Expression of immune-related genes in rainbow trout (Oncorhynchus mykiss) induced by probiotic bacteria during Lactococcus garvieae infection Tania Pérez-Sánchez a, * , José Luis Balcázar b , Daniel L. Merrifield c , Oliana Carnevali d , Giorgia Gioacchini d , Ignacio de Blas a , Imanol Ruiz-Zarzuela a a Laboratory of Fish Pathology, Faculty of Veterinary, Universidad de Zaragoza, c/. Miguel Servet 177, 50013 Zaragoza, Spain b Catalan Institute for Water Research (ICRA), Scientific and Technological Park of the University of Girona, c/. Emili Grahit 101,17003 Girona, Spain c Aquaculture and Fish Nutrition Research Group, School of Biomedical and Biological Sciences, University of Plymouth, Devon PL48AA, Plymouth, UK d Department of Marine Sciences, Polytechnic University of Marche, Via Brecce Bianche, 60131 Ancone, Italy article info Article history: Received 1 April 2011 Received in revised form 23 April 2011 Accepted 8 May 2011 Available online 19 May 2011 Keywords: Lactobacillus plantarum Lactococcus lactis Leuconostoc mesenteroides Lactococcus garvieae Cytokines abstract The aim of the present study was to investigate the effect of lactic acid bacteria (LAB) on the control of lactococcosis as well as to assess the impact of probiotics on the expression of immune-related genes in the head kidney and intestine of rainbow trout (Oncorhynchus mykiss). Lactobacillus plantarum, Lacto- coccus lactis and Leuconostoc mesenteroides, were administered orally at 10 6 CFU g 1 feed to fish for 36 days. Twenty-one days after the start of the feeding period, fish were challenged with Lactococcus gar- vieae. Only the fish fed the diet containing Lb. plantarum showed significantly (P < 0.05) improved protection against L. garvieae compared to the control. Subsequently, real-time PCR was employed to determine the mRNA levels of IL-1b, IL-8, IL-10 and TNF-a in the head kidney, and IL-8, Tlr5 and IgT in the intestine of the control and Lb. plantarum groups. IL-1b, IL-10 and TNF-a gene expression were signifi- cantly up-regulated by Lb. plantarum. Moreover, the mRNA levels of IL-10, IL-8 and IgT were significantly higher in the Lb. plantarum group after L. garvieae infection, suggesting that Lb. plantarum can stimulate the immune response of rainbow trout. PCR-DGGE revealed no detectable levels of the probiotics or the pathogen present on the distal intestinal mucosa. These findings demonstrate that direct probioticehost interactions with the intestine are not always necessary to induce host stimulatory responses which ultimately enhance disease resistance. Furthermore, as L. garvieae did not colonise the intestinal tract, and therefore likely did not infect via this route, the antagonistic properties of the probiotic candidate towards L. garvieae were likely of little influence in mediating the improved disease resistance which could be attributed to the elevated immunological response. Ó 2011 Elsevier Ltd. All rights reserved. 1. Introduction Lactococcus garvieae is the causal agent of lactococcosis, a disease that causes severe economic losses in farmed marine and freshwater fish species, particularly during the summer months, given its association with high water temperatures [1]. The disease is characterised by a haemorrhagic septicaemia and meningoen- cephalitis in several species of marine and freshwater fish [1,2] and mammals [3]. Moreover, L. garvieae has also been isolated from humans in several cases, suggesting that this bacterium could be catalogued as a potential zoonotic agent [4]. In fish farming, bacterial disease outbreaks are typically treated with antibiotics; however, they are often ineffective and their indiscriminate and prophylactic use has led to an increase in anti- biotic resistances [1]. Commercial vaccines are available for some pathogens [5], but vaccination cannot prevent disease development in immunologically immature fish. In the case of lactococcosis, immunity after vaccination gives a good level of protection, but it only lasts for a short period of time [6]. Over the last decade there has been a growing interest in the use of beneficial microorganisms to prevent or control pathogenic microorganisms as an alternative to traditional disease control treatments [7,8]. Probiotics have been defined as a viable microbial feed supplements which beneficially influence the health of the host [9], and they offer potential alternatives by providing benefits to the host primarily via the direct or indirect modulation of the gut microbiota. Suggested modes of action resulting from increased * Corresponding author. Tel.: þ34 976 764 110; fax: þ34 976 761 612. E-mail address: taniaper@unizar.es (T. Pérez-Sánchez). Contents lists available at ScienceDirect Fish & Shellfish Immunology journal homepage: www.elsevier.com/locate/fsi 1050-4648/$ e see front matter Ó 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.fsi.2011.05.005 Fish & Shellfish Immunology 31 (2011) 196e201