IN-VITRO CULTURE OF THE EASTERN OYSTER PARASITE PERKINSUS-MARINUS - OPTIMIZATION OF THE METHODOLOGY
IN-VITRO CULTURE OF THE EASTERN OYSTER PARASITE PERKINSUS-MARINUS - OPTIMIZATION OF THE METHODOLOGY
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DOI：
10.1006/jipa.1995.1079
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发表时间：
1995-09-01
影响因子：
3.4
通讯作者：
VASTA, GR
中科院分区：
文献类型：
--
作者：
GAUTHIER, JD;VASTA, GR
我们已经成功地建立了一个体外连续培养的东部牡蛎（Crassostrea virginica）寄生虫Perkinsus marinus（Gauthier和Vasta，1993），现在已经表征了一些变量，如温度，盐度，pH值，播种密度，并选择营养需求，影响寄生虫增殖。最适温度、盐度和pH值范围分别为28-32 ℃、25-30 ppt和6.6-6.8。在培养的第一个48小时期间的增殖速率与接种物的大小直接相关。将Ham’s F12营养混合物（血清替代物）添加到Dulbecco改良的Eagle’s（DME）基础培养基中显著增强了增殖，特别是在DME：Ham’s F12为1：2的比例下。最佳生长需要5%胎牛血清，但较高浓度（10-20%）具有抑制作用。牡蛎血浆的存在仅在低浓度的胎牛血清（0-0.1%）和双倍添加的Ham's F12（DME：Ham's 1：2）下增强生长。在6个月内通过约30次后，培养的寄生虫表现出与新鲜采集的标本相似的形态和毒力特征。优化的培养基显然提供了与宿主细胞内条件相当的环境，因为经常观察到在牡蛎血细胞内增殖的小（约4 μ m）滋养体阶段得以维持。(C)出版社：Academic Press
We have succeeded in establishing an in vitro continuous culture of the eastern oyster (Crassostrea virginica) parasite Perkinsus marinus (Gauthier and Vasta, 1993) and have now characterized a number of variables, such as temperature, salinity, pH, seeding density, and selected nutritional requirements that affect parasite proliferation. Optimum temperature, salinity, and pH ranges were 28-32 degrees C, 25-30 ppt, and 6.6-6.8, respectively. Proliferation rates during the first 48 hr of culture were directly related to the size of the inoculum. The addition of Ham's F12 nutrient mixture (a serum replacement) to Dulbecco modified Eagle's (DME) base medium substantially enhanced proliferation, particularly at a DME:Ham's F12 ratio of 1:2. Five percent fetal bovine serum was required for optimal growth, but higher concentrations (10-20%) were inhibitory. The presence of oyster plasma enhanced growth only at low concentrations of fetal bovine serum (0-0.1%) and doubled addition of Ham's F12 (DME:Ham's 1:2). After approximately 30 passes over a 6-month period, the cultured parasite exhibited morphological and virulence features that are similar to those from the freshly collected specimens. The optimized medium apparently provides an environment comparable to the intracellular conditions of the host since the small (approximately 4 mu m) trophozoite stage frequently observed to proliferate within the oyster hemocyte is maintained. (C) 1995 Academic Press, Inc.