Full length article In vitro and in vivo evaluation of lactic acid bacteria of aquatic origin as probiotics for turbot (Scophthalmus maximus L.) farming Estefanía Mu ~ noz-Atienza a , Carlos Araújo a, b , Susana Magad  an c, 1 , Pablo E. Hern  andez a , Carmen Herranz a , Ysabel Santos d , Luis M. Cintas a, * a Grupo de Seguridad y Calidad de los Alimentos por Bacterias L acticas, Bacteriocinas y Probi oticos (Grupo SEGABALBP), Departamento de Nutrici on, Bromatología y Tecnología de los Alimentos, Facultad de Veterinaria, Universidad Complutense de Madrid, 28040-Madrid, Spain b Institute for Biotechnology and Bioengineering, Centre of Genomics and Biotechnology, University of Tr as-os-Montes and Alto Douro, 5001-801 Vila Real, Portugal c Centro Oceanogr afico de Vigo, Instituto Espa~ nol de Oceanografía (IEO), 36390 Vigo, Pontevedra, Spain d Department of Microbiology and Parasitology, Faculty of Biology, University of Santiago de Compostela,15782-Santiago de Compostela, Spain article info Article history: Received 24 July 2014 Received in revised form 2 October 2014 Accepted 4 October 2014 Available online 16 October 2014 Keywords: Turbot (Scophthalmus maximus L.) Probiotics Lactic acid bacteria Functional properties Immunity-related gene expression abstract Turbot (Scophthalmus maximus L.) is an important commercial marine flatfish. Its production may be affected by bacterial diseases that cause severe economical losses, mainly tenacibaculosis and vibriosis, provoked by Tenacibaculum maritimum and Vibrio splendidus, respectively. An alternative or comple- mentary strategy to chemotherapy and vaccination for the control of these diseases is the use of pro- biotics. In this work, we report the in vitro and in vivo potential of eight lactic acid bacteria (LAB), previously isolated from fish, seafood and fish products intended for human consumption, as turbot probiotics. Seven out of the eight LAB exerted direct antimicrobial activity against, at least, four strains of T. maritimum and V. splendidus. All LAB survived in seawater at 18  C for 7 days, and withstood exposure to pH 3.0 and 10% (v/v) turbot bile; however, they differed in cell surface hydrophobicity (8.2e21.7%) and in their ability to adhere to turbot skin (1.2e21.7%) and intestinal (0.7e2.1%) mucus. Most of the tested strains inhibited the binding of turbot pathogens to the mucus. Leuconostoc mesenteroides subsp. cremoris SMM69 and Weissella cibaria P71 were selected based on their strong antimicrobial activity against T. maritimum and V. splendidus, good probiotic properties, and different adhesion ability to skin mucus and capacity to inhibit the adhesion of turbot pathogens to mucus. These two LAB strains were harmless when administered by bath to turbot larvae and juveniles; moreover, real-time PCR on the transcription levels of the immunity-related genes encoding IL-1b, TNF-a, lysozyme, C3, MHC-Ia and MHC-IIa in five organs (head-kidney, spleen, liver, intestine and skin) revealed the ability of these LAB to stimulate their expression in turbot juveniles, especially the non-specific immunity associated genes in mucosal tissues. Based on our results, Lc. cremoris SMM69 and W. cibaria P71 may be considered as suitable probiotic candidates for turbot farming. © 2014 Elsevier Ltd. All rights reserved. 1. Introduction Turbot (Scophthalmus maximus L.) aquaculture is a mature technology with a global production of 77,118 tons in 2012; how- ever, a continued research and development effort is required for the prevention and control of fish diseases [1]. In this respect, tenacibaculosis and vibriosis are considered the main turbot dis- eases, causing severe economic losses in turbot farming. Tenaci- baculosis (or flexibacteriosis) is a bacterial disease caused by Tenacibaculum maritimum (formerly, Flexibacter maritimus), a Gram-negative filamentous biofilm-forming microorganism that affects cultured adults and juvenile marine fish, with the latter suffering the most severe form of the disease [2]. Vibriosis is one of the main bacterial diseases that causes most of the economical problems in fish marine culture, and it is provoked by several species of fish-pathogenic vibrios [2,3]. One of the most relevant pathogenic vibrio species is Vibrio splendidus, a Gram-negative microorganism linked with high rates of mortality in larval turbot rearing fed with live feed [3]. In the fish farming sector, antibiotics * Corresponding author. Departamento de Nutrici on, Bromatología y, Tecnología de los Alimentos, Facultad de Veterinaria, Universidad Complutense de Madrid, Avda. Puerta de Hierro s/n, 28040-Madrid, Spain. Tel.: þ34 913943751; fax: þ34 913943743. E-mail address: lcintas@vet.ucm.es (L.M. Cintas). 1 Current address: Virologie et Immunologie Mol eculaires, Institut National de la Recherche Agronomique (INRA), 78352 Jouy-en-Josas, France. Contents lists available at ScienceDirect Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi http://dx.doi.org/10.1016/j.fsi.2014.10.007 1050-4648/© 2014 Elsevier Ltd. All rights reserved. Fish & Shellfish Immunology 41 (2014) 570e580