Comparision of a serological potency assay for furunculosis vaccines (Aeromonas salmonicida subsp. salmonicida) to intraperitoneal challenge in Atlantic salmon (Salmo salar L.) Anne Berit Romstad a, * , Liv Jorun Reitan b , Paul Midtlyng c , Kjersti Gravningen d , Vibeke Emilsen e , Øystein Evensen c a PHARMAQ AS, Skogmo Industriområde, 7863 Overhalla, Norway b Norwegian Veterinary Institute, P.O. Box 750, 0106 Oslo, Norway c Norwegian School of Veterinary Science, P.O. Box 8146 Dep, 0033 Oslo, Norway d PHARMAQ Vietnam Co. Ltd., 58 Vo VanTan, D 3, Ho Chi Minh City, Viet nam e VESO Vikan, 7800 Namsos, Norway article info Article history: Received 28 December 2012 Received in revised form 9 October 2013 Accepted 18 November 2013 Keywords: Atlantic salmon Aeromonas salmonicida Batch potency test ELISA 3R abstract Batch potency testing of salmonid vaccines is mainly performed by in vivo challenge, which requires a lot of animals and causes severe pain. Due to the animal welfare concerns associated with in vivo immunization challenge tests, methods which could refine, reduce or replace (3Rs) these tests are needed. The aim of this study was to assess the use of serological assay (immunization & antibody estimation with an enzyme-linked immunosorbent assay (ELISA) for batch potency testing of oil adjuvanted, inactivated commercial furunculosis vaccines. In total ten vaccines were included in the study: two commercial multi-component vaccines and two experimental single-component furunculosis vaccines with 5% and 20% antigen content (relative to the commercial vaccine), from two manufacturers. In addition two experimental single component vaccines based on A-layer positive and A-layer negative Aeromonas salmonicida respectively were included. Challenge and blood sampling were conducted 9 weeks post vaccination. There was a correlation between antibody response against A. salmonicida as measured by ELISA and protection in i.p. challenge. This study shows that the ELISA assay can be used for testing different vaccine formulations and can potentially replace in vivo challenge tests for batch potency testing of furunculosis vaccines. Ó 2014 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved. 1. Introduction Aeromonas salmonicida subspecies salmonicida (A. salmonicida), the causative agent of furunculosis, is one of the most widely distributed pathogenic bacteria in salmonid aquaculture [1]. For the past 20 years the disease has been successfully controlled by the use of oil adjuvanted vaccines [2], and efficacious vaccines are one of the keys to the success of salmon farming. Presently, 100% of all farmed Atlantic salmon (Salmo salar L.) in Norway, approximately 320 million fish per year, are vaccinated prior to sea transfer [3]. Furunculosis vaccine batches are tested for potency by in vivo tests before release to the market, according to the European Pharmacopeia (Ph. Eur.) monograph [4], which requires the use of a high number of animals (minimum 60 fish per antigen). Earlier reports have revealed that most of the fish are used for batch po- tency testing [5,6]. Most companies perform their batch testing as immunization-challenge assays. The monograph [4] advices that for each antigen, a minimum of 30 vaccinated and 30 control fish are challenged and for a multi-component vaccine, containing seven antigens, this would result in a minimum of 420 fish per in vivo batch potency test. An alternative serological potency test as described in the Ph. Eur. monograph [4] would require a minimum of 25 vaccinated and 10 control fish and, therefore would have the potential to reduce the number of fish by over 90% and would also minimize the pain for the experimental animals [6,7]. Results from earlier studies demonstrated that salmonids give a good antibody response to A. salmonicida antigen [7e9] and that there is a close dose-response correlation between the antigen * Corresponding author. Tel.: þ47 91683971. E-mail address: anne-berit.romstad@pharmaq.no (V. Emilsen). Contents lists available at ScienceDirect Biologicals journal homepage: www.elsevier.com/locate/biologicals 1045-1056/$36.00 Ó 2014 The International Alliance for Biological Standardization. Published by Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.biologicals.2013.11.007 Biologicals 42 (2014) 86e90