对虾暴发性病害的流行病学研究及养殖扇贝大规模死亡的病原病理分析

IOCAS-IR > 海洋环流与波动重点实验室

	对虾暴发性病害的流行病学研究及养殖扇贝大规模死亡的病原病理分析
	王运涛
学位类型	博士
	1999
学位授予单位	中国科学院海洋研究所
学位授予地点	中国科学院海洋研究所
学位专业	海洋生物学
关键词	中国对虾栉孔扇贝海湾扇贝白斑综合症病毒类立克次氏体
摘要	本文主要从病理病原的角度研究了中国对虾、栉孔扇贝和海湾扇贝大规模死亡的原因，同时，对中国对虾暴发性病害的流行病学特点及传播机制进行了探讨。中国对虾暴发性病害主要由对虾白斑综合症病毒（WSSV）引起。利用病虾的血淋巴，进行病毒的分离纯化，可以最大限度地降低对虾组织中有机物的干扰，提高WSSV的纯度，同时减少了提取步骤和处理时间，病毒粒子更完整、活力更高。带病毒的组织液经过TritonX-100处理后，可得到较完整，纯度较高的病毒核酸。对虾白斑综合症病毒在灭菌海水中经过15天（22 ℃）便失去感染力；WSSV对紫外线比较敏感，经过紫外线（15W，波长253.7nm，20cm距离）照射20min后，全部失活。在对虾病毒人工感染试验中，采用LC＿（50）法和紫外分光光度计法对感染病毒进行定量，其中，LC＿（50）能够较准确地反映病毒粒子的活力，而紫外分光光度计测得的病毒悬液浓度易受病毒悬液中杂质的干扰，误差相对较大。环境温度对对虾白斑病的暴发有重要影响，水温在9－13 ℃时，感染病毒的健康虾可以成为病毒的携带者，但不发病；18－22 ℃时，人工感染的健康虾无论采取投喂、浸泡还是注射的侵染方式，均发病死亡。WSSV对不同种类对虾具有不同的致病力，通过试验发现，在常见的对虾种类中，南美白对虾对WSSV的抵抗力大于中国对虾和鹰爪虾。本文利用PCR及电镜技术，对中国对虾染病亲虾的卵巢和各期卵及其刚孵化出的无节幼体进行了病毒检测，染病亲虾刚产的卵及未产出的卵均没有发现病毒存在，在卵巢的结缔组织中检测到病毒存在，刚孵化出的无节幼体PCR检测结果呈阳性，电镜下观测到有病毒样颗粒侵染无节幼体的上层表皮组织。在卵黄内及卵膜的内外层均未检测到病毒，没有发现病毒经卵内及卵膜传播的证据。病毒很可能是在无节幼体期进入对虾体内的。通过PCR及电镜检测发现，对虾养殖环境中的脊尾白虾、日本美人虾、野生螺类、小杂蟹体内均有WSSV存在，说明这些环境生物可能是对虾病毒的中间宿主或保存宿主，在虾病的暴发中起重要传播作用。通过对对虾的养殖海水进行不同级别的过滤发现，虾池中微型生物的数量对WSSV的传播也有一定的影响，在对虾养殖过程中，利用20cm绢网过滤海水，可有效降低WSSV的传播。通过组织病理分析和电镜观察，发现在濒死的栉孔扇贝和海湾扇贝体内有寄生原核生物－类立克次氏体（RLO），同时还发现少量的病毒粒子。病理学研究表明，类立克次氏体（RLO），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。
其他摘要	The causality of the mass mortality of marine cultured shrimps and scallops were maily studied in the etiology and pathology; moreover, the characteristics of epizoology and transmission mechanisms of the explosive epidemic disease of Penaeus chinensis. were investigated, in particular. It was found that the explosive epidemic disease of cultured penaeid shrimp (penaeus chinensis) was principal caused by White Spot Syndrome Virus (WSSV). When WSSV was isolated and purified from the hemolymph of experimentally infected shrimps, the interference of organic substances in the shrimp tissue could be reduced the lowest limit and the virus purity would be greatly raised. As extract time shortening and procedure simplifying, more intact and active virions could be obtained compared with the common methods; so more undamaged nucleic acid with high purity could be got after the virions were treated with TritonX-100. In sterilized seawater at 22 ℃, WSSV would loss its infectious activity 15 days later and the virus was sensitive to ultraviolet (U. V) irradiation; it was inactivated in 20 minutes under U. V. lamp (15W, wave length: 253.7nm, distance: 20cm). The infecting virus amount was determined by LC_(50) or spectrophotometer. The activity of virions could be accurately measured by LC_(50); but done with spectrophotometer, the measurements had a bigger erro owing to the interference of organic substances in the shrimp tissue. The outbreak of White Spot Syndrome of penaeid shrimps was remarkablely affected by environmental temperature. The infected shrimps had became carrier of the virus, but had no syndrome at 9-13 ℃ of seawater. However, When seawater temperature rose to 18-22 ℃, all infected shrimps began to have the syndrome and soon died no matter how they were infected by feeding, immersion or injection. The results showed that the pathogenicity of WSSV was different for various shrimp, discovered by experiment, the WSSV-resistance of P. vannamei is stonger than that of P. chinensis and Trachypenaeus curvirostris PCR and electron microscope were used to detect the virus in the ovaries, eggs at different development stages, and naupliuses of infected broodstocks of P. chinensis. The virus was not found in eggs just spawned or not, but in the connective tissue of ovary. The detected results of nauplius were positive, and virus-like organisms were fond to invade the epidermis tissue of nauplius. The virus was not detected in the yolk as well as inner and outer membrane of egg. It had not been found that the virus was transmitted through egg contents and membrane. Suggesting the possibility of the virus invading shrimp bady in the nauplius stage. WSSV was also detected by PCR and with electron microscope to exist in Exopalaemon carinicauda, Callianassa japonica, spiral shells and crabs in shrimp ponds, and they might be intermediate or reservoir hosts of the virus and play important transmitting role in the outbreak of shrimp disease. The quantity of microbita in shrimp ponds could affect the WSSV transmission, and when cultured seawater was filtered with membrances(φ = 20um), WSSV transmission would be be effectively reduced. Observed by light and electron microscopes, a lot of parasitic prokaryots and a few of virions also were found in moribund scallops. These parasitic prokaryots were identified to be Rickettsia-like Organisms(RLO). Demonstrated by research on pathology, RLO caused seriously pathological changes in the tissue and cell of chlamys farreri and Argopecten irradians, It would be possible that they might be important alleged "pathogens" to initiate the mass mortality of scallops.
页数	88
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/1207
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	王运涛. 对虾暴发性病害的流行病学研究及养殖扇贝大规模死亡的病原病理分析[D]. 中国科学院海洋研究所. 中国科学院海洋研究所,1999.

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