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Editor-in-Chief Published under auspices of Dan Mires The Society of Israeli Aquaculture and Marine Biotechnology (SIAMB), Editorial Board University of Hawaii at Manoa Library Sheenan Harpaz Agricultural Research Organization and Beit Dagan, Israel University of Hawaii Aquaculture Zvi Yaron Dept. of Zoology Program in association with Tel Aviv University AquacultureHub Tel Aviv, Israel http://www.aquaculturehub.org Angelo Colorni National Center for Mariculture, IOLR Eilat, Israel Rina Chakrabarti Aqua Research Lab Dept. of Zoology University of Delhi Ingrid Lupatsch Swansea University Singleton Park, Swansea, UK Jaap van Rijn The Hebrew University Faculty of Agriculture Israel Spencer Malecha Dept. of Human Nutrition, Food and Animal Sciences University of Hawaii Daniel Golani The Hebrew University of Jerusalem Jerusalem, Israel Emilio Tibaldi Udine University Udine, Italy ISSN 0792 - 156X Israeli Journal of Aquaculture - BAMIGDEH. Copy Editor Ellen Rosenberg PUBLISHER: Israeli Journal of Aquaculture - BAMIGDEH - Kibbutz Ein Hamifratz, Mobile Post 25210, ISRAEL Phone: + 972 52 3965809 http://siamb.org.il The Israeli Journal of Aquaculture – Bamidgeh 59(2), 2007, 111-116. 111 Immunogenic and Antigenic Profiles of Nine Lactococcus garvieae Strains from Different Rainbow Trout Farms S. Altun1, A.K. Adiloglu2, A. Kubilay1, O. Diler1, N. Delibas3 and R. Sutcu3 1 Faculty of Fisheries, Department of Aquaculture, Suleyman Demirel University, 32500, Egirdir, Isparta, Turkey 2 Department of Medical Microbiology, Faculty of Medicine, Suleyman Demirel University, 32260 Isparta, Turkey 3 Department of Medical Biochemistry, Faculty of Medicine, Suleyman Demirel University, 32260 Isparta, Turkey (Received 5.8.06, Accepted 8.1.07) Key words: Oncorhynchus mykiss, Lactococcus garvieae, immunization, Western blot Abstract The aims of this study were to determine differences with respect to immunogenic potency in the antigenic profiles of nine Lactococcus garvieae strains from Turkey, Spain, and England, to develop a bacterin, and to examine the immunological response of rainbow trout to the bacterin. The strains had identical Western blot patterns with 30, 37, 40, 46, 52, and 66 kDA mw protein bands. After culturing the bacteria, proteins were separated by SDS-PAGE and transferred to Immobilon membranes. The membranes were incubated with hyperimmune rabbit sera obtained by immunizing a rabbit against L. garvieae. One group of 50 fish was immunized with formalin- killed bacterin prepared from the most immunogenic strain of L. garvieae. A second group of 50 fish served as an unimmunized control. Four weeks after vaccination, both groups were chal- lenged intraperitoneally with a homologous strain. The protection rate of the bacterin was judged by the relative percent survival (RPS) of the groups. A significantly high level of protection was achieved in the vaccinated group (88.8% RPS). Introduction The gram-positive bacterium Lactococcus quinqueradiata) in Japan and rainbow trout garvieae is an important pathogen that caus- (Oncorhynchus mykiss) in Europe and es septicemia and meningoencephalitis in Australia (Kusuda et al 1991; Eldar et al., freshwater and marine fish (Eldar et al. 1996; 1996; Bercovier et al., 1997; Eldar and Barnes et al., 2002). It has been isolated from Ghittino, 1999; Ravelo et al., 2003), as well as cultured species such as yellow tail (Seriola from homeothermic and poikilothermic ani- * Corresponding author. Tel.: +90-246-3133447, fax: +90-246-3133452, e-mail: [email protected] 112 Altun et al. mals such as cows, buffalos, the freshwater molecular weights of proteins in L. garvieae prawn Macrobrachium rosenbergii (Collins et from different farms by analyzing extracted al., 1984; Teixeira et al., 1996; Chen et al. bacterial proteins by blotting with anti-L. 2001), and humans (Elliott et al., 1991; Fefer garvieae antibodies from a rabbit immunized et al., 1998). In Turkey, L. garvieae was first with L. garvieae bacterin. One of the advan- isolated from rainbow trout farms in 2001 tages of whole-cell protein profile analysis (Diler et al., 2002; Altun et al., 2004). Since over conventional physiological tests is that then, it has spread rapidly, causing substan- once bacteria are isolated and identified to the tial economic losses, and is one of the impor- genus level, proteins can be prepared and tant pathogens in the trout industry, causing SDS-PAGE results can be determined within infections especially in summer. one day. In contrast, physiologic tests for Numerous commercially available vac- species identification can require incubation of cines against fish pathogenic bacteria pro- at least seven days (Elliott et al., 1991). We duce good results. However, chemotherapeu- also aimed to develop a bacterin and examine tic agents such erythromycin, oxytetracycline, the immunological response of rainbow trout and enrofloxacine may be sensitive in in vitro to the bacterin. susceptibility tests but ineffective in vivo (Austin and Van Pouce, 1993; Bercovier et al., Materials and Methods 1997), probably due to anorexia of infected Fish. Rainbow trout (initial wt 20 g) were held farmed fish. Pathogenic properties of anti- in 600-l tanks filled with 12°C fresh water at a genic variations of L. garvieae isolated from flow rate of 1-1.5 l/min and continuous aera- yellow tail in Japan have been described (Alim tion. Fish were fed daily at 2% of their body et al., 1996; Yoshida et al., 1997; Ooyama et weight and water quality was monitored daily. al., 2002). In several attempts to develop vac- Bacterial strains, virulent strains, hyperim- cines against L. garvieae, only intraperitoneal mune serum. Nine isolated L. garvieae strains administration achieved effective responses from different trout farms were studied (Table (Bercovier et al., 1997 ). Therefore, it is nec- 1). The reference strain NCDO-2155 served essary to test adapted solutions on local as a control. Isolated strains were incubated strains of the aquatic pathogen L. garvieae on trypticase soy agar (TSA; Difco (Romalde et al., 2004). Laboratories, Detroit, MI) at 25°C for 24-48 h The aim of this study was to determine and pure cultures were stored at -80°C in tryp- Table 1. Origin of Lactococcus garvieae strains. No. Strain Origin Year of isolation Comments 1A3 Antalya, Turkey 2001 2B1 Isparta, Turkey 2003 3 279-00 Spain 2000 4 00-21 England 2000 5 NCDO-2155 (ATCC-43921) UK 1973 Control 6K1 Konya, Turkey 2001 7C1 Mugla, Turkey 2001 8M1 Mugla, Turkey 2001 Virulence experiment 9D1 Denizli, Turkey 2001 Immunogenic and antigenic profiles of Lactococcus garvieae 113 ticase soy broth ( Difco Laboratories, Detroit, with a computerized image analysis system MI) with 15% glycerol. Lactococcus garvieae (Uviphoto MW V.99, Ultra-Violet Products Ltd, M1, cultivated from rainbow trout in Turkey, is Cambridge, UK). Sera from control fish (trout the most virulent strain (80% loss rates) and injected intraperitoneally with a saline solu- was used for the virulence experiment (Diler tion) served for control blots. SDS-PAGE and et al., 2002). LD50 was calculated by deter- Western blot analyses were triplicated on all mining the cumulative mortality rates one nine strains. month after injection of M1 bacteria at two Preparation of bacterin. A formalin-killed doses. Lactococcus garvieae (NCDO-2155) bacterin was prepared as described by Eldar hyperimmune serum was obtained from the et al. (1997). Briefly, after L. garvieae strains Department of Fish Disease in Ege University. were incubated in trypticase soy broth for 24 Electrophoresis (SDS-PAGE) and Western h, the bacteria were killed by adding formalin blotting. Each strain was sonicated 5 min at to a final concentration of 0.7% (v/v). The maximum amplitude (Bandelin Electronic product was incubated at 25°C for 3 h and at UW2070, Germany) and centrifuged 30 min at 4°C overnight. The formalin was removed by 14,000 x g at 4°C. The resulting pellet was dialyzing three times with phosphate buffered stained with Gram stain. Detection of gram-neg- saline (PBS, pH 7.4) and the pellet was resus- ative cocci indicated that the bacteria underwent pended in PBS at final concentration of 1011 complete lysis and that the supernatant consist- cells/ml. ed predominantly of cell wall components. The Immunization and challenge of fish. To supernatant was precipitated by adding five vol- evaluate the protective effects of the bacterin, umes of ice cold acetone. The amount of protein two groups of 50 fish (avg wt 20 g) were was determined in sonicated bacteria by the injected intraperitoneally, one with 0.1 ml of technique of Bradford (1976) and treated formalin-killed bacterin (1010 cells/fish) and according to the method of Laemmli