Journal of Ocean University of Qingdao (Oceanic and Coastal Sea Research) Review ISSN 1671-2463, October 31,2003, Vol.2, No.2, pp.117-128 http:// www. ouc. edu. cn/xbywb/ E-mail: xbywb@mail, ouc. edu. cn

Pathogenicity of in Fish: an Overview

LI Jun, Norman Y.S. Woo*

Department of Biology, The Chinese University of Hong Kong, Shatin, N. T., Hong Kong SAR , P. R. China

(Received ,July 8, 2003; accepted August 28, 2003)

Abstract of the genus are ubiquitously distributed in the marine environment. Due to the rapid expansion of intensive mariculture and the consequent deterioration of culture conditions, more and more Vibrio spp. have been recog- nized as pathogenic agents in outbreaks of vibriosis, a serious epizootic disease affecting most wild and farmed fish species worldwide, which has become the most important limiting factor for the development of intensive mariculture industry. At- tempts have been made to understand the pathogenicity of vibrios in host fish with the ultimate aim of elucidating the best means for disease control. After an extensive literature survey of the recent advances in the field of fish vibriosis, the patho- logical changes, virulence factors and associated potential pathogenic mechanisms, transmission routes and related environ- mental factors involved in outbreak of vibriosis, as well as the controlling strategies are reviewed in the present paper.

Key words Vibrio spp.; vibriosis; pathogenicity; fish

Number ISSN 1671-2463(2003)02-117-12

et al., 2003). Based on an extensive literature survey, 1 Vibriosis and Associated Vibrio Spe- details of potential pathogenic vibrios and associated susceptible fish hosts are compiled and presented in cies in Cultured Fish Table 1. Vibrio species are the most dominant heterotrophic V. anguillarum (now as Listonella anguillarum ) bacteria in the marine environment and are widely dis- is by far the best known and widespread of all fish tributed in the coastal seawaters and/or brackish wa- pathogens, and is responsible for the majority of fish ters. They are also found on the surface and/or in the loss worldwide (Austin and Austin, 1993). For ex- gastrointestinal tract of marine animals or other organ- ample, vibriosis due to V. anguillarum has been de- isms (Colweli and Grimes, 1984; Austin and Austin, scribed in over 40 fish species, both wild and cul- 1993). Due to the rapid expansion of intensive mari- tured, throughout the world (Anderson and Conroy, culture and the consequent deterioration of culture 1970). In addition to V. anguillarum, V. damsela, conditions, vibriosis, the disease caused by a bacteri- V. vulnificus, V. salmonicida and V. harveyi have al- um of the genus Vibrio, occurs frequently worldwide, so been recognized to be severely pathogenic to a wide which affects a large number of fish species (Austin range of fish species (see Table 1), more details of and Austin, 1993). which have been reviewed elsewhere (Egidius, 1987; In their review, Colwell and Grimes (1984) considered Austin and Austin, 1993 ; Thune et al., 1993 ; Toranzo V. alginolyticus, V. parahaemolyticus, V. and Barja, 1993). (non-O1), V. vulnificus (Biotype 2), V. anguilla- V. alginolyticus is another major fish pathogen rum, V. ordalii, V. damsela, V. carchariae and V. within the genus Vibrio and has caused severe vibrio- salmonicida as fish pathogens. Since then, more and sis with massive mortalitiy in various fish species more pathogenic Vibrio species, including V. harveyi, throughout the world (Colorni et al., 1981 ; Austin et V. marinus, V. furnissii, V. rnimicus, V. pelagius, al., 1993; Lee, 1995; Saeed, 1995; Woo, 1995; AI- V. splendidus and V. tapetis, have been reported in varez et al., 1998; Balebona et al., 1998; Zhu et al., relation to epizootic diseases of various fish species 2000). However, the virulent properties of V. algi- (Austin and Austin, 1993; Angulo et al., 1994; Es- nolyticus to fish still cannot be firmly established be- teve et al., 1995; Saeed, 1995; Benediktsdottir et aL, cause virulence appears to vary from species to species, 1998; Alvarez et al., 1998; Diggle et aL, 2000; Wu and in some cases, virulence even varies within the and Pan, 1997, 2000; Villamil et al., 2003a; Jensen same fish species. Moreover, the onset of vibriosis caused by this bacterium is always associated with de- Corresponding author. E-mail: normanwoo@ cuhk. edu. hk teriorating culture conditions or physical damage of Tel : 852-2609-6148 ; Fax : 852-2603-5646 cultured fish; and therefore it is always considered as 118 Journal of Ocean University of Qingdao 2003, Vol. 2, No. 2 an opportunistic pathogen (Colorni et al., 1981; Aus- tin et al., 1993; Balebona et al., 1998).

Table 1 Vibrio species and associated susceptible fish Vibrio species Susceptible fish References Grouper, Epinephelus alabaricuss Lee, 1995 Gilthead sea bream, Sparus aurata Balebona et al., 1998 V. alginolyticus Turbot juvenile, Scophthalmus maccirnus Austin et al., 1993 Silver sea bream, Sparus sarba Li et al., 1998; Li, 2002 V. alginolyticus V. parahaernolyticus Silver sea bream, Sparus sarba Li et al., 1999 V. vulnificus Woo et al., 1995 alginolyticus Grouper, Epinephelus salmoides Ong, 1988 V. parahaemolyticus V. alginolyticus parahaemolyticus Gilthead sea bream, Sparus aurata Colorni et al., 1981 V. anguillarum Rainbow trout, Salmo gairdneri Atlantic salmon, Salmo salar Tiainen et al., 1994 Sea trout Turbot, Scophthalmus maa'imus Coho salmon, Oncorhynchus kisutch Toranzo et al., 1987 Rainbow trout, Salmo gairdneri V. anguillarum Sea bream, Acanthopagrus cuvieri Rasheed, 1989 Turbot, Scophthalmus maMmus Coho salmon, Oncorhynchus kisutch Santos et al., 1991 Rainbow trout, Onco74~ynchus mykiss Rainbow trout, Sa[mo gairdneri Lamas et al., 1994 Atlantic salmon, Salmo salar Svendsen and Bogwald, 1997 Sea perch, Lateolabraz japonicus Xiao et al., 1999 V. anguillarum Pacific salmon Chart and Trust, 1984; V. ordalii Ransom et al., 1984 Summer flounder, Paralichthys dentatus Soffientino et al., 1999 V. carchariae Brown shark, Carcharkinus plumbeus Bertone et al., 1996 Grouper, Epinephelus coioides Lee et al., 2002 V. carchariae Brown shark, Carcharhinus plumbeus damsela Spiny dogfish, Squalus aczznthias Grimes et al., 1984, 1985 Lemon sharks, Negaprion brevirostris Turbot, Scophthalmus maMmus Fouz et al., 1992b Damselfish, Chromis punctipinnis Love et al., 1981 17. damsela Sea bream, Sparus aurata Vera et al., 1991 Yellowtail, Seriola quiniqueradiata Sakata et al., 1989 Sole, Solea senegalensis Zorrilla et al., 1999 V. furnissii European eel, Anguilla anguilla Esteve et al., 1995 Silver mullet, Mugil curema Alvarez et al., 1998 Atlantic spadefish, Chaetodipterus faber Silver black porgy, Acanthopagrus cuvieri Saeed, 1995 V. harveyi Brown spotted grouper, Epinephelus tauvina Rainbown trout, Salrno gairdneri Zhang and Austin, 2000 Atlantic salmon, Salrno salar Yellowtail, Seriola dumerili Wu and Pan, 1997 Sole, Solea senegalensis Zorrilla et al., 2003 !7. marinus Atlantic salmon, Salrno salar Benediktsdottir et al., 1998 mimicus Red sea bream, Pagrus major Wu and Pan, 2000 V. parahaemolyticus Iberian toothcarp, Aphanius iberus Alcaide et aL, 1999 V. pelagius Turbot, Scophthalmus maximus Villamil et al., 2003 a, b V. salmonicida Atlantic salmon, Salmo salar Totland and Nylund, 1988 Turbot, Scophthalmus maMmus Angulo et al., 1994 V. spIendidus ( I ) Rainbow trout, Oncorhynchus mykiss !7. splendidus and Turbot, Colistium nudipinnis Diggles et al., 2000 V. campbellii-like Brill, Colistium guntheri V. splendidus and Corking-wrasse, Symphodus melops Jensen et al., 2003 V. tapetis Salte et al., 1994; V. viscosus Atlantic salmon, Salmo salar Lunder et al., 1995; Bruno et al., 1998; Greger and Goodrich, 1999 Grouper, Epinephelus sp. Liu et aI., 1994 Grouper, Epinephelus awoara Qin and Pan, 1996 V. vulnificus Eel, AnguilIa anguilla Tison et al., 1982; Dalsgaard et al., 1999 European eel, Anguilla anguilla Biosca et al., 1991; Amaro et aL, 1995; Collado et aL, 2000; Fouz et al., 2000 LI J. et al.: Pathogenicity of Vibrios in Fish : an Overview 119

In the coastal regions of China, outbreaks of serious hatchery-reared turbot (Colistium mudioinnis ) and vibriosis caused by V. alginolyticus (Lee, 1995; Woo brill (C. guntheri) infected respectively by V. splen- et al., 1995; Li et al., 1998; Li, 2002), V. anguil- didus and V. campbellii-like variants (Diggles et aL, larum (Xiao et al., 1999), V. carchairae (Lee et aZ., 2000). In most of these cases, numerous rod-shaped 2002), V. harveyi (Wu and Pan, 1997), V. mimicus bacteria were found in the sections of affected kidney (Wu and Pan, 2000), and V. vulnificus (Liu et aL, and liver under electron microscopy. 1994; Qin and Pan, 1996) have been reported in cul- In addition to live vibrio, the extracellular products tured marine fish, such as sea bream, sea perch, (ECPs) secreted by Vibrio spp. are also toxic and grouper and yellowtail (Seriola dumeril) (Table 1). generally cause similar pathological changes as those caused by live bacteria (De la Cruz and Muroga, 1989; Fouz et al., 1992a; Zhang and Austin, 2000). How- 2 Pathological Changes of Infected Fish ever, in the study of Fouz et al. (1995), it was found 2.1 Clinical Symptoms that injection of the ECPs of V. damsela elicited more Although the causative pathogen in different out- rapid and severe histopathological changes than injec- break episodes of vibriosis may be different in various tion of live cells. However, Lamas et al. (1994) found fish species, the diseased fish always exhibit similar that injection of live V. anguillarum into rainbow clinical symptoms (vibriosis syndrome). Most of the trout caused more acute lesions than injection of ECPs infected fish display darkened body coloration, loss of because the bacteria multiplied extensively in the kid- appetite and exhibit abnormal swimming behavior ney and spleen. with head floating near the surface of water. Severely From the above description, it can be concluded infected fish display serious symptoms of vibriosis such that the spleen, kidney and liver are the main target as skin ulceration, hemorrhage at fin base and body organs of vibrio infection (Ransom et aL, 1984; Bru- surface, rotted fins and tails, and abdominal disten- no et al., 1986; Lamas et al., 1994). The severely sion. Internally, the liver is pale and with petechiae, infected organs are the principal sites of proliferation and the kidneys and spleen are congested. In most of bacteria and subsequent accumulation of toxins cases, accumulation of reddish ascitic fluid is also ob- (Fouz et aL, 1995). In Atlantic salmon (Salmo sal- served in the peritoneal cavity of severely infected fish at) suffering from cold water vibriosis, a close rela- (Toranzo et al., 1987; Rasheed, 1989; Fouz et aI., tionship between the number of bacteria detected in 1992b; Li et aL, 1998, 1999; Diggles et al., 2000). the tissues and the degree of ultrastructural damage Very often, the exact clinical symptoms exhibited by has been clearly demonstrated (Totland et al., 1988). the infected fish mostly depend on the severity of the Pronounced organ damage may seriously affect normal disease and the virulence of the pathogenic agents. physiological processes, such as osmoregulation in kid- ney and metabolism in liver of the infected fish. 2.2 Histopathological Changes 2.3 Hematological Changes Infected fish generally exhibit marked pathological symptoms, and lesions are particularly prominent in Changes in hematological parameters have been tissues such as muscle, liver, kidney, spleen, intesti- widely used for assessing the health status Of fish in nal tract and brain (Egidius, 1987; Austin and Aus- response to microbial infections. Significant decreases tin, 1993; Fouz et al., 1995). For example, Love et al. in red blood cell (RBC) counts, hematocrit and hemo- (1981) demonstrated strong muscle lysis in the gradu- globin concentrations are often characterized in severe- lomatous ulcerative lesions and presence of histiocytes ly diseased fish upon either natural or experimental in- in the dermis and skeletal muscles of damsel fish natu- fection of vibrios (De la Cruz and Muroga, 1989; Ma- rally infected by V. damsela. Histopathological exami- honey and McNulty, 1992; Austin et aL, 1993; La- nation of the kidneys revealed that the glomeruli were mas et al., 1994; Li, 2002). Similarly, significant extensively infiltrated with blood cells and the archi- lower levels of RBC counts, hematocrit and hemoglo- tecture of the tubules totally disrupted, changing the bin values were also recorded in fish which received in- shape of the renal cell from cylindrical to cuboidal. jection of ECPs, suggesting that the ECPs play impor- The hepatic sinusoids were also infiltrated with eryth- tant roles in the hemolysis of blood cells (De la Cruz rocytes, and severe necrosis was found in the intersti- and Muroga, 1989; Mahoney and McNulty, 1992; tial tissues of both kidney and liver. In the seabream Lamas et aL, 1994; Zhang and Austin, 2000). De- ( Acanthopagrus cuvieri ) infected by V. anguiHarum tails about the hemolytic mechanisms of ECPs and (Rasheed, 1989), typical histopathological lesions, their roles in the pathogenesis of vibriosis will be dis- such as necrosis and atrophy of hepatocytes, necrosis cussed in the following section. In addition, the erythro- of sheathed arteries in the spleen and necrosis of renal cyte sedimentation rate increased significantly in fish tubules and glomeruli in the kidney occur. Significant that had been injected with live vibrios or their ECPs pathological lesions in the liver, kidney, stomach, in- (Lamas et al., 1994). Extensive damage of vascular testine, brain, and spinal cord were also observed in tissues, especially those with rich blood supply has al- 120 Journal of Ocean University of Qingdao 2003, Vol. 2, No. 2 so been observed in the vibrio-infected fish (Totland kidney and spleen, as well as intestine (Muiswinkel, et al., 1988; Fouz et al., 1995). 1992; Zapata etal., 1996; Dalmo etal., 1997). Leu- In fish, microbial infection always results in a sig- kocytes associated with immune function include lym- nificant elevation in total white blood cell (WBC) cou- phocytes (B-cells and T-cells), phagocytes (granulo- nts (Haney et al., 1992; Pathiratne and Rajapakshe, cytes, thrombocytes and macrophages), and some 1998). However, little information is available on the non-specific cytotoxic cells (Scapigliati et aL, 1999). leukocyte changes of diseased fish associated with vibrio- The severely damaged lymphoid organs and hemopoi- sis. In our recent study on vibriosis of sea bream, sig- eric tissues in infected fish are expected to invoke con- nificant increase in total leukocyte count was observed siderable changes in immunological function (Fouz at the early stages of infection, but leukocyte count et al., 1995). declined significantly during the advanced infective Macrophage phagocytosis is considered as the first stages. Similar results were also obtained for fish chal- and often the most important immunological response lenged by experimental inoculations of V. aZgino- in teleost fish in response to the invasion of pathogenic lyticus or its ECPs (Li, 2002). In the same study, microorganisms (Secombes and Fletcher, 1992). The the percentage of lymphocyte within the total WBC invasion of microorganisms causes an inflammatory re- population significantly declined in seabream experi- action in the fish, and both tissue maerophages and mentally injected with vibrio, in accordance with the circulatory monocytes migrate to the invasive sites to results obtained from other severely infected fish spe- phagocytose and destroy the intruders. Other phago- cies (Haney et aL, 1992; Pathiratne and Rajapakshe, cytes such as neutrophils and granulocytes are also at- 1998). Significant changes were observed in eosino- tracted to the abrasion sites, where they release de- phil counts, but these changes were opposite in natu- structive enzymes for lysis of invasive microorganisms rally infected fish and experimentally infected fish. (Anderson, 1990). However, most studies on the de- Moreover, an elevation of monocyte count in vibrio- fensive mechanism of phagocytes are obtained from the infected sea bream has also been demonstrated (Li, responses of fish to inoculation of killed bacteria or 2002). However, the exact roles of these differential other avirulent antigens (Sakai et al., 1991), and lit- ieukocytes in the defensive response of sea bream are tle is known about the phagocytic responses to viable still unknown. bacterial cells in fish. In our recent report, clear evi- In addition to the changes in blood cells, bacterial dence has been obtained from phagocytic activity of disease in fish commonly results in major alterations in macrophage isolated from the pronephros and spleen of blood biochemical composition (Iwama et aZ., 1986; vibrio-infected silver sea bream; phagocytic activity Moyner, 1993), a phenomenon that is a reflection of was significantly stimulated at the early stages of in- metabolic and immune dysfunction. Haney et al. (1992) fection, but declined significantly in moribund fish reported that Chum salmon (Oncorhynchus keta ) in (Deane et al., 2001). Similar changes have been dem- response to severe viral infection showed a significant onstrated in fish that received experimental inocula- decrease in pIasma glucose levei. Similarly, signifi- tions of V. a[ginolyticus or its toxic ECPs (Li, cantly lower level of serum glucose was demonstrated 2002). More recently, significant alteration of mac- in sea bream infected by vibrios, regardless whether rophage phagocytic activities has been reported in tur- the infection was induced naturally or experimentally bot experimentally infected by pathogenic V. pelagius (Li, 2002). In addition, significant decrease in serum (Villamil et aL, 2003b). Once the first line of defens- protein has been reported in fish during infection with es is broken down, invasive bacteria may quickly gain live vibrios or ECPs (De la Cruz and Muroga, 1989; control, multiply, and ultimately kill the fish. Signif- Mahoney and McNulty, 1992; Li, 2002). However, icant increase in bacterial numbers was demonstrated there are studies in which no significant effect or ele- in both spleen and head kidney, which paralleled the vation of serum protein level in vibrio-infected fish has decline in macrophage phagocytosis of the injected fish been reported (Lamas et al., 1994). These diverse re- (Li, 2002). sponses of fish to vibrio infection indicate that differ- In addition, the humoral immune response is also ent pathogenic mechanisms are involved. Therefore, important in the defense against microbial infection; characterization of serum proteins, especially the changes however, activation of the specific immune system and in total protein level and protein pattern in infected secretion of specific antibodies into serum and/or mu- fish could provide insight into the underlying mecha- cus are time consuming. Thus, significant elevation of nisms of pathogenesis of vibrio. immunoglobulin M (IgM) level was seen in salmonids chronically infected by Aeromonas salrnonicida ( Oles- en and Jorgensen, 1986 ; Magnadottir and Gudmunds- 2.4 Immunological and Hormonal Changes dottir, 1992). However, in the case of acute infec- It has been well recognized that the immune system tion, IgM level was greatly reduced in carp which had of teieost fish is composed of lymphoid organs/tissues been experimentally injected with A. salmonicida and functional immune leukocytes. The principal lym- (Evenberg et al., 1986). Despite these reports, de- phoid organs/tissues in adult teleost fish are thymus, tails on the specific immune responses in fish to vibrio LI J. et al.: Pathogenicity of Vibrios in Fish : an Overview 121 infection remain unclear. 1998; Wang and Leung, 2000). The hydrophobicity Hormonal modulatory mechanisms are essential of the microbial surface has been suggested to be a de- physiological processes for fish to regain homeostasis termining factor in the adherence of bacteria to host when they contract disease. There have been a num- surface, which especially enables the bacteria to inter- ber of studies attempting to associate the diseased con- act with animal cells and to survive within the host dition with altered hormonal responses, although the (Lee and Yii, 1996; Balebona et al., 1998). For ex- association is far from conclusive (Haney et al., 1992; ample, in V. salmonicida, the presence of a hydro- Mesa et aL, 1998). More recently, our laboratory has phobic surface antigen, called VS-P1 (40 kd), has been reported changes in circulating levels of several key described as a possible virulence determinant protect- hormones in response to vibriosis in silver sea bream ing the bacteria against the bactericidal activity of host (Deane et aL, 2001). It is clear that circulating levels serum (Espelid et al., 1987; Hjelmeland et al., 1988). of cortisol and thyroid hormones are only significantly Similarly, it was reported that the hydrophobic nature changed in moribund fish, whereas testosterone and of the cell surface would be advantageous to pathogen- estradiol are significantly altered at early stages of vibri- ic V. anguillarurn, allowing its survival and multipli- osis (Deane et aL, 2001). Similarly, Mesa et al. (1998) cation in tissues of diseased fish (Horne and Baxen- also reported that plasma cortisol is substantially ele- dale, 1983). However, Santos et al. (1991) found that vated during the later stages of bacterial kidney disease hydrophobic interactions were not essential for the col- in juvenile Chinook salmon. onization in host tissues by fish pathogens. Similar ob- Ample evidence is available for a role of hormones in servations showing that a lack of a direct relationship modulating immune function in fish (Narnaware and between cell surface properties and virulence of fish Woo, 1999; Harris and Bird, 2000). It has been well pathogen have been reported in V. alginolyticus (Bale- documented that steroid hormones, such as estradiol, bona et al., 1998) and V. harvyei (Lee and Yii, 1996). testosterone and glucocorticoids are immunosuppressive Such discrepancies indicate the unreliability of these in fish (see review of Narnaware and Woo, 1999). properties as virulence markers in seroepizootiological High levels of circulating steroid hormones have been studies, unless experimental protocols are standardized demonstrated to affect lymphocyte proliferation, de- (Toranzo and Barja, 1993). creasing the number of antibody-producing cells and Endotoxins (lipopolysaccharides, LPSs), the main other cellular functions of the immune system, and cell-wali components of Gram-negative bacteria, cont- subsequent immunosuppression and further decrease of ribute to the toxicity of V. anguillarum (Aoki et al., disease resistance may result (Maule et al., 1989; 1985). However, different investigators have report- Anderson, 1990; Wang and Belosevic, 1994). For ed contradictory results regarding the properties of en- example, administration of physiological levels of cor- dotoxins from V. anguillarum in different fish species tisol causes a clear reduction in circulating thrombo- (see review of Toranzo and Barja, 1993). Therefore, cytes and lymphocytes, and increases the susceptibility more evidence is needed to determine the role of bacte- of Atlantic salmon, Salrno salar, to V. samonicida rial endotoxins in the pathogenesis of vibrio. (Wiik et aL, 1989). Direct evidence of the immuno- In addition, it has been reported that the multifla- supressive effects of serum cortisol on non-specific im- gella of V. anguillarum enable it to locate, adhere mune responses has been clearly obtained in diseased to, and penetrate mucosal surfaces, all of which cont- silver sea bream (Deane et aL, 2001). We found that ribute to the high virulence of V. anguillarum to fish during the progression of vibriosis, from mild to se- (Chart, 1983). vere infection, serum cortisol remained unchanged, whereas the phagocytic indices for both spleen and 3.2 Iron Uptake Systems pronephros macrophages increased. For moribund fish Iron is an essential element for growth and multipli- these indices were significantly decreased from stimu- cation of microorganisms, but its concentrations are lated levels in parallel with a substantial elevation in very low in the host fluids and most of the iron is fre- serum cortisol. In addition, significant decrease of Ig quently chelated with proteins, such as transferrin and M levels in both plasma and skin mucus caused by ad- lactoferrin (Otto et al., 1992). As a result, iron ac- ministration of sex steroid hormones and/or cortisol quisition mechanisms are generally present in microor- has been reported in rainbow trout (Hou et al., 1999) ganisms, enabling them to obtain the necessary iron and masu salmon (Nagae et aL, 1994). from the iron-binding proteins. High affinity iron up- take systems are thus well recognized as important vir- 3 Virulence Mechanisms ulence factors in various and fish pathogenic vibrios (Crosa, 1980, 1984; Toranzo and 3.1 Bacterial Surface Properties and Endotoxins Barja, 1993). Adhesion of bacteria to host surface has been de- The iron uptake mechanism of V. anguillarum has scribed as one of the initial steps in microbial patho- been well investigated (Crosa et al., 1980, 1984; Tol- genesis (Home and Baxendale, 1983; Wang et aL, masky et al., 1985). In some strains of V. anguillarum, 122 Journal of Ocean University of Qingdao 2003, Vol. 2, No. 2 the iron uptake system is composed of an iron chela- osis in several fish species (Lee, 1995; Farto et al., tor, siderophore (named anguibactin), and a 86 kDa 2002). receptor protein located on the outer membrane of the Hemolytic and cytotoxic activities of ECPs have also cell wall (called OM2). Both the siderophore and the been considered as important virulence factors contrib- receptor are coded for by a 47 MDa plasmid (pJM1) uting to the pathogenicity of the infection process (Es- (Crosa, 1980 ; Tolmasky et al., 1985 ; Conchas et al., teve et al., 1995; Biosca and Amaro, 1996; Zhang 1991). However, in those pathogenic strains that lack et al., 2001). Marked hemolytic activity has been this plasmid, a different siderophore and different clearly demonstrated in the ECPs in vitro and in vivo, iron-regulated membrane proteins that are encoded by a phenomenon which is responsible for the severe chromosomal genes are found (Lemos et al., 1988). hemorrhaging symptoms found in vibrio-infected fish The presence of siderophores has also been reported to (Thune et aL, 1993; Fouz et aL, 1994; Lamas et aL, be related to the pathogenicity of other fish pathogenic 1994 ; Biosca and Amaro, 1996 ; Balebona et al., 1998 ; vibrios, including V. ordalii, V. vulnificus, and V. Zhang and Austin, 2000; Li, 2002). Regarding the aIginolyticus (Biosca et al., 1996; Balebona et al., cytotoxic activity present in the ECPs, its precise con- 1998). However, V. anguillarum is currently the tribution to the pathogenicity of vibrios is still ob- only fish pathogen, and it has been clearly demon- scure, because cytotoxicity of the ECPs is highly de- strated that the ability to scavenge iron from the host pendent on the cell lines chosen for the test (Toranzo is a crucial virulence determinant (Tolmasky and Cro- et al., 1987). Moreover, cytotoxicity to fish cell lines sa, 1984; Wolf and Crosa, 1986). has also been reported in the ECPs isolated from some In addition, V. anguillarum is known to acquire non-pathogenic reference strains (Toranzo et al., 1983). iron from other sources, such as hemoglobin and free For example, V. salmonicida and V. ordalii are poor heme groups present in serum. These auxiliary iron- producers of proteases and hemolysins, and the mech- uptake mechanisms do not require the synthesis of sid- anisms of their hemorrhagic activity are still unknown erophores, and these mechanisms also contribute to (Toranzo and Barja, 1993). the virulence of this pathogen (Mazoy et aL, 1992). However, in vivo experiments indicated only those 4 Transmission Routes of Vibriosis strains that possess siderophore-mediated iron uptake systems could multiply in fish and cause mortality It has been well documented that water-borne infec- (Toranzo and Barja, 1993). tion is the primary mode of transmission for V. an- guillarum infestation in fish, and the skin and the in- testinal tract are the main portals of bacterial entry 3.3 Extracellular Products (Exotoxins) (Muroga and De la Cruz, 1987; Kanno et al., 1989; It has been well documented that extracellular prod- Spanggaard et al., 2000). In their review, Toranzo ucts (ECPs) play important roles in the pathogenesis and Barja (1993) have provided evidence that V. an- of vibrio in fish. The ECPs from various vibrios are guiZlarum was able to penetrate the host through the highly toxic to a great number of fish species and cause skin, fins, gills, and anus. In addition, a water trans- pathological changes which are similar to those elicited mission route of vibriosis has been substantiated in after inoculation of live bacteria (Inamura et al., 1984, other Vibrio species, such as V. vulnificus (Biotype 1985; Fouz et aL, 1992a; Lamas et aZ., 1994; Esteve 2) (Amaro et aL, 1995; Marco-Noales et al., 2001) et al., 1995; Lee, 1995; Biosca and Amaro, 1996; and V. damsela (Fouz et al., 2000) in which the bac- Balebona et al., 1998; Wang et al., 1998; Zhang and tericidal activity of the skin mucus has been overcome. Austin, 2000). Using different challenge methods, Nordmo et al. Regarding the pathogenic mechanisms of ECPs, (1997) demonstrated that V. salmonicida, the patho- proteolytic activity and various hydrolytic activities of gen of cold water vihriosis, could be transmitted them have been considered as important virulence fac- through water among Atlantic salmon ( Salmo salar). tors for the invasion, survival and proliferation of In addition, V. anguillarum was stable in seawater vibrios in host animals (Biosca and Amaro, 1996; and viable cells of this bacterium could be detected af- Balebona et al., 1998). For example, the ulceration ter one year (Toranzo et al., 1982; Hoff, 1989). and degradation of tissue surrounding the site of injec- Moreover, V. vulnificus (Biotype 2), the obligate eel tion could have been due to the hydrolytic action of pathogen, has been reported to be able to survive ECPs (Balebona et al., 1998). It was Inamura et al. alone in brackish water or attach to eel surface for at (1985) who first purified a 36 kDa protease from the least 14 days (Amaro et al., 1995). All these findings ECPs of V. anguillarurn and demonstrated its toxic suggest that the pathogens are able to spread over long properties by inoculation into goldfish and mouse. distances through water and infect healthy fish by us- Consectuently, similar toxic components have been pu- ing the skin as a portal of entry. rified from the ECPs of pathogenic V. alginolyticus Since the skin mucus possesses high bactericidal ac- (34kDa protease) and V. pelagius (39 kDa metallo- tivity (Harrel et al., 1976; Takashahi et al., 1987; protease) that were responsible for outbreaks of vibri- Fouz et al., 1990), both the skin and the mucous LI J. et al.. Pathogenicity of Vibrios in Fish : an Overview 123

layer on the skin surface form the first barriers against bream (Sparus sarba ) was postulated to be associated bacterial invasion. The integrity of skin/mucus barrier with overcrowding stress and an abrupt increase in is very important for protecting the fish against vibrio- seawater temperature (Li, 2002). We thought that sis. For example, Balebona et al. (1998) found that the overcrowding of fish in floating cages might lead to the mortality of gilthead sea bream when they were abrasions of the otherwise intact integument, and this challenged by V. alginolyticus by immersion depend- could trigger pathogen infestation. It has been gener- ed on the integrity of the fish skin. In their study, the ally accepted that higher water temperature enhances LD50 s were more than 2 • 10?cfu mL-1 for fish with specific immune responses, whereas lower temperature intact surface layers and less than 2 • 103cfu mL -1 for adversely affects immunoglobulin production in teleost fish with the mucus layer removed or the skin dam- fish (Bly and Clem, 1992; Morvan et al., 1997, 1998; aged. Similar results have been observed in silver sea Magnadottir et al., 1999). However, if the tempera- bream infected by V. alginolyticus (Li, 2002), in ture is beyond the physiological range of fish, immu- grouper (Epinephelus sp. ) infected by V. vulnificus nosuppresive effects will appear. On the other hand, (Liu et al., 1994), and in Atlantic salmon (Salmo higher seawater temperatures ( e.g. 28-30~ ) will tend salar ) infected by V. anguillarum and A. salmonici- to favor the multiplication of Vibrio spp. da (Svendsen and Bogwald, 1997). In addition, there are reports about Vibrio sp. invading the fish host 6 Strategies for Control of Vibriosis through skin lesions caused by ectoparasites (Grimes et al., 1985; Rodgers and Burke, 1988). These find- For the control of vibriosis, antibiotics and other ings indicate that fish skin and associated substances chemotherapeutic agents are commonly used in fish on the surface can keep the animal free from disease farms either as feed additives or as components in im- infection. mersion baths to achieve either prophylaxis or thera- Another mode of infection occurs through contami- py. For example, many antimicrobial compounds, in- nation of eggs of the parent host fish, an example be- cluding chloramphenicol, nitrofurazone, oxolinic acid, ing an outbreak of 'red head' disease caused by an un- oxytetracycline and sulphamerazine, have been proved identified Vibrio species in freshwater sheatfish (Si- to be very useful in controlling fish diseases (Austin lurs glanis). Alternatively, the authors also speculat- and Austin, 1993). Similarly, in our studies, about 51 ed that the bacteria may spread by direct contact when Vibrio isolates from diseased silver sea bream dis- fish fry were bathed in contaminated water (Farkas played similar drug-resistant patterns, and were sus- and Malik, 1986). ceptible to ceftriaxone, streptomycin, nalidixic acid and rifampicin (Li et al., 1999), suggesting these 5 Relationship between Outbreaks of Vibri- drugs could possibily be used for disease control in practice. However, extensive use of antibiotics and osis and Environmental Conditions other chemotherapeutic agents has resulted in an in- Although a pathogen possessing the virulence prop- crease in drug-resistant bacteria in aquatic environ- erties necessary to produce disease may be present in a ments. More and more antimicrobial compounds population of fish, the nutritional or environmental (amikacin, ampicillin, kanamycin, penicillin G, strep- conditions of the fish are usually more important than tomycin and tetracycline etc. ), which have been among other conditions in determining the severity of a dis- the most commonly used drugs in clinical and agricul- ease outbreak (Hedrick, 1998). Several excellent re- tural treatments, are now partially or completely inef- views are available to address the effect of environ- fective in controlling vibriosis, as well as other bacte- mental stresses on triggering disease outbreaks in fish rial diseases of fish (Austin, B. and D.A. Austin, (Snieszko, 1974; Anderson, 1990; Muiswinkel et al., 1993; Li et al., 1999; Li, 2002). 1999). Various stresses such as sudden increase in In Aeromonas spp., multiple resistance towards an- water temperature (Angulo et al., 1994; Magarinos tibiotics has been recognized to be associated with et aL, 2001), poor water quality and inadequate diet plasmids, especially transferable ones (e.g. R-factor) (Diggles et aL, 2000) would tend to debilitate fish be- (Saitanu et al., 1994). We have observed the exist- fore vibrio infection. For example, Rodgers and Burke ence of transferable plasmids encoding multiple antibi- (1988) surveyed the incidence of red-spot disease in otic resistance by experimental conjugation of resistant sea mullet (Mugi! cephalus ) caused by V. anguilla- vibrio strains with recipients. However, the transfer rum, and the epidemic appeared to be related to envir- frequencies were very low, ranging from 10 -11 to 10 .9 , onmental parameters such as rapidly changing temper- suggesting that drug-resistance is mainly chromosomal atures and prolonged exposure to low estuarine salini- (Li et aL, 1999). ties. The onset of schooling behavior prior to spawn- In practice, a considerable amount of antibiotics, ing was also assumed to be of importance for the administered in the feed, are unabsorbed by the fish spread of the disease. Similarly, in our recent studies, and enter into the environment in antimicrobially ac- an epizootic outbreak of vibriosis in juvenile silver sea tive forms. In addition, spatial and temporal differ- 124 Journal of Ocean University of Qingdao 2003, Vol. 2, No. 2

ences always result in significant discrepancies in anti- gill disease leading to progressive low-level mortalities microbial susceptibilities of vibrios (French et aL, 1986; among juvenile turbot, Scophthalmus maximus L., in a Li et al., 1999). Therefore, in order to reduce the in- Scottish aquarium. J. Fish Dis., 16: 277-290. Austin, B., and D.A. Austin, 1993. Bacterial Fish Patho- discriminate use of these antibiotics, drug susceptibili- gens. 2nd edition. Ellis Horwood Ltd., Chichester. pp. ty tests should be carried out prior to the use of any 265 -307. antibiotics in fish farms (Li et al., 1999). Balebona, M.C., M.J. Andreu, M.A. Bordas, I. Zorril- Although chemotherapy is currently the most com- la, M.A. Morinigo, et al., 1998. Pathogenicity of Vibrio monly used method in controlling and treating bacteri- alginolyticus for cultured gilthead sea bream (Sparus au- al diseases in fish farms, limitations such as posing ex- rata L. ). Appl. Environ. Microbiol., 64: 4269-4275. tra economical problem to fish farmers and influencing Benediktsdottir, E., S. Helgason, and H. Sigurjonsdottir, surrounding ecosystem do exist (Li et al., 1999). There- 1998. Vibrio sp. isolated from salmonids with shallow skin lesions and reared at low temperature. J. Fish Dis., fore, development of methods other than chemothera- 21: 19-28. py is desired. Prophylactic treatment of vibriosis by Bertone, S., C. Gili, A. Moizo, and L. Calegari, 1996. vaccination is highly recommended, and recent reports Vibrio carchariae associated with a chronic skin ulcer on a have shown that this area is highly promising (Qin shark, Carcharhinus plumbeus (Nardo). J. Fish Dis., 19: and Pan, 1996 ; Woo et al., 2001; Li, 2002). In ad- 429 -434. dition, increasing evidence indicates that application of Biosca, E.O., and C. Amaro, 1996. Toxic and enzymatic probiotics in aquaculture is effective against vibriosis activities of biotype 2 with respect to host specificity. Appl. Environ. Microbiol., 62: 2331- by improving culture conditions, inhibiting potential 2337. pathogens, enhancing nutrition, and stimulating host Biosca, E.G., C. Amaro, C. Esteve, E. Alcaide, and E. immunity. Details on this subject have been adequate- Garay, 1991. First record of Vibrio vulnificus biotype 2 ly reviewed elsewhere (Iranto and Austin, 2002). from diseased European eel, AnguiIIa anguitla L., J. Fish Dis., 14: 103-109. Biosca, E., B. Fouz, E. Alcaide, and C. Amaro, 1996. Acknowledgements Siderophore-mediated iron acquisition mechanisms in Vibrio vulnificus biotype 2. Appl. Environ. Microbiol., 62:928 This study was supported by Earmarked Research -935. Bly, J.E., and L.W. Clem, 1992. Temperature and teleost Grants (CUHK 4135/98M, CUHK 4168/99M, CU- immune function. Fish Shellfish Immunol., 2: 159-171. HK 4146/01M) awarded to NYSW by the Research Bruno, D.W., J. Griffiths, J. Petrie, and T.S. Hastings, Grants Council of Hong Kong. 1998. 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