Bacteria Isolated from Diseased Wild and Farmed Marine Fish in Greece

World production of sea bream and sea bass farming has been rising over time. The total production of gilthead sea bream (Sparus aurata L.) and European sea bass (Dicentrarchus labrax, L.) in Europe has been increased. These marine fish species have high economic value in the aquaculture industry. More specifically, the total production of 92,310 tonnes in 1999 amounted to 175,196 tonnes in 2006, representing an average annual growth rate of 9.6%. Greece is the country with the largest production of euryhaline fish (sea bream and sea bass) between Mediterranean countries. In 2008, Greece held its largest production (130,000 tonnes of which 95,000 tonnes sea bass and sea bream). Climatic and geomorphologic conditions of Greece promoting the cultivation of euryhaline fish, grants and projects given by the European Union, the decline of fish stocks and the restrictions have been imposed last yeas in fishing, contributed significantly in the development of the industry of fish farming. Today the industry fully covers the needs of the Greek market and most of the quantity is exported to foreign markets, the main destination countries Italy, Spain, France, England and Portugal. Currently, additional species have entered in the farming, belonging mainly to the family Sparidae, such as Puntazzo pntazzo, Diplodus sargus, Lithognathus mormyrus, Pagrus pagrus, Pagellus erythrinus and Dentex dentex. Bacterial diseases of fish origin have become one of the major agents of economical losses since the beginning of marine farming (Kubota & Takakuwa, 1963; Anderson & Conroy, 1970). The development of intensive marine fish farming in the form of the concentration of large quantities of biomass in a relatively small volume water leads-under certain conditions (combination of factors) – to the emergence of diseases which lead to losses in the population. The occurrence of a disease can lead to death or symptoms both refer to deviation from the normal structure or function of the host (Hedrick, 1998). Most diseases of farmed fish originate from wild populations. The close contact between farmed and wild fish results in exchange of pathogens. The clinical symptoms caused by any pathogen depend on the type of host, age of the fish and stage of disease (acute, chronic, subclinical form). Moreover, in some cases, there is no correlation between internal and external injuries. In fact, systemic diseases (eg. pasteurellosis) with high mortality rates, causing internal damage to infected fish, but often have a healthy appearance. Conversely, other diseases with relatively low mortality cause significant physical damage, including ulcers, necrosis, exophthalmos, making the fish unfit for the market.The diseases, the number and


Introduction
World production of sea bream and sea bass farming has been rising over time.The total production of gilthead sea bream (Sparus aurata L.) and European sea bass (Dicentrarchus labrax, L.) in Europe has been increased.These marine fish species have high economic value in the aquaculture industry.More specifically, the total production of 92,310 tonnes in 1999 amounted to 175,196 tonnes in 2006, representing an average annual growth rate of 9.6%.Greece is the country with the largest production of euryhaline fish (sea bream and sea bass) between Mediterranean countries.In 2008, Greece held its largest production (130,000 tonnes of which 95,000 tonnes sea bass and sea bream).Climatic and geomorphologic conditions of Greece promoting the cultivation of euryhaline fish, grants and projects given by the European Union, the decline of fish stocks and the restrictions have been imposed last yeas in fishing, contributed significantly in the development of the industry of fish farming.Today the industry fully covers the needs of the Greek market and most of the quantity is exported to foreign markets, the main destination countries Italy, Spain, France, England and Portugal.Currently, additional species have entered in the farming, belonging mainly to the family Sparidae, such as Puntazzo pntazzo, Diplodus sargus, Lithognathus mormyrus, Pagrus pagrus, Pagellus erythrinus and Dentex dentex.Bacterial diseases of fish origin have become one of the major agents of economical losses since the beginning of marine farming (Kubota & Takakuwa, 1963;Anderson & Conroy, 1970).The development of intensive marine fish farming in the form of the concentration of large quantities of biomass in a relatively small volume water leads-under certain conditions (combination of factors) -to the emergence of diseases which lead to losses in the population.The occurrence of a disease can lead to death or symptoms both refer to deviation from the normal structure or function of the host (Hedrick, 1998).Most diseases of farmed fish originate from wild populations.The close contact between farmed and wild fish results in exchange of pathogens.The clinical symptoms caused by any pathogen depend on the type of host, age of the fish and stage of disease (acute, chronic, subclinical form).Moreover, in some cases, there is no correlation between internal and external injuries.In fact, systemic diseases (eg.pasteurellosis) with high mortality rates, causing internal damage to infected fish, but often have a healthy appearance.Conversely, other diseases with relatively low mortality cause significant physical damage, including ulcers, necrosis, exophthalmos, making the fish unfit for the market.The diseases, the number and types of bacterial pathogens have been well documented in several farmed fish species, such as global production of sea bream and sea bass farming has been rising over time.Also the incidence of diseases and the number and types of bacterial pathogens have been well documented in farmed fish species, such as salmonids and turbot.However, for farmed and wild fish species in Greece the number and type of bacteria associated with pathology have been described sporadically (Athanassopoulou et al., 1999;Bakopoulos et al., 1995;Varvarigos 1997;Yiagnisis et al., 1999Yiagnisis et al., , 2007)).The purpose of this chapter is to give an accurate, as possible, description of the main bacterial pathogens isolated from farmed and wild fish in Greece and to give information of their occurrence in relation to age and season.This is the first integrated study from Greece including a lot of farmed and wild fish species.

Clinical signs
Signs of diseases include anorexia, lethargies, pale gills, disorientation, darkening in colour, abdominal retention, exophthalmos, abdominal swelling, and external haemorrhages in the head, eyes, skin, gills and at the bases of the fins as well as skin ulcers.

Post-mortem findings
Visceral petechiation, pale kidneys, enlarged spleen and kidney, liquefactive renal necrosis, lesions in the kidney, tubercles in the spleen, are some of the post-mortem findings.

Laboratory testing
Diseased fish collected from Greek fish farms.Dead (recently) or moribund fish, showing any symptoms, were transported to the laboratory under refrigeration (2 ºC -8 ºC) and opened aseptically.Sampling was carried out mainly from headkidney, but sometimes from the spleen, liver and the brain (especially in small fry).Tryptic Soy agar (Oxoid), Tryptic Soy Broth (TSB Oxoid) enriched with 2% NaCl (TSAS, TSBS), Marine agar (MA, Difco) and Thiosulfate-Citrate-Bile salt-Sucrose agar (TCBS, Oxoid) were used as culture media for the bacteria.All inoculated culture media incubated at 22 º C for 2-5 days.A representative number of different types of colonies detected on culture media was collected from plates and streaked on TSAS plates for purity and to carry out identification.Pure cultures of the isolates, obtained by repeated plating on TSAS, were identified by biochemical characterization.Bacteria, identified as Listonella anguillarum and Photobacterium damselae subsp.piscicida,were confirmed serologically by agglutination test (system BIONOR).

Identification of bacteria
When identifying bacteria, certain characteristics are selected and used for this purpose.Pure cultures of the isolates, obtained by repeated plating on TSAS are used for identification.Primary identification usually involves simple tests such as morphology, motility, growth on various types of culture media, catalase and oxidase tests.Gram staining reveals the morphology and divides bacteria in two categories -the Gram-positive and the Gram-negative bacteria.For morphological appearance it is preferable to examine young cultures from non-selective media.Bacterial colonies of a single species, when grown on specific media under controlled conditions are described by their characteristic size, shape and pigment.The fermentation of glucose may also be used to distinguish between groups of bacteria.Using these few simple tests it is usually possible to place bacteria, provisionally, in one of the main groups.Then the isolated bacterium is subjected to a battery of tests.Some of them are found in commercial identification systems.

Growth of bacteria at different temperatures
The growth medium used, for these tests, was the Tryptic Soy Broth with 2% NaCl.The temperatures chosen were 4, 35 and 40 º C for 7, 2 and 1-2 days.The growth of microorganisms at different temperatures is used as an identification key.For example Vibrio aestuarinus does not grow at 40 º C but Vibrio alginolyticus and V.parahaemolyticus can grow.

Growth of bacteria in different concentration of sodium chloride
The concentrations of sodium chloride were selected 0,3,6,8 and 10%.The growth medium used for these tests was the Tryptic Soy Broth with 2% NaCl.This culture medium is a differential medium because it allows the investigator to distinguish between different types of bacteria based on the trait of NaCl tolerance.Thus a selective, differential medium for the isolation of Vibrio alginolyticus, contains a high concentration of salt (10%) that inhibits the growth of Vibrio harveyi (it grows until 8% NaCl).

Using the system API 20E (Biomerieux)
Many commercially available diagnostic kits have been introduced into routine laboratory diagnostics of fish pathogens, such as API 20E, API ZYM, API 20NE, API 50 CH, API Rapid ID 32 (bioMerieux,Marcy-l'Etoile, France), Biolog MicroPlatesGN2, GP2, AN (Biolog, Inc., Hayward, CA, USA), Enterotubes, BBL Crystal E/NF (Becton-Dickinson& Company, Franklin Lakes, NJ, USA), and some others.Of these, API 20E rapid identification system has been the most widely used for identification of fish pathogenic bacteria (Popovic et al., 2007).The API-20E test kit, a very specific means of bacterial identification (for Enterobacteriaceae and other non-fastidious Gram-negative rods), is a collection of mini test tubes, each with a reagent that test for a different aspect of bacterial metabolism.It provides an easy way to inoculate and read tests.After incubation with an unknown Gram-negative bacterium, the interpretation of positive and negative tests allows for identification to the species level.The preparation of inoculum for the API 20E, was the addition of pure-developed colonies in 5 ml of 2% NaCl.The solutions used for inoculation of cells as indicated respectively by the manufacturer.The inoculation of cells of the tiles and the addition of paraffin oil was made with the help of sterile pipettes volume of 1ml, under sterile conditions.Incubation of plates was at 22-25 º C for 24-48 h.Reading the results was made according to the instructions.It is known that the information from the database of microorganisms API20E identification system is based on results from the study of human clinical strains.For this reason, the identification through the database API 20E system was not used in mind completely.The results of the reactions, however, were considered for identification of bacteria.

Using the system API Staph (Biomerieux)
For identification of Gram-positive cocci, that give a positive reaction of catalase and negative oxidase reaction, the Api Staph identification system was used (Biomerieux) according to manufacturer's instructions.Staphylococci are gram-positive cocci occurring most often in irregular "grape-like" clusters.API-Staph consists of a strip containing dehydrated test substrates in individual microtubes.The tests are reconstituted by adding an aliquot of the API-Staph medium to each tube inoculated with the strain to be studied.The tests included acid production from d-glucose, d-trehalose, d-mannitol, d-mannose, xylose, maltose, lactose, sucrose, N-acetylglucosamine, raffinose, d-fructose, d-melibiose, xylitol, and α-methyl-glucosamine; nitrate reduction; and alkaline phosphatase, arginine dihydrolase, urease, and acetoin production.Coagulase production is a significant additional key.It is the ability to clot plasma.A rapid slide coagulase test may be performed.Most Staphylococcus species isolated from fish are coagulase negative.The data have been processed using the software program ApiLab Plus.API Staph (Biomerieux) is an established identification system for Staphylococci, used for many years.

Identification of isolated bacteria: Present work
Pure cultures of the isolates, obtained by repeated plating on TSAS, were identified by biochemical characterization following the criteria described by Austin et al., 1995.Biochemical keys, described by Alsina & Blanch, 1994a, 1994b, were used for identification of Vibrio species.These biochemical keys are designed for environmental and clinical isolates and can be used for strains that are Gram negative, giving a positive oxidase reaction, grow in TCBS medium.Some trials were included in the system identification Api 20E.The following biochemical and morphological tests were made: Gram stain, cell morphology, oxidase reaction, catalase reaction, motility, test glucose O-F, growth in TCBS agar, swarming ability of colonies in TSAS, growth in 0, 3,6,8 and 10% NaCl, growth at 4 º C, 35 º C, 40 º C, resistance at 10 and 150 mg O/129, resistance to 10μg ampicillin and tests of API 20E system as the ortho-nitro-phenyl-BD-galaktopyranozidium, hydrolysis of arginine, the decarboxylation of lysine and ornithine, assimilation of citrate, production H 2 S, production of urease, deamination of tryptophan, indole production and aketoinis, hydrolysis of gelatin, fermentation of glucose, mannitol, inositol, sorbitol, rhamnose, sucrose, meliviozis, amygdalin and arabinose as carbon sources.Two thousands one hundred twenty four bacterial isolates obtained from diseased farmed and wild fish species in Greece were identified.During the nine years (1997)(1998)(1999)(2000)(2001)(2002)(2003)(2004)(2005) (Yiagnisis and Athanassopoulou, 2011).
Listonella anguillarum was the most frequent species isolated from farmed sea bass during the 9-year sample period.

Bacteria isolated from gilthead sea bream (Sparus aurata)
Figure 2.5.2.1 shows the isolation rates of bacterial groups from diseased farmed gilthead sea bream in Greece from 1997-2005 (Yiagnisis and Athanassopoulou, 2011).The lower numbers of bacteria from diseased farmed fish were isolated during the winter.These results are in agreement with those of other researchers (Company et al., 1999) where in a bacteriological and parasitological study of farmed dentex conducted in the Mediterranean region, it was reported a relationship between high mortality and high temperature water.These results however are not in agreement with the results of Zorilla et al, 2003, who (Yiagnisis and Athanassopoulou, 2011).
M o s t o f t h e b a c t e r i a , i s o l a t e d f r o m b o t h farmed and wild fish, were Gram-negative.Specifically 77% (for farmed sea bass), 73% (for farmed sharpsnout sea bream) and 48% (for farmed sea bream) of the isolated bacteria were identified as Vibrio species (including Listonella anguillarum).For farmed species Sparus aurata (sea bream) and Diplodus puntazzo (sharpsnout sea bream) the most frequent type was Vibrio alginolyticus.This high incidence of Vibrio spp.have was found in previous studies done in Spain by Balebona et al, 1998b andZorilla et al, 2003.Vibrio alginolyticus isolated frequently with other Vibrio species and Photobacterium damselae subsp.piscicida.In fact, from our results it appears that the incidence of Vibrio alginolyticus in sea bream and sea bass is similar to that of Photobacterium damselae subsp.piscicida.V. alginolyticus was isolated from water of fish farms and live food.Other authors have reported the isolation of this species from water aquaculture (Angulo et al., 1993, Blanch et al., 1997).

Conclusion
European sea bass (Dicentrarchus labrax) is the fish species with the majority of isolated bacterial strains in this study.Bacterial species most frequently isolated from sea bass are Listonella anguillarum, Vibrio alginolyticus, Photobacterium damselae subsp.piscicida, Vibrio splendidus II and Vibrio parahaemolyticus.Listonella anguillarum is the main bacterial species isolated.L. anguillarum constitute 26% of total bacteria isolated from diseased sea bass, with the greater isolation frequency occured in April.Photobacterium damselae subsp.piscicida is an obligate fish pathogen, isolated also from sea bass fry.Vibrio alginolyticus is an opportunistc vibrio, isolated from sea bass.Photobacterium damselae subsp.piscicida and Vibrio alginolyticus increased frequencies of isolation were observed in September.Another opportunistic vibrio, Vibrio splendidus II is isolated mainly from sea bass fry and its greater isolation

Table 2 .
a total of 2124 strains of bacteria were isolated from 430 cases, as shown in Table2.5.1.5.1 Origin and number of 2124 identified bacterial isolates from diseased farmed and wild fish in Greece(Yiagnisis and Athanassopoulou, 2011).Bacteria Isolated From Diseased Wild and Farmed Marine Fish in Greece 157 Bacteria were identified initially at the genus level and then at the species level.Vibrio splendidus II has the highest incidence in fry.The majority of bacteria were isolated during spring, as season but on September, as month (Figure2.5.1.2).As shown in Figure2.5.1.3,the greater isolation frequency of Listonella anguillarum occurred in April while the greater isolation frequency of Vibrio splendidus II occurred in May and Photobacterium damselae subsp.piscicida and Vibrio alginolyticus increased frequencies of isolation were observed in September.For Vibrio parahaemolyticus the most observed incidence was in October.It is observed that the bacterium Vibrio alginolyticus was isolated simultaneously with Photobacterium damselae subsp.piscicidae.
www.intechopen.comDifferentspecies of Vibrios together with Listonella (Vibrio) anguillarum constitute 77% of total bacteria isolated from diseased European sea bass.The percentage of isolated Photobacterium spp. is 17% while Photobacterium damselae subsp.piscicida is the main species.The remaining groups of bacteria were isolated at low rates.Table2.5.1.1 shows the bacteria isolated from sea bass divided into age groups.Bacterial species most frequently isolated from sea bass are Listonella anguillarum, Vibrio alginolyticus, Photobacterium damselae subsp.piscicida, Vibrio splendidus II and Vibrio parahaemolyticus.L. anguillarum is the most frequent bacterial isolated species from European sea bass.L. anguillarum and Photobacterium damselae subsp.piscicida were isolated mainly from larger fish.
Number of bacterial strains isolated from diseased farmed gilthead sea bream in correlation to the month of isolation(Yiagnisis and Athanassopoulou, 2011).In Figure2.5.2.1 it is observed that different Vibrio species along with Listonella anguillarum constitute 48% of total bacteria isolated from diseased sea bream.The percentage of isolated Photobacterium spp. is 34% while Photobacterium damselae subsp.piscicida is the main species.The incidence of Aeromonas spp. was 9%, of Pseudomonas spp.3%, of Shewanella putrefaciens 2%, of Gram positive cocci 3% and of Cytophaga like bacteria 1%.As it is shown in the table 2.5.2.1, the majority (692) of total isolated bacteria (887) belongs to sea bream fry.Bacteria with the highest incidence in the sea bream is Photobacterium damselae subsp.piscicida,Vibrioalginolyticus,Photobacteriumdamselaesubsp.damselae,Vibriocosticola,VibriosplendidusII, Vibrio parahaemolyticus and Vibrio vulnificus.Listonella anguillarum was isolated from fry only, with very low frequency.The majority of bacteria were isolated during summer, especially June (Figure2.5.2.2).Figure2.5.2.3 shows the number and species of bacterial species, most frequently isolated from sea bream in relation to month of isolation.Vibrio alginolyticus, Photobacterium damselae subsp.piscicidae, Photobacerium damselae subsp.damselae, Vibrio vulnificus, Vibrio parahaemolyticus and Vibio costicola are most frequently isolated from diseased farmed sea bream in the summer and Vibrio splendidus II in May.It is observed that the bacterium Vibrio alginolyticus was isolated almost simultaneously with Photobacterium damselae subsp.piscicida, as in sea bass.

3 Bacteria isolated from sharpsnout sea bream (Diplodus puntazzo)
in a bacteriological study of farmed sea bream (Sparus aurara, L.) held in southwest Spain reported the lower numbers of bacterial isolates during summer.Figure2.5.3.1 shows the isolation rates of bacterial groups from diseased farmed sharpsnout sea bream in Greece from 1997-2005.It is observed that different Vibrio species along with Listonella anguillarum constitute 73% of total bacteria (99) isolated from diseased sharpsnout sea bream.The percentage of isolated Photobacterium damselae subsp.damselae is 12% while Vibrio alginolyticus is the main isolated species.The incidence of Aeromonas spp. was 4%, of Pseudomonas fluorescens/putida 4% and of Staphylococcus spp.7%.The table 2.5.3.1 shows the bacteria isolated from sharpsnout sea bream divided into age groups.Most of bacterial strains have been isolated from larger fish than fry.

Table 2 .
5.5.1.Number of bacterial strains, isolated, from diseased wild fish species in relation to month of isolation