Abstract：
Two monoclonal in vitro isolates of Perkinsus beihaiensis were established from tissues of infected Mediterranean mussels Mytilus galloprovincialis, using a lipid concentrate and yeast extract medium (LpcYM) to induce enlarged prezoospoangia from histozoic trophozoites, and Perkinsus broth medium (PBM) to support subsequent zoosporulation and schizogonic proliferation from those cells. Zoospores of the isolates developed into uninucleate signet-ring trophozoites followed by schizonts typical of Perkinsus spp., but an additional type of unknown botryoidal cell clusters appeared and increased in density. These botryoidal cell clusters had second-generation daughter schizonts within which further internal cells were produced. Although botryoidal cell clusters in primary cultures rarely proliferated in PBM, cells contained by botryoidal clusters enlarged as prezoosporangia in LpcYM, and a large number of proliferative stages (i.e. trophozoites and schizonts typical for Perkinsus spp.) were obtained from these prezoosporangia in PBM. At lower cell density, trophozoites transformed into the botryoidal cell clusters in PBM, and the number of cells increased little, while trophozoites rapidly increased by typical schizogony at higher density in PBM. Based on these observations, we determined an optimal cell density for continuous culture of P. beihaiensis isolates, confirmed their ability to proliferate and survive after cryopreservation, and describe an experimental protocol to culture this most recently described species in the genus Perkinsus.
摘要：
从被感染的地中海贻贝Mytilusgalloprovincialis的组织中建立了两个北PerkinsusBeihaiensis的单克隆体外分离株，使用脂质浓缩物和酵母提取物培养基（LpcYM）从组织生代滋养体诱导扩大的前孢子虫，和Perkinsus肉汤培养基（PBM）以支持随后的动物孢子形成和从这些细胞的分裂增殖。分离物的动物孢子发育成单核印戒滋养体，随后是典型的Perkinsusspp分裂体。,但是出现了另一种未知的肉毒杆菌细胞簇，并且密度增加。这些肉毒杆菌细胞簇具有第二代子裂殖体，在其中产生了更多的内部细胞。尽管原代培养物中的肉毒杆菌细胞簇很少在PBM中增殖，在LpcYM中，肉毒杆菌团簇所包含的细胞被扩大为前孢子虫，以及大量的增殖阶段（即Perkinsusspp典型的滋养体和裂殖体。)是从PBM中的这些前孢子虫中获得的。在较低的细胞密度，滋养体转化为PBM中的肉毒杆菌细胞簇，细胞数量几乎没有增加，而在PBM中，典型的分裂体在较高密度下迅速增加。基于这些观察,我们确定了连续培养的最佳细胞密度。证实了它们在冷冻保存后的增殖和生存能力，并描述了在Perkinsus属中培养这种最新描述的物种的实验方案。
