The contribution of Aeromonas salmonicida extracellular products to the induction of inflammation in Atlantic salmon (Salmo salar L.) following vaccination with oil-based vaccines

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.