Mutoloki Stephen, Brudeseth Bjørn, Reite Ola B, Evensen Oystein
Department of Basic Sciences and Aquatic Medicine, Norwegian School of Veterinary Science, Oslo, Norway.
Fish Shellfish Immunol. 2006 Jan;20(1):1-11. doi: 10.1016/j.fsi.2005.01.005.
Intraperitoneal injection of Atlantic salmon with oil-based vaccines often results in severe side effects. Aeromonas salmonicida subspecies salmonicida, a primary antigen in the vaccines, produces extracellular products (ECPs) that are included in the formulation but the role of ECPs in inducing side effects is not well understood. In the present study, we evaluated the contribution of ECPs to early inflammatory reactions since early events determine the outcome of inflammation. Five groups of Atlantic salmon pre-smolts were injected intraperitoneally with one of the following preparations: (1) A. salmonicida water-in-oil (w/o) containing standard amounts of ECPs; (2) A. salmonicida (w/o) with ECPs concentrated five times; (3) A. salmonicida (w/o) without ECPs (ECPs were removed by washing and re-suspension of the bacteria prior to formulation); (4) w/o only (without antigens), and (5) physiological saline. Tissue sections of the injection site (pyloric caeca and surrounding areas) were collected at monthly intervals for 4 months in phosphate buffered formalin and processed for light microscopy. Computer-assisted microscopy with the help of Image Pro analysis program was used to measure the area of inflammation on H&E stained sections. Differential cell counts of leucocytes involved in the inflammatory reaction were also done based on morphology. Overall results show that fish injected with vaccines containing concentrated amounts of ECPs displayed a higher average area of inflammation compared to all other groups. In contrast, washed preparations induced mild reactions compared to vaccines containing either standard or concentrated ECPs. Mild, non-persistent reactions were observed in the group injected with oil adjuvant only. Neutrophils were persistent in inflammations induced by all preparations except w/o only. No inflammatory reactions were observed in the group injected with PBS. The results suggest that ECPs are pro-inflammatory in Atlantic salmon. It is anticipated that ECPs are more readily exposed to inflammatory cells than the bacterial cells themselves during early stages of inflammation because of their orientation at the water-oil interface. The results indicate that ECPs of A. salmonicida play an important role in the induction of early inflammatory reactions. It is also documented that the combination of antigens with oil adjuvants, and not the adjuvants alone, is the inducer of strong inflammatory reactions in Atlantic salmon.
给大西洋鲑鱼腹腔注射油基疫苗常常会导致严重的副作用。鲑鱼气单胞菌杀鲑亚种是疫苗中的主要抗原，它会产生细胞外产物（ECPs），这些产物包含在疫苗制剂中，但ECPs在引发副作用中所起的作用尚未得到充分了解。在本研究中，我们评估了ECPs对早期炎症反应的影响，因为早期事件决定了炎症的结果。将五组大西洋鲑鱼幼鱼腹腔注射以下制剂之一：（1）含有标准量ECPs的鲑鱼气单胞菌水包油（w/o）疫苗；（2）ECPs浓度浓缩五倍的鲑鱼气单胞菌（w/o）疫苗；（3）不含ECPs的鲑鱼气单胞菌（w/o）疫苗（在制剂前通过洗涤和重悬细菌去除ECPs）；（4）仅w/o（不含抗原），以及（5）生理盐水。在4个月内，每月采集注射部位（幽门盲囊及周围区域）的组织切片，置于磷酸盐缓冲福尔马林中，并进行光学显微镜处理。借助Image Pro分析程序的计算机辅助显微镜用于测量苏木精-伊红（H&E）染色切片上的炎症面积。还根据形态对参与炎症反应的白细胞进行了分类计数。总体结果表明，与所有其他组相比，注射含有浓缩量ECPs疫苗的鱼显示出更高的平均炎症面积。相比之下，与含有标准或浓缩ECPs的疫苗相比，洗涤后的制剂引发的反应较轻。在仅注射油佐剂的组中观察到轻微、非持续性的反应。除了仅注射w/o的组外，所有制剂诱导的炎症中嗜中性粒细胞都持续存在。在注射磷酸盐缓冲盐水（PBS）的组中未观察到炎症反应。结果表明，ECPs在大西洋鲑鱼中具有促炎作用。预计在炎症早期，由于ECPs在水油界面的定位，它们比细菌细胞本身更容易接触到炎症细胞。结果表明，鲑鱼气单胞菌的ECPs在诱导早期炎症反应中起重要作用。还记录到，抗原与油佐剂的组合，而非单独的佐剂，是大西洋鲑鱼中强烈炎症反应的诱导因素。
