ICAR-National Bureau of Fish Genetic Resources, Canal Ring Road, P.O. Dilkusha, Lucknow, 226002, Uttar Pradesh, India.
ICAR-National Bureau of Fish Genetic Resources, Canal Ring Road, P.O. Dilkusha, Lucknow, 226002, Uttar Pradesh, India.
Fish Shellfish Immunol. 2018 Jul;78:195-201. doi: 10.1016/j.fsi.2018.04.041. Epub 2018 Apr 21.
Infection with Aphanomyces invadans is one of the most destructive diseases of freshwater fishes. Indian major carps, the dominant cultured species in the Indian sub-continent are highly susceptible to this disease. Till date, there is no effective treatment for control of this disease and immunization can be one of the strategies to reduce disease-related losses. In the present study, inactivated germinated zoospores of A. invadans were evaluated as antigen in conjunction with and without adjuvant Montanide™ ISA 763 A VG, for assessing their efficacy in rendering protection against A. invadans infection. For the experiment, rohu Labeo rohita, (n = 160, 74 ± 12 g) were divided into 4 groups (C, A, G and GA) with 40 fish in each group. The fish in groups i.e., C, A, G and GA were injected intraperitoneally with PBS, adjuvant emulsified with PBS, inactivated germinated zoospores, and inactivated germinated zoospores emulsified with adjuvant, respectively. After 21 days of immunization, the fish were given a booster dose as above. After 7 days of the booster dose, the fish were challenged with zoospores of A. invadans to determine the relative percent survival (RPS). The results revealed that all the fish in C, A and G group succumbed to infection (0% RPS), although there was delayed mortality in fish from A and G groups in comparison to the C group. However, the fish in GA group showed significantly higher (P < 0.05) protection (66.7% RPS). In addition, following booster immunization (before challenge), the antibody level in the GA group was significantly higher (P < 0.05) than the control group. In western blotting, sera from G and GA groups showed reactivity with peptides of about 54 KDa. Following challenge (on 14th day), the antibody level as well as total antiprotease activity in fish of all the groups was significantly decreased in comparison to pre-challenge, except fish in GA group not exhibiting any gross lesions. In addition, sera of surviving fish of GA group showed significant inhibition of germination of zoospores and germlings growth in comparison to other groups (P < 0.05). Further, histopathological examination of the muscle tissue revealed that, in fish of GA group without any gross lesions, there were well developed granulomas and extensive mononuclear cell infiltration restricted to the site of injection, whereas in other groups, there was extensive myonecrosis with proliferating hyphae. These preliminary findings indicate that inactivated germinated zoospores of A. invadans in combination with adjuvant could stimulate good immune response and confer remarkable protection in rohu.
感染无隔尾孢虫是淡水鱼类最具破坏性的疾病之一。印度主要鲤鱼是印度次大陆主要养殖的品种，对这种疾病高度敏感。迄今为止，尚无有效的治疗方法来控制这种疾病，而免疫接种可以是减少与疾病相关损失的策略之一。在本研究中，评估了无隔尾孢虫的灭活萌发游动孢子作为抗原，与佐剂 Montanide™ISA763AVG 联合使用或不联合使用，以评估其在提供针对无隔尾孢虫感染的保护方面的功效。对于实验，将罗非鱼（Labeo rohita）（n=160，74±12g）分为 4 组（C、A、G 和 GA），每组 40 条鱼。用 PBS 注射组 C、用 PBS 乳化佐剂的组 A、用灭活萌发游动孢子的组 G 和用佐剂乳化的灭活萌发游动孢子的组 GA。免疫接种 21 天后，给鱼注射上述增强剂量。增强剂量 7 天后，用无隔尾孢虫的游动孢子对鱼进行攻毒，以确定相对存活率（RPS）。结果表明，C、A 和 G 组的所有鱼都因感染而死亡（0%RPS），尽管 A 组和 G 组的鱼比 C 组的鱼死亡时间稍晚。然而，GA 组的鱼表现出显著更高的（P<0.05）保护率（66.7%RPS）。此外，在增强免疫接种（攻毒前）后，GA 组的抗体水平明显高于对照组（P<0.05）。在 Western blot 中，G 组和 GA 组的血清与约 54kDa 的肽反应。攻毒后（第 14 天），与攻毒前相比，所有组的鱼的抗体水平和总抗蛋白酶活性均显著降低，但 GA 组的鱼没有表现出任何明显的病变。此外，GA 组存活鱼的血清显示出对游动孢子萌发和幼体生长的显著抑制，与其他组相比（P<0.05）。此外，肌肉组织的组织病理学检查表明，在没有明显病变的 GA 组鱼中，有发达的肉芽肿和广泛的单核细胞浸润仅限于注射部位，而在其他组中，有广泛的肌坏死和增殖的菌丝。这些初步发现表明，无隔尾孢虫的灭活萌发游动孢子与佐剂联合使用可以刺激良好的免疫反应，并在罗非鱼中提供显著的保护。
