Table 1_Protective immune response in rainbow trout (Oncorhynchus mykiss) against the parasitic nematode Anisakis simplex.docx

IntroductionParasitic nematodes are prevalent in fish populations. The parasites are pathogenic but depress host responses, which limit clearance of the pathogens from the invasion sites. We hypothesized that one of several control strategies, which could augment protection, is immunization of the fish host with parasite antigens prior to live pathogen exposure.
MethodsWe used rainbow trout Oncorhynchus mykiss as a host model and third stage larvae (L3) Anisakis simplex (Nematoda, Ascaridoidea, Anisakidae) as pathogen model. We used a total of 120 fish and immunized 40 of the fish with a homogenate (adjuvanted) of parasite larvae (i.p. injection), 40 fish received adjuvant only and 40 PBS. Following 38 days (d) half of the fish in each group were exposed to infection with live worms (oral administration), and after an additional 25 d the infection success was evaluated together with antibody responses in the different groups.
ResultsInjection of A. simplex L3 antigens induced a series of adaptive and innate host responses. ELISA and Western blot analyses indicated specific IgM reactions in immunized trout against worm antigens with molecular weights (MW) of approximately 39, 103 and 119 kilodalton (kDa). Fish immunized and subsequently infected with live larvae reacted to those three and six additional antigens with MW approximately 61, 73, 84, 152, 186 and 277 kDa. The immunized fish showed a significantly lower worm burden following exposure to live parasite larvae (when compared to naïve fish), but no full protection was achieved. Expression analyses of both adaptive and innate immune genes in fish showed a general down-regulation following infection.
DiscussionPrior immunization with A. simplex L3 homogenate induced a strong antibody response, but the protection was incomplete. It was noteworthy that an infection period (25 d) with live parasites merely induced an insignificant antibody response. It may be explained by the immunosuppressive compounds released by live worm larvae. With the aim of increasing the protective response, we suggest in future immunization experiments to target immunosuppressive worm antigens by immunizing the host organisms with excretory/secretory (ES) proteins and extracellular particles from A. simplex L3.

引言
寄生性线虫（Parasitic nematodes）在鱼类种群中分布广泛。该类寄生虫具有致病性，但会抑制宿主免疫反应，从而阻碍病原体从入侵位点被清除。我们提出假设：在活体病原体暴露前，使用寄生虫抗原对鱼类宿主进行免疫接种，是可增强防护效果的若干防控策略之一。

方法
本研究以虹鳟（Oncorhynchus mykiss）作为宿主模型，以简单异尖线虫第三期幼虫（L3）（Anisakis simplex，线虫纲，蛔目，异尖科）作为病原体模型。共使用120尾虹鳟，其中40尾经腹腔注射（i.p. injection）接种了经佐剂乳化的寄生虫幼虫匀浆，40尾仅接种佐剂，剩余40尾注射磷酸盐缓冲液（PBS）。接种38天后，每组各取半数个体经口投喂活体幼虫进行感染，再经过额外25天后，评估各组的感染成功率与抗体应答水平。

结果
简单异尖线虫L3抗原的接种可触发宿主一系列适应性与先天性免疫应答。酶联免疫吸附实验（ELISA）与蛋白质免疫印迹（Western blot）分析显示，免疫组虹鳟体内产生了针对蠕虫抗原的特异性免疫球蛋白M（IgM），所识别抗原的分子量约为39、103及119千道尔顿（kDa）。经免疫接种后再感染活体幼虫的虹鳟，除识别上述三种抗原外，还可识别另外六种分子量约为61、73、84、152、186及277 kDa的抗原。与未致敏对照鱼相比，免疫组虹鳟在暴露于活体寄生虫幼虫后的虫荷量显著降低，但未实现完全防护。对鱼类适应性与先天性免疫基因的表达分析显示，感染后免疫基因普遍呈现下调表达。

讨论
预先使用简单异尖线虫L3幼虫匀浆进行免疫，可诱导强烈的抗体应答，但防护效果并不完全。值得注意的是，长达25天的活体寄生虫感染仅引发了微弱的抗体应答。这一现象可通过活体幼虫释放的免疫抑制性化合物得到解释。为增强防护效果，我们建议在未来的免疫接种实验中，以简单异尖线虫L3的排泄/分泌（ES）蛋白及胞外颗粒作为靶标免疫宿主，从而靶向干预寄生虫的免疫抑制性抗原。