Full length article Optimisation and standardisation of functional immune assays for striped catfish (Pangasianodon hypophthalmus) to compare their immune response to live and heat killed Aeromonas hydrophila as models of infection and vaccination Wanna Sirimanapong a, b, * , Kim D. Thompson a , Kan Kledmanee b , Prawporn Thaijongrak b , Bertrand Collet c , Ei Lin Ooi d , Alexandra Adams a a Institute of Aquaculture, School of Natural Sciences, University of Stirling, Stirling FK9 4LA, Scotland, United Kingdom b Faculty of Veterinary Sciences, Mahidol University, Salaya Campus, Nakornpathom 73170, Thailand c Marine Scotland Science, Aberdeen AB11 9DB, Scotland, United Kingdom d Novus International, Novus Aqua Research Center, Linh Trung Ward, Thu Duc District, Ho Chi Minh City, Viet Nam article info Article history: Received 9 May 2014 Received in revised form 9 July 2014 Accepted 10 July 2014 Available online 24 July 2014 Keywords: Pangasianodon hypophthalmus Aeromonas hydrophila Live and killed bacteria Innate and adaptive immunity abstract Aquaculture production of Pangasianodon hypophthalmus is growing rapidly in South East Asia, especially in Vietnam. As it is a relatively new aquaculture species there are few reports evaluating its immune response to pathogens. Thus, functional assays for P. hypophthalmus were optimised to evaluate both innate and adaptive immune responses, and were then used to examine immune response following stimulation with live and heat-killed Aeromonas hydrophila. These were used as models of infection and vaccination, respectively. Four treatment groups were used, including a control group, a group injected intraperitonally (IP) with adjuvant only, a group injected with heat-killed A. hydrophila (1  10 9 cfu ml 1 mixed with adjuvant), and a group injected with a subclinical dose of live A. hydrophila. Samples were collected at 0, 1, 3, 7, 14 and 21 days post-injection (d.p.i.) to assess their immune response. The results indicated that challenge with live or dead bacteria stimulated the immune response in P. hypophthalmus significantly above the levels observed in control groups with respect to specific antibody titre, plasma lysozyme and peroxidase activity, and phagocytosis by head kidney macrophages at 7 or/and 14 d.p.i. At 21 d.p.i., total and specific antibody (IgM) levels and plasma lysozyme activity in fish injected with either live or dead A. hydrophila were significantly different to the control groups. Differential immune responses were observed between fish injected with either live or dead bacteria, with live A. hydrophila significantly stimulating an increase in WBC counts and plasma peroxidase activity at 3 d.p.i., with the greatest increase in WBC counts noted at 21 d.p.i. and in phagocytosis at 14 d.p.i. By 21 d.p.i. only the macrophages from fish injected with dead A. hydrophila showed significantly stimulation in their respiratory burst activity. This study provides basic information on the immune response in pangasius catfish that can be useful in the health control of this species. © 2014 Elsevier Ltd. All rights reserved. 1. Introduction Pangasianodon hypophthalmus is the most important fish spe- cies currently cultured in Vietnam. Aquaculture production of pangasius is also developing in other Asian countries such as Bangladesh, India, Indonesia, Malaysia, Myanmar, the Philippines and Thailand, however Vietnam still remains the largest global producer [1]. Outbreaks of bacterial diseases in P. hypophthalmus culture have a significant impact on production. Two of the main problems reported are bacillary necrosis of Pangasianodon (BNP) [2] caused by Edwardsiella ictaluri, and motile aeromonas septicaemia (MAS) [3] caused by Aeromonas hydrophila. A. hydrophila vaccine preparations based on dead bacterial preparations, killed using formalin or heat (i.e. whole bacterial * Corresponding author. Faculty of Veterinary Sciences, Mahidol University, Sal- aya Campus, Nakornpathom 73170, Thailand. Tel.: þ66 8 1991 1567. E-mail addresses: wanna.sir@mahidol.ac.th, vswsmu@gmail.com (W. Sirimanapong). Contents lists available at ScienceDirect Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi http://dx.doi.org/10.1016/j.fsi.2014.07.021 1050-4648/© 2014 Elsevier Ltd. All rights reserved. Fish & Shellfish Immunology 40 (2014) 374e383