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Carriage of Potentially Fish-Pathogenic Bacteria in Sparus Aurata Cultured in Mediterranean Fish Farms

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Carriage of Potentially Fish-Pathogenic Bacteria in Sparus Aurata Cultured in Mediterranean Fish Farms DISEASES OF AQUATIC ORGANISMS Vol. 54: 119–126, 2003 Published March 31 Dis Aquat Org Carriage of potentially fish-pathogenic bacteria in Sparus aurata cultured in Mediterranean fish farms M. J. Pujalte1, 2, A. Sitjà-Bobadilla3, P. Álvarez-Pellitero3, E. Garay1, 2,* 1Instituto Cavanilles de Biodiversidad y Biología Evolutiva, and 2Departament de Microbiología i Ecología, Facultad de Biologia, Campus de Burjassot, Universitat de València, 46100 Valencia, Spain 3Instituto de Acuicultura de Torre de la Sal, Consejo Superior de Investigaciones Científicas (CSIC), Torre La Sal, Ribera de Cabanes, 12595 Castellón, Spain ABSTRACT: A bacteriological survey of gilthead sea bream Sparus aurata from different fish farms and culture systems on the Spanish Mediterranean coast was conducted. Three different studies were performed. Study A included hatchery-reared larvae; Study B, periodic examination of ran- domly sampled growing fish; and Study C, growing fish sampled only during mortality/morbidity events. In Studies B and C, sea cages, earth ponds and indoor tanks were surveyed, and in both cases diseased (showing clinical signs) and non-diseased fish were included. In Study A, a shift from Vibrio spp. (30 d after hatching) to oxidative species (60 d after hatching) was detected, and no mor- tality events were registered. The percentage of fish yielding bacterial growth were similar in Stud- ies B and C, reaching 57.4 and 61.3%, respectively. A statistically significant relationship between the bacterial carriage and the type of facility was only found in Study B, showing that fish from sea cages had a higher bacterial occurrence than fish from other facilities. A statistically significant rela- tionship between bacterial carriage and signs of disease was found, although the pattern differed in each study. Thus, in Study B only 36.2% of fish yielding abundant bacterial growth were diseased, versus 68.0% in Study C. In total, 25.0% of the fish examined were diseased. Bacterial species com- position was similar in asymptomatic and diseased fish, except for a group of V. ichthyoenteri-like isolates that occurred almost exclusively in asymptomatic fish. Dominant bacterial species were V. harveyi and V. splendidus, followed by V. ichthyoenteri-like isolates, Photobacterium damselae ssp. damselae and V. fisheri. Non-fermenters were less frequent but, among them, unidentified halophilic Cytophaga-Flavobacterium isolates and Pseudoalteromonas haloplanktis were the most abundant. An association of individual species with disease was not clear, which suggests the involvement of mixed infections. KEY WORDS: Sparus aurata · Vibrio harveyi · Vibrio splendidus · Photobacterium damsela · Pseudoalteromonas haloplanktis · Vibrio ichthyoenteri · Mediterranean aquaculture Resale or republication not permitted without written consent of the publisher INTRODUCTION associated with disease outbreaks in marine fish farms, but the pathological role of those normally inhabiting Mediterranean aquaculture has experienced a rapid the marine environment is unclear. Such bacteria can expansion in the last decade and the number of facili- cause problems when conditions favour their prolifera- ties dedicated to the growth of marine finfish has tion (Rodgers & Furones 1998), thus making the differ- dramatically increased. European sea bass Dicentrar- entiation between primary pathogens and opportunis- chus labrax and gilthead sea bream Sparus aurata tic pathogens difficult. Disease outbreaks in cultured are presently the dominant species cultured on the gilthead sea bream include Vibrio spp., Photobac- Spanish Mediterranean coast and are accompanied by terium damselae, Pseudomonas spp., and Flexibacter specific disease problems related to poor on-site condi- maritimus (Breuil & Haffner 1990, Vera et al. 1991, tions, poor husbandry, or adverse environmental fac- Balebona et al. 1995, 1998b, Berthe et al. 1995, Bovo et tors. Different bacterial species have been found to be al. 1995, Sedano et al. 1996, Domenech et al. 1997, *Corresponding author. Email: [email protected] © Inter-Research 2003 · www.int-res.com 120 Dis Aquat Org 54: 119–126, 2003 Baptista et al. 1999). These species have been events were reported by fish farmers. In addition to recovered from moribund or recently dead fish show- Farms F1, F2 and F3, another sea cage farm (F4) and ing clinical signs. However, studies on the bacterial the facilities (intensive indoor tanks) of the Instituto de flora present in asymptomatic fish are scarce. Acuicultura de Torre de la Sal (IATS), both on the West- Several studies have been conducted along the ern Mediterranean coast (Castellón), were studied. Spanish Mediterranean coast to identify the main bac- Fish in Studies B and C were reared under natural terial groups present in water and bivalves (Ortigosa et temperature and photoperiod, and disease signs and al. 1994a,b, Arias et al. 1999, Pujalte et al. 1999). Some mortalities were recorded. of these bacteria exhibit clear seasonal variation, as Sampling procedure and bacteriological analysis. Vibrio harveyi dominated with temperatures above Study A: At each sampling, larvae were pooled, 20°C and V. splendidus dominated at temperatures be- washed and homogenised in sterile seawater with a low 20°C (Arias et al. 1999, Pujalte et al. 1999). These glass homogeniser. Both larval homogenates and sea- and other bacterial species showing seasonal occur- water from the rearing tank were serially diluted in rence have been reported as opportunistic pathogens sterile seawater, plated on marine agar (MA; Sea Agar, for sea bass and sea bream (Balebona 1998b). Scharlau Chemie), and incubated at 22 to 25°C for up In the present study, bacterial carriage in gilthead to 10 d. Colonies were counted, and 30 to 40 random sea bream, cultured in the Spanish Mediterranean colonies resulting from the highest dilution were iso- area, from larval to commercial size fish was studied. lated and identified according to the methods des- Fish samples included both diseased (with clinical cribed below. signs) and asymptomatic animals. Periodic routine Study B: At each sampling, 20 randomly collected samplings throughout the growing period as well as live fish were killed by overexposure to the anaesthetic special samplings, mainly coinciding with disease out- MS-222 (Sigma). The fish were weighed, measured breaks, were carried out at different fish culture facili- and bacteriological samples were taken aseptically ties. The aim of this study was to determine the occur- with a sterile loop from the head kidney (occasionally rence, relative abundance and significance of the from the liver in small fish) and streaked on MA and different bacterial species associated with healthy and tryptone soya agar plus 1% NaCl (TSA1) (Scharlau diseased fish during their culture. Chemie) plates. Study C: The number of fish per sampling was vari- able, but the same procedure described for Study B MATERIALS AND METHODS was followed. In Studies B and C, a selection of all the different Fish groups. The bacteriological survey was con- types of colonies was done after the MA and TSA1 ducted for 2 yr in several aquaculture facilities along plates had been incubated at 20 to 25°C for 48 to 72 h. the Spanish Mediterranean coast. Samplings were Plates were re-incubated for up to 10 d and examined carried out as follows: for growth of slow developing colonies. Growth on Study A: Hatchery-reared larvae were sampled on both media from each individual sampled was scored Days 30 (before weaning) and 60 (after weaning) post- according to growth, as follows: 0 = no growth, T = hatch. At each sampling, 40 larvae, along with sea- traces of growth (1 to 9 colonies); A = abundant growth water from the rearing tank, were analysed. Water (10 or more colonies). Fish with at least traces of temperature at the hatchery was 18 to 20°C. growth were considered positive (P) for bacteria (P = Study B: A total of 397 fish were examined from T+ A). 3 farms with 2 different growing systems: sea cages In both studies (B and C), sampled fish included both (Farms F1 and F2) and intensive earth ponds (Farm diseased (with clinical signs) and asymptomatic (with- F3). Fish were randomly sampled just before entering out clinical signs) individuals. However, in Study C, the facilities, and then at 3 mo intervals until commer- diseased fish were more abundant due to the nature of cial size was attained. In F1 (Western Mediterranean the samplings. When external wounds appeared in Sea, Castellón, Spain), fish entered with an average diseased fish, additional samples were taken after weight of 21.6 g and were delivered to the market at washing with 70% ethanol. 339.8 g. In F2 (Western Mediterranean Sea, Tarragona, Water temperatures varied from 11 to 18°C between Spain), fish average weight increased from 8.5 to December and May (considered as the cold season) 281.3 g throughout the study, whereas in F3 (Western and from 26 to 15°C between June and November Mediterranean Sea, Tarragona, Spain) average weight (considered as the warm season). ranged from 2.1 to 414.1 g. Identification: All colonies obtained from Studies A, Study C: A total of 150 fish from different farms were B and C were checked for purity by repeated streaking sampled when mortalities, morbidities or abnormal on MA plates, and then submitted to phenotypic tests, Pujalte et al.: Bacterial carriage in Sparus aurata 121 in accordance with Ortigosa et al. (1994a,b), which in- RESULTS cluded: determination of Gram reaction; oxidase; Na+ requirement; luminescence; pigmentation; fermenta- Study A: larvae tive metabolism of glucose; arginine dihydrolase; ly- sine and ornithine decarboxylases; indole production; As shown in Table 1, colony forming units per gram Voges-Proskauer; growth and sucrose fermentation on (cfu g–1) of larvae were always 2 orders of magnitude thiosulphate-citrate-bile-sucrose (TCBS) agar; growth higher than those from the surrounding water at 4 and 40°C; hydrolysis of casein; alginate and starch, (cfu ml–1). Vibrio spp. were dominant in 30 d larvae and the ability to use the following substrates as sole (mainly Vibrio splendidus and V.
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