The contribution of Aeromonas salmonicida extracellular products to the induction of inflammation in Atlantic salmon (Salmo salar L.) following vaccination with oil-based vaccines Stephen Mutoloki a , Bjørn Brudeseth b , Ola B. Reite a , Øystein Evensen a, ) a Department of Basic Sciences and Aquatic Medicine, Norwegian School of Veterinary Science, PO Box 8146 Dep. 0033, Oslo, Norway b Animal Health Division, Alpharma AS, PO Box 158, Skøyen, N-0212 Oslo, Norway Received 27 August 2004; revised 17 November 2004; accepted 10 January 2005 Available online 27 April 2005 Abstract 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 Ò 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 ) Corresponding author. Tel.: C47 22 964500; fax: C47 22597103. E-mail address: oystein.evensen@veths.no (Ø. Evensen). 1050-4648/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2005.01.005 Fish & Shellfish Immunology 20 (2006) 1e11 www.elsevier.com/locate/fsi