Toxicity and dose determination of quillaja saponin, aluminum hydroxide and squalene in olive flounder (Paralichthys olivaceus)

Adjuvants are substances added to vaccines to enhance the immune response of a given antigen. Most of the adjuvants are toxic at certain doses, and toxicity varies in different species. Moreover, there are no standard dosage limits set for adjuvant use in fish vaccines. We evaluated the acute toxicity, serum enzymes (AST/ALT) indicating hepatic injury and histopathological changes due to intra-peritoneal administration of different concentrations of a panel of adjuvants including quillaja saponin, aluminum hydroxide, squalene emulsion and Freund's incomplete adjuvant (FIA) with a dose ranging study of saponin (500, 160, 50, 16 and 5 μg fish−1), aluminum hydroxide (5000, 1600, 500, 160 and 50 μg fish−1), squalene emulsion (20, 10 and 5%), and FIA to determine the acceptable dosage for vaccination in olive flounder (Paralichthys olivaceus) fingerlings measuring 4.66 ± 0.41 g, 8.47 ± 0.42 cm. Saponin was highly toxic with a LD50 of approximately 105 μg fish−1 (22.4 mg kg−1) causing severe histological damage and AST level was high at dose above 16 μg fish−1 and ALT, specific for liver damage was high only at 160 μg fish−1 (11 U/L) and was safe at 5 μg fish−1. Aluminum hydroxide was toxic at 5000 μg fish−1 and was acceptable at dose below 1600 μg fish−1 with moderate histology and AST/ALT levels similar with control. Squalene emulsion showed increased inflammation at 20% and 10% emulsions and the inflammatory response was mild at a concentration of 5% oil emulsion and AST/ALT levels being similar to control in 10% and 5% emulsions and elevated in 20% on both sampling days. FIA was not lethal, but induced severe inflammation at injection site and around blood vessels. In comparison to FIA, saponin found to be safe at dose of 5 μg fish−1, aluminum hydroxide below 1600 μg fish−1, and squalene at 5% emulsion and could be accepted for vaccination studies. These results provide an insight for the selection of safer dose of adjuvants for intra-peritoneal vaccination of olive flounder.