西哥湾扇贝(Argopecten irradians concentricus)稚贝作为实验材料,控制水温在16和24℃条件下进行对盐度的耐受力实验.实验结果表明,当温度在24℃时,稚贝存活的盐度范围为18.5～44.4,低盐度对稚贝的存活影响明显.稚贝可生长的盐度范围是21.2～41.0(日增长率r>4.0),适宜的生长盐度为24.0～32.0(日增长率r>8.0),其中26.0～28.0的盐度为最佳生长盐度.在16℃条件下,最初8h内,其生存盐度范围为17.4～46.4,但是48h后稚贝的存活盐度范围与24℃温度条件时无明显差别,显示出低温条件会使稚贝对极限盐度的反应速率减缓.当盐度处于接近极限范围时,盐度变化幅度过大易导致稚贝死亡.墨西哥湾扇贝对盐度的适应能力与原生境盐度有关,稚贝预先在盐度为19.5和44.9中适应9d后,再过度到14.3和48.5中,48h后,存活率为78.3%和100%,而若是从盐度为32中直接移到3和48.5中,48h后存活率分别为0和27.3%.显然,通过中间驯化或适应过程,稚贝会提高耐盐能力.此结果表明,在高温多雨的南方海域,应避免在易受淡水中冲击的河口、海湾区域开展墨西哥湾扇贝养殖.     

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.     

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.     

温度、盐度和密度交互作用对波纹巴非蛤幼贝生长的影响

为查明波纹巴非蛤(Paphia undulata)幼贝最适生长条件,采用响应面法研究了温度、盐度和密度交互作用对波纹巴非蛤幼贝生长的影响。利用Box-Behnken中心组合进行3因素3水平的实验设计(3因素为温度(A),盐度(B),密度(C);3水平为-1,0,1),以幼贝生长率为响应值,以A(26℃,28℃,30℃)、B(28,30,32)和C(0.56,1.4,2.24个/cm2)为影响因子。实验结果表明：(1)盐度对幼贝生长影响最显著(P<0.05),3种生态因子对幼贝生长率影响程度大小顺序是：B>A>C;(2)通过SAS软件分析,得出波纹巴非蛤幼贝最适生长环境组合是温度为26.93℃、盐度为31.39、密度为1.12个/cm2,理论最佳生长率为41.47%。为验证响应面法预测结果的准确性,以预测结果为实验条件,相同实验时间后,得到的生长率为42.30%,结果与理论预测值相近,响应面方法可靠。     

几种环境因子对皱纹盘鲍的胁迫作用及其阈值

针对福建等海域养殖皱纹盘鲍度夏死亡率高的现象,试验研究了不同盐度、温度和溶解氧条件,对皱纹盘鲍幼鲍和1龄鲍死亡率的影响,并根据试验结果设定了皱纹盘鲍海上养殖盐度、温度和溶解氧预警阈值。结果表明:低盐胁迫对皱纹盘鲍的死亡率影响显著,在水温24～27℃时,盐度20是幼鲍和1龄鲍试验组和对照组死亡率有显著差异的临界点,盐度预警阈值为22;高温胁迫对皱纹盘鲍的死亡率影响显著,温度28℃是幼鲍和1龄鲍试验组和对照组死亡率有显著差异的临界点,温度预警阈值为27℃;皱纹盘鲍有较强的耐低氧能力,在水温26℃,盐度为30,溶解氧低于2.0mg/L时,开始大量死亡,溶解氧预警阈值为3.5mg/L;高温胁迫和低盐胁迫对皱纹盘鲍的死亡率有显著的交互作用,温度升高,低盐耐受能力下降。     

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…     

A prokaryotic mycoplasma-like organism infection in the scallop Argopecten irradians

Intracytoplasmic infection with a prokaryotic mycoplasma-like organism (MLO) were characterized in the scallop Argopecten irradians, and the first report of such infection in this scallop was represented. Ultrastructurally these microorganisms are usually pleomorphic and variable in morphology and size, and appear in several cell types such as small spherical body, rod-shaped bodies and longer filament-shaped body. They lack a cell wall. These observations revealed that these types of the prokaryote showed some characteristics of a mycoplasma-like organism (MLO). MLO reproduced in two ways: binary fission and budding. The results of isolation and purification showed that a large number of MLOs existed in the tissues of diseased scallops. The results of experimental infection revealed that the MLO is pathogenic to the scallop Argopecten irradians.     

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．