ORIGINAL ARTICLE Bacillus subtilis AB1 controls Aeromonas infection in rainbow trout (Oncorhynchus mykiss, Walbaum) A. Newaj-Fyzul 1,2 , A. A. Adesiyun 2 , A. Mutani 2 , A. Ramsubhag 3 , J. Brunt 1 and B. Austin 1 1 School of Life Sciences, Heriot-Watt University, Edinburgh, Scotland, UK 2 School of Veterinary Medicine, The University of the West Indies, St Augustine, Trinidad, West Indies 3 Department of Life Sciences, The University of the West Indies, St Augustine, Trinidad, West Indies Introduction Recently, there has been an increasing practice of man- aging bacterial fish diseases by using naturally antagonis- tic micro-organisms to control populations of potential pathogens, either by competitive inhibition, enhancement of fish immunity or by the microbial enhancement of the environment. Such organisms have been usually referred to as probiotics (Salminen et al. 1999), and are usually incorporated into the fish feed. Lactic acid bacteria (LAB) are among the most common probiotics used in aquacul- ture, and have been proposed to function as nonspecific immunostimulants and in environmental enhancement (Vadstein 1997; Ringø and Gatesoupe 1998; Skjermo and Vadstein 1999; Robertson et al. 2000). However, a greater variety of micro-organisms has been considered for use as probiotics in aquaculture than in other areas of agri- culture (Irianto and Austin 2002). In this study, Bacillus subtilis AB1 was isolated and evaluated as a putative pro- biotic in preventing disease in rainbow trout caused by a highly virulent strain of Aeromonas sp. Materials and methods Fish Rainbow trout, Oncorhynchus mykiss (Walbaum) (average weight = 30 g) were obtained from a commercial fish farm in Scotland. The fish were maintained in continuo- usly aerated free-flowing dechlorinated fresh water at 17°C and fed with a commercial pellet diet (Trouw, Wincham, UK). Representative samples from the fish stock were rou- tinely examined microbiologically and physically to ensure the absence of bacterial diseases and parasites following methods described by Austin and Austin (1989). Bacterial pathogen Aeromonas sp. ABE1 originally isolated from diseased til- apia (Oreochromis sp.) was obtained from the culture collection of the School of Veterinary Medicine, The University of the West Indies, St. Augustine, Trinidad. Pathogenicity of Aeromonas sp. ABE1 against rainbow Keywords Aeromonas, Bacillus subtilis AB1, fish disease, innate immunity, probiotic, specific immunity. Correspondence B. Austin, School of Life Sciences, John Muir Building, Heriot-Watt University, Riccarton, Edinburgh, EH14 4AS, Scotland, UK. E-mail: b.austin@hw.ac.uk 2006 ⁄ 1652: received 27 November 2006, revised 13 March 2007 and accepted 13 March 2007 doi:10.1111/j.1365-2672.2007.03402.x Abstract Aim: To develop a probiotic with effectiveness against Aeromonas sp., which was pathogenic to rainbow trout. Methods and Results: When Bacillus subtilis AB1, which was obtained from fish intestine, was administered for 14 days to rainbow trout in feed at a con- centration of 10 7 cells per gram either as viable, formalized or sonicated cells or as cell-free supernatant, the fish survived challenge with the pathogen. AB1 stimulated immune parameters, specifically stimulating respiratory burst, serum and gut lysozyme, peroxidase, phagocytic killing, total and a1-antiprotease and lymphocyte populations. Conclusions: Bacillus subtilis AB1 was effective as a probiotic at controlling infections by a fish-pathogenic Aeromonas sp. in rainbow trout. Significance and Impact of the Study: Disease control in fish is possible by means of the oral application of live and inactivated cells and their subcellular components with the mode of action reflecting stimulation of the innate immune response. Journal of Applied Microbiology ISSN 1364-5072 ª 2007 The Authors Journal compilation ª 2007 The Society for Applied Microbiology, Journal of Applied Microbiology 103 (2007) 1699–1706 1699