Columnaris disease, instigated by Gram-negative Flavobacterium covae (F. covae), is a dreadful disease of many different genera of freshwater fish with alarmingly high mortality rates. Ictalurus punctatus (I. punctatus) is extremely prone to this disease. Despite the terrible implications of this disease, very little is known about how the host and the bacterium's virulence factor interact. Secreted extracellular proteins (SEPs) are key bacterial virulence agents that frequently provide immunogenicity. In a previous study, the major secreted extracellular proteins of F. covae were identified and their protective efficacy was verified in I. punctatus, and the results were published. In the current study, an endeavor has been made to examine the progressive shifts in I. punctatus immunity following immunization with F. covae SEPs in order to more thoroughly comprehend the host's intrinsic and acquired immune responses against F. covae infection. An enzyme-linked immunosorbent test (ELISA) is operated to analyze specific humoral immune responses against SEPs of F. covae. Moreover, the expressions of immune-associated genes that are related to inherent and acquired immunity were monitored in the anterior kidneys and spleens of the vaccinated I. punctatus. Marked rise in immunoglobulin M (IgM) levels were detected in the serum of I. punctatus vaccinated with different SEP formulations, including crude SEPs, heat-deactivated SEPs, and SEPs combined with adjuvant, at 14 and 21 days post-vaccination. At the same time points, a marked increase in the expression levels of clusters of differentiation (CD4-1 and CD8-β) and major histocompatibility complex (MHC-II) genes was detected in the vaccinated fish's anterior kidneys and spleens, while the highest levels of interleukin-8 (IL-8) and tumor necrosis factor alpha (TNF-α) up-regulations were detected in the immunized fish's anterior kidneys 14 days following vaccination. This work revealed that SEPs are an effective basis for vaccination against F. covae infection, eliciting humoral and inherent immune responses of I. punctatus after SEPs immunization. Improvements are needed to further enhance the I. punctatus immunological responses to SEPs of F. covae since these antigens remain a prospective candidate for future optimization and clinical trials in aquaculture settings.

中文翻译：

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斑点叉尾鱼黄杆菌胞外蛋白的免疫原性

柱状线虫病是由革兰氏阴性 Covae 黄杆菌 (F. covae) 引起的，是许多不同属淡水鱼的可怕疾病，死亡率高得惊人。马尾鮰（I. punctatus）极易患此病。尽管这种疾病具有可怕的影响，但人们对宿主和细菌毒力因子如何相互作用知之甚少。分泌型细胞外蛋白 (SEP) 是关键的细菌毒力因子，经常提供免疫原性。在之前的研究中，鉴定了 Covae 主要分泌的胞外蛋白，并在 I. punctatus 中验证了它们的保护功效，并发表了结果。在当前的研究中，我们努力检查用 F. covae SEP 免疫后 I. punctatus 免疫力的逐渐变化，以便更彻底地了解宿主针对 F. covae 感染的内在和获得性免疫反应。酶联免疫吸附试验 (ELISA) 用于分析针对 F. covae SEP 的特异性体液免疫反应。此外，在接种疫苗的马尾鱼的前肾和脾脏中监测了与固有免疫和获得性免疫相关的免疫相关基因的表达。接种不同SEP制剂（包括粗制SEP、热灭活SEP和与佐剂结合的SEP）的马尾伊氏菌，在接种后14天和21天时，其血清中免疫球蛋白M（IgM）水平显着升高。同时，在接种疫苗的鱼的前肾和脾脏中检测到分化簇（CD4-1和CD8-β）和主要组织相容性复合体（MHC-II）基因的表达水平显着增加，而最高接种后 14 天，在免疫鱼的前肾中检测到白细胞介素 8 (IL-8) 和肿瘤坏死因子 α (TNF-α) 水平上调。这项工作表明，SEP 是针对 F. covae 感染的疫苗接种的有效基础，在 SEP 免疫后引发点状 I. punctatus 的体液和固有免疫反应。需要进行改进以进一步增强 I. punctatus 对 F. covae SEP 的免疫反应，因为这些抗原仍然是水产养殖环境中未来优化和临床试验的潜在候选者。