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The Israeli Journal of Aquaculture The Open Access Israeli Journal of Aquaculture – Bamidgeh As from January 2010 The Israeli Journal of Aquaculture - Bamidgeh (IJA) will be published exclusively as an on-line Open Access (OA) quarterly accessible by all AquacultureHub (http://www.aquaculturehub.org) members and registered individuals and institutions. Please visit our website (http://siamb.org.il) for free registration form, further information and instructions. This transformation from a subscription printed version to an on-line OA journal, aims at supporting the concept that scientific peer-reviewed publications should be made available to all, including those with limited resources. The OA IJA does not enforce author or subscription fees and will endeavor to obtain alternative sources of income to support this policy for as long as possible. Editor-in-Chief Published under auspices of Dan Mires The Society of Israeli Aquaculture and Marine Biotechnology (SIAMB) Editorial Board & University of Hawaiʻi at Mānoa Rina Chakrabarti Aqua Research Lab, Dept. of & Zoology, University of Delhi, India AquacultureHub Angelo Colorni National Center for Mariculture, http://www.aquaculturehub.org IOLR, Eilat, Israel Daniel Golani The Hebrew University of Jerusalem, Israel Hillel Gordin Kibbutz Yotveta, Arava, Israel ! Sheenan Harpaz Agricultural Research Organization, Beit Dagan, Israel Gideon Hulata Agricultural Research Organization Beit Dagan, Israel George Wm. Kissil National Center for Mariculture, IOLR, Eilat, Israel ! ! Ingrid Lupatsch Swansea University, Singleton Park, Swansea, UK ! Spencer Malecha Dept. of Human Nutrition, Food & Animal Sciences, CTAHR, University of Hawaii ISSN 0792 - 156X Constantinos Mylonas Hellenic Center for Marine Research, Crete, Greece © Israeli Journal of Aquaculture - BAMIGDEH. Amos Tandler National Center for Mariculture, IOLR, Eilat, Israel PUBLISHER: Emilio Tibaldi Udine University, Udine, Italy Israeli Journal of Aquaculture - BAMIGDEH - Jaap van Rijn Faculty of Agriculture, The Hebrew Kibbutz Ein Hamifratz, Mobile Post 25210, University of Jerusalem, Israel ISRAEL Phone: + 972 52 3965809 Zvi Yaron Dept. of Zoology, Tel Aviv University, Israel http://siamb.org.il Copy Editor Miriam Klein Sofer The Israeli Journal of Aquaculture - Bamidgeh, IJA_68.2016.1317, 8 pages The IJA appears exclusively as a peer-reviewed on-line open-access journal at http://www.siamb.org.il/. To read papers free of charge, please register online at registration form. Sale of IJA papers is strictly forbidden. In Vitro Testing of Potential Probiotic Bacteria against Vagococcus salmoninarum in Rainbow Trout (Oncorhynchus mykiss, Walbaum 1792) Behire Işıl Didinen1* Ertan Emek Onuk2 Hatice Sayı1 1Egirdir Fisheries Faculty, Suleyman Demirel University, Isparta, Turkey 2Department of Aquatic Animal Diseases, Faculty of Veterinary Medicine, Ondokuz Mayıs University, Samsun, Turkey Keywords: Oncorhyncus mykiss; probiotic, Vagococcus salmoninarum; aquaculture; Lactococcus garvieae; Lactococcus lactis Abstract In this study, probiotic properties of endogenous microbiota of rainbow trout against Vagococcus salmoninarum isolated during an outbreak of vagococcosis in a trout farm in the Mediterranean region were evaluated. The candidate probiotic bacteria were isolated from rainbow trout intestines. A total of 157 isolates were obtained and screened for antagonistic activity against V. salmoninarum via the Well Diffusion Agar method. Six isolates were determined for antagonistic activity against V. salmoninarum. Conventional microbiological tests and API 20 Strep tests (bioMe´rieux) were used for further phenotypic characterization of all six antagonistic isolates. For molecular identifications of isolates, L. garvieae specific PCR and 16S rRNA gene sequence analysis were used. Antagonistic strains were identified including TUB/2013/V47 (L. garvieae), TUB/2013/V27 (L.garvieae), TUB/2013/V10 (L. garvieae), TUB/2013/V2 (L. garvieae) TUB/2013/V1 (L. lactis) and TUB/2013/V4 (L. lactis). The strains were then tested for hydrophobicity, bile salts and acid tolerance and antimicrobial activity. All isolates were congo red-positive, indicating the presence of hydrophobic structures in their cell walls. It was determined that whole antagonistic strains were resistant to low pH conditions and 0.6-1.5% bile concentrations. Antimicrobial test results showed that most of the strains are susceptible in vitro to amoxicillin/clavulanic acid ampicillin, doxycycline, erythromycin and florfenicol, which are frequently used in aquaculture. As a result, it was found that strains have in vitro probiotic properties (hydrophobic, tolerant to bile salts and low pH conditions). Further study is needed to explore their in vivo probiotic effects against vagococcosis. * Corresponding author. [email protected] 2 Didinen et al. Introduction Streptococcosis has been defined as a complex of diseases caused by agents belonging to different genera and species of gram-positive cocci. Streptococcal infections can be divided into two major groups: warm water infections (caused by Lactococcus garvieae, Streptecoccus iniae, S. agalactiae and S. parauberis pathogenic for both cultured freshwater and marine fish at water temperatures above 15 °C); and coldwater infections (caused by streptococcal species such as Vagococcus salmoninarum pathogenic for salmonids at water temperatures below 12°C) (Eldar & Ghittino, 1999; Ghittino et al., 2003). Vagococcosis caused by V. salmoninarum is an important bacterial disease in the European rainbow trout industry, with mortality rates of 20-80% in sub adult or adult fish. Outbreaks occur at water temperatures of 10-12oC and are associated with post- spawning stress in broodstock (Michel et al., 1997; Ruiz-Zarzuela et al., 2005). V. salmoninarum has been reported in rainbow trout in Australia (Schmidtke and Carson, 1994), France (Michel et al.,1997), Italy (Ghittino et al. 2004), Spain (Ruiz-Zarzuela et al., 2005), and Turkey (Didinen et al., 2011; Tanrikulu et al., 2014; Ozcan et al., 2014). Antibiotic treatment of vagococcosis in the field has not been effective, although V. salmoninarum isolates are susceptible to several drugs such as ampicillin, amoxycillin, erythromycin, oxytetracycline and doxycycline tested in vitro (Michel et al,. 1997; Ruiz- Zarzuela et al., 2005; Didinen et al., 2011). Treatments with antibiotics were effective only for short periods (5-7 days) and continuous delivery was necessary to reduce mortality, followed by a probable enhancement of antibiotic resistance (Ruiz-Zarzuela et al., 2005). Vaccination has also not been successful (Michel et al., 1997; Ruiz-Zarzuela et al., 2005). It is necessary to determine and develop effective protection against V. salmoninarum. Probiotic lactic acid bacteria (such as Lactococcus spp., Pediococcus spp. or Lactobacillus spp.) have been proposed as biological control agents in aquaculture (Merrifield et al., 2010). These bacteria have been used as probiotics in aquaculture due to their low or no virulence properties, outcompeting harmful bacteria by producing antimicrobial substances, adherence capacity to colonize the digestive tract and competing with pathogens for adhesion sites (Farzanfar, 2006). Reports about screening and use of lactic acid bacteria against lactococcosis in rainbow trout have been documented (Balcázar et al. 2007;2008; Vendrell et al., 2008; Pérez-Sánchez et al., 2011a; Pérez-Sánchez et al., 2011b; Didinen et al., 2016). There is only one study about screening of LAB against V. salmoninarum in vitro conditions (Balcázar et al. 2007). The aim of this study was to isolate candidate probiotic bacteria from the intestines of rainbow trout and determine in vitro probiotic properties, their inhibitory activity against V. salmoninaru, pH, and bile tolerance that enables them to survive in the gastrointestinal tract of the host, hydrophobicity that enables intestinal colonization, and antibiotic resistance. Materials and Methods Isolation and initial screening of potential probiotic bacteria. Bacteria were isolated from the hindgut of 20 healthy rainbow trout (250 g) using the spread plate method. They were kept at 25oC for 2 days on Triptic Soy Agar (TSA, Merck). Each isolate was stored at -80°C in Triptic Soy Broth with 15% (v/v) glycerol. V. salmoninarum was previously isolated during a natural vagococcosis outbreak in rainbow trout (Didinen et al. 2011). All potential probiotic bacteria were tested for antagonistic activity against V. salmoninarum by a well diffusion agar assay (WDAA). The pathogen was grown in 4mL TSB for 1 d at 25°C, and 10 μl of each culture was mixed into 10 mL of melted TSA. After solidifying and drying for 15-20 minutes, wells were punched (diameter = 3 mm) and 10 μl of a 2 day old potential probiotic bacteria culture (approx. 108-109 CFU/mL) grown in TSB at 25°C was added to the wells in triplicate. Plates were incubated at 25°C for one day and clearing zones were measured around the wells. Strains causing clearing zones in the WDAA were tested once more in TSA to ensure that the antagonistic activity was stable after storage and sub-culture (Hjelm et al., 2004). Hydrophobicity test. A hydrophobicity test using Congo red stain to determine the hydrophobicity of the bacteria was carried out using plates prepared in TSA with 0.03% Potential Probiotic Bacteria against Vagococcus Salmoninarum in Rainbow Trout 3 Congo red. Congo red was added after sterilization of TSA. Using the cross-streak method each isolate was spread on plates, and incubated at 25°C
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