Disease Problems Affecting Fish in Tropical Environments
Disease Problems Affecting Fish in Tropical Environments
Disease Problems Affecting Fish in Tropical Environments
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This article was downloaded by: [Central Institute for BrackishwaterAquaculture-ICAR]On: 17 August 2012, At: 05:12Publisher: Taylor & FrancisInforma Ltd Registered <strong>in</strong> England and Wales Registered Number: 1072954Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH,UKJournal of Applied AquaculturePublication details, <strong>in</strong>clud<strong>in</strong>g <strong>in</strong>structions forauthors and subscription <strong>in</strong>formation:http://www.tandfonl<strong>in</strong>e.com/loi/wjaa20<strong>Disease</strong> <strong>Problems</strong> <strong>Affect<strong>in</strong>g</strong> <strong>Fish</strong><strong>in</strong> <strong>Tropical</strong> <strong>Environments</strong>Iddya Karunasagar a , Indrani Karunasagar a &Subhendu Kumar Otta aa Department of <strong>Fish</strong>ery Microbiology, College of<strong>Fish</strong>eries, Mangalore-575 002, IndiaVersion of record first published: 04 Oct 2008To cite this article: Iddya Karunasagar, Indrani Karunasagar & Subhendu Kumar Otta(2003): <strong>Disease</strong> <strong>Problems</strong> <strong>Affect<strong>in</strong>g</strong> <strong>Fish</strong> <strong>in</strong> <strong>Tropical</strong> <strong>Environments</strong>, Journal of AppliedAquaculture, 13:3-4, 231-249To l<strong>in</strong>k to this article: http://dx.doi.org/10.1300/J028v13n03_03PLEASE SCROLL DOWN FOR ARTICLEFull terms and conditions of use: http://www.tandfonl<strong>in</strong>e.com/page/termsand-conditionsThis article may be used for research, teach<strong>in</strong>g, and private study purposes.Any substantial or systematic reproduction, redistribution, resell<strong>in</strong>g, loan,sub-licens<strong>in</strong>g, systematic supply, or distribution <strong>in</strong> any form to anyone isexpressly forbidden.The publisher does not give any warranty express or implied or make anyrepresentation that the contents will be complete or accurate or up todate. The accuracy of any <strong>in</strong>structions, formulae, and drug doses should be<strong>in</strong>dependently verified with primary sources. The publisher shall not be liablefor any loss, actions, claims, proceed<strong>in</strong>gs, demand, or costs or damages
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Downloaded by [Central Institute for Brackishwater Aquaculture-ICAR] at 05:12 17 August 2012<strong>Disease</strong> <strong>Problems</strong> <strong>Affect<strong>in</strong>g</strong> <strong>Fish</strong><strong>in</strong> <strong>Tropical</strong> <strong>Environments</strong>Iddya KarunasagarIndrani KarunasagarSubhendu Kumar OttaSUMMARY. Aquaculture produce <strong>in</strong> Asian countries contributes a majorshare to total global fish production. However, disease due to bioticand abiotic factors results <strong>in</strong> considerable losses to the <strong>in</strong>dustry. Themost frequently encountered bacterial agents associated with fish diseases<strong>in</strong> tropical environments are Vibrio sp. <strong>in</strong> mar<strong>in</strong>e and brackishwatersystems and motile aeromonads <strong>in</strong> freshwater systems. Virulencemechanisms of these bacterial species have been widely studied. Thougha few viral agents such as rhabdovirus, reo-like virus, <strong>in</strong>fectious pancreaticnecrosis virus, picornavirus, and irido-like virus have been recorded<strong>in</strong> different parts of Asia, such reports are scanty. Among fungal pathogens,Aphanomyces <strong>in</strong>vaderis is the most virulent and has been reportedto be associated with epizootic ulcerative syndrome. Other fungal agentssuch as Achlya sp. and Saprolegnia sp. have been recorded <strong>in</strong> variousdisease conditions. In addition to various conventional microbiologicaland serological methods, nucleic acid-based methods are be<strong>in</strong>g widelyadopted for diagnosis of different fish diseases. Few successful vacc<strong>in</strong>eshave been developed to protect aga<strong>in</strong>st disease conditions, and some ofIddya Karunasagar, Indrani Karunasagar, and Subhendu Kumar Otta, Departmentof <strong>Fish</strong>ery Microbiology, College of <strong>Fish</strong>eries, Mangalore-575 002, India.Address correspondence to: Iddya Karunasagar, Department of <strong>Fish</strong>ery Microbiology,University of Agricultural Sciences, College of <strong>Fish</strong>eries, Mangalore-575 002,India.[Haworth co-<strong>in</strong>dex<strong>in</strong>g entry note]: “<strong>Disease</strong> <strong>Problems</strong> <strong>Affect<strong>in</strong>g</strong> <strong>Fish</strong> <strong>in</strong> <strong>Tropical</strong> <strong>Environments</strong>.”Karunasagar, Iddya, Indrani Karunasagar, and Subhendu Kumar Otta. Co-published simultaneously <strong>in</strong> Journalof Applied Aquaculture (Food Products Press, an impr<strong>in</strong>t of The Haworth Press, Inc.) Vol. 13, No. 3/4,2003, pp. 231-249; and: Susta<strong>in</strong>able Aquaculture: Global Perspectives (ed: B. B. Jana, and Carl D. Webster)Food Products Press, an impr<strong>in</strong>t of The Haworth Press, Inc., 2003, pp. 231-249. S<strong>in</strong>gle or multiple copies ofthis article are available for a fee from The Haworth Document Delivery Service [1-800-HAWORTH, 9:00a.m. - 5:00 p.m. (EST). E-mail address: get<strong>in</strong>fo@haworthpress<strong>in</strong>c.com].© 2003 by The Haworth Press, Inc. All rights reserved. 231
Downloaded by [Central Institute for Brackishwater Aquaculture-ICAR] at 05:12 17 August 2012232 SUSTAINABLE AQUACULTURE: GLOBAL PERSPECTIVESthe better known ones are aga<strong>in</strong>st vibriosis and furunculosis. Many othervacc<strong>in</strong>es are still <strong>in</strong> the experimental stages. Early diagnosis of diseaseand development of successful vacc<strong>in</strong>es are important for the future developmentof aquaculture. [Article copies available for a fee from TheHaworth Document Delivery Service: 1-800-HAWORTH. E-mail address: Website: © 2003 by The Haworth Press, Inc. All rights reserved.]KEYWORDS. Aquaculture, tropics, bacteria, fungi, virus, diagnosis,immunoprophylaxisINTRODUCTIONMany tropical countries <strong>in</strong> Asia have a long tradition of aquaculture,and accord<strong>in</strong>g to FAO statistics, over 80% of fish produced by aquaculturecomes from Asia, where the production was 31.07 million metrictons valued US$38.855 billion (FAO 1996). N<strong>in</strong>e of the 14 topaquaculture producers <strong>in</strong> the world are from Asia. Cypr<strong>in</strong>ids, tilapia,and catfishes are among the major freshwater fishes cultured and contributeas much as 72% of the total aquaculture production <strong>in</strong> Asia(Csavas 1994). The share of cultured diadromous fish <strong>in</strong> Asia, which <strong>in</strong>cludesalmonids, eels, milkfish, and hilsa, is 42.9%. Major mar<strong>in</strong>e fishthat are cultured <strong>in</strong>clude red fish, basses, congers, jacks, mullets, andsauries. However, culture of such mar<strong>in</strong>e fish is not on a big scale, andhence their contribution is low. One of the major problems limit<strong>in</strong>g fishproduction <strong>in</strong> Asia is loss due to diseases. Aquatic animal disease andenvironmental related problems may cause annual losses of more thanUS$3 billion annually to aquaculture production <strong>in</strong> Asian countries(FAO 1996). <strong>Fish</strong> <strong>in</strong> freshwater, brackishwater, and mar<strong>in</strong>e environmentsare susceptible to a number of bacterial, viral, and parasitic diseases.In this review, the major disease problems of tropical fish arepresented.Major Bacterial Groups InvolvedBACTERIAL DISEASESMass mortalities of fish due to bacterial diseases have been reportedfrom a number of countries <strong>in</strong> Asia. Among the various bacterial agents
Downloaded by [Central Institute for Brackishwater Aquaculture-ICAR] at 05:12 17 August 2012Karunasagar et al. 233associated with fish disease <strong>in</strong> tropical environments, motile aeromonads<strong>in</strong> fresh water and Vibrio spp. <strong>in</strong> brackish and mar<strong>in</strong>e water aremost frequently encountered. Gopalakrishnan (1961) reported massmortalities <strong>in</strong> India is major carps, catla, Catla catla; rohu, Labeorohita; and mrigala, Cirrhenius mrigala; due to motile aeromond <strong>in</strong>fectionand noted that catla is most susceptible, followed by mrigal androhu. <strong>Disease</strong>s due to motile aeromonads may manifest <strong>in</strong> a number ofways. Snieszko and Axelrod (1971) classified disease signs caused byAeromonas hydrophila <strong>in</strong>to four categories: (1) acute, rapidly fatal septicaemiawith a few gross symptoms–an acute form with dropsy; (2) blisters,abscesses, and scale protrusion; (3) a chronic ulcerous form withfuruncles and abscesses; and (4) a latent form with no signs. In tropicalfish, motile aeromonad <strong>in</strong>fections manifest<strong>in</strong>g <strong>in</strong> all these forms havebeen reported (Gopalakrishnan 1961; Karunasagar et al. 1986, 1989).Motile aeromonads are commonly found <strong>in</strong> freshwater ecosystems, <strong>in</strong>clud<strong>in</strong>gfish farms, and it is not yet established whether stra<strong>in</strong>s associatedwith fish disease are different from other environmental stra<strong>in</strong>s.The taxonomy of aeromonads is receiv<strong>in</strong>g considerable attention, dueto the <strong>in</strong>creased recognition of the members of this genera as humanpathogens (Carnahan 1993). Currently ten different species and 14DNA hybridization groups or biovars are recognized (Joseph andCarnahan 1994), but <strong>in</strong> moribund tropical fish, most commonly isolatedspecies are A. hydrophila and A. sobria. Though A. caviae are also isolatedfrom aquaculture systems, this species has low virulence <strong>in</strong> experimentalstudies and may represent a secondary flora (Karunasagar et al.1995).Motile aeromondads are also the most commonly associated bacteria<strong>in</strong> cases of epizootic ulcerative syndrome (Llobrera and Gacutan 1987;Pal and Pradhan 1990; Torres et al. 1990; Lio-Po et al. 1991; Karunasagarand Karunasagar 1994; Karunasagar et al. 1995). However, the preciserole of motile aeromonads <strong>in</strong> this syndrome is not understood. Stra<strong>in</strong>swith vary<strong>in</strong>g virulence are commonly found associated with the syndrome(Karunasagar et al. 1995), but motile aeromonads could be isolatedfrom <strong>in</strong>ternal organs of fish show<strong>in</strong>g reddish lesions of sk<strong>in</strong> beforeulceration (Karunasagar and Karunasagar 1994), and therefore, it ispossible that these bacteria have a role <strong>in</strong> caus<strong>in</strong>g ulcerations. Dur<strong>in</strong>grecent years, some workers have suggested that epizootic ulcerativesyndrome (EUS) is a fungus-associated syndrome with an <strong>in</strong>vasive fungus,Aphanomyces <strong>in</strong>vaderis, the predom<strong>in</strong>ant species (Roberts et al.1992, 1993; Willoughby et al. 1995). However, it is not yet establishedthat the fungus is the primary causative agent. In fact, the isolation rate
Downloaded by [Central Institute for Brackishwater Aquaculture-ICAR] at 05:12 17 August 2012234 SUSTAINABLE AQUACULTURE: GLOBAL PERSPECTIVESof fungus <strong>in</strong> cases of EUS is rather low, and the presence of the fungus is<strong>in</strong>ferred from histological sections (Roberts et al. 1993). It has beensummarized that the fungus is killed by host tissue reactions (Ch<strong>in</strong>abutand Roberts 1999). On the other hand motile aeromonads isolated fromEUS showed dermonecrotic properties, and produced a number ofproteolytic enzymes associated with virulence (McGarey et al. 1991;Karunasagar et al. 1995; Leano et al. 1996); therefore, it is possible thatmotile aeromonads have a role <strong>in</strong> caus<strong>in</strong>g ulcers, though they may notbe the primary causative agent of EUS.Among pathogens of brackishwater and mar<strong>in</strong>e fish, Vibrio spp. arethe most important agents caus<strong>in</strong>g <strong>in</strong>fections <strong>in</strong> a number of culturedfish species (Table 1). However, the species <strong>in</strong>volved have not beenproperly characterized. In temperate waters, V. anguillarum, V. ordalii,and V. salmonicida are important pathogens (Aust<strong>in</strong> and Aust<strong>in</strong>, 1999).Though the former two species have been reported from mar<strong>in</strong>e fishesof Japan (Muroga et al. 1986), it has been shown that the susceptibilityof the Asian mar<strong>in</strong>e fish to V. anguillarum is much lower compared tothat of salmonids and ayu, Plecoglossus altivelis, and that this bacteriumcould be categorized as a facultative pathogen (Muroga 1997). Vibriosisis generally observed <strong>in</strong> stressed fish, and the species observed arema<strong>in</strong>ly V. parahaemolyticus <strong>in</strong> grouper, Ep<strong>in</strong>ephalus salmoides and V.parahaemolyticus and V. alg<strong>in</strong>olyticus <strong>in</strong> seabass, Lates calcarifer(Cheong et al. 1982). The disease may occur <strong>in</strong> the form of dermatitis orenteritis <strong>in</strong> a variety of cultured mar<strong>in</strong>e f<strong>in</strong>fish, regardless of size (Leong1992). The cl<strong>in</strong>ical signs of dermatitis are hemorrhages, lesions, and progressivedevelopment of deep ulcerative myositis, while <strong>in</strong>ternally,congestion, hemorrhage of liver, and enlargement and liquifaction ofkidney might occur (Leong 1992). Enteritis is the less common form ofvibriosis manifest<strong>in</strong>g as hemorrhage of the <strong>in</strong>test<strong>in</strong>e and destruction ofthe tunica mucosa (Leong 1992).Other bacterial pathogens such as Flavobacterium spp., Flexibacterspp., Pseudomonas spp., Citrobacter freundii, Mycobacterium spp.,Edwardsiella tarda, and Staphylococcus aureus have been reported tocause diseases <strong>in</strong> tropical fishes (Table 1). These are generally <strong>in</strong>volved<strong>in</strong> sporadic cases.Bacterial Pathogenicity and Virulence FactorsMost of the bacterial pathogens described above are commonlyfound <strong>in</strong> aquatic systems, and whenever these bacteria are isolated frommoribund fish, the question rema<strong>in</strong>s as to whether they are primary
Downloaded by [Central Institute for Brackishwater Aquaculture-ICAR] at 05:12 17 August 2012Karunasagar et al. 235TABLE 1. Important bacterial diseases reported from tropical fish.Causative agent <strong>Fish</strong> affected ReferenceAeromonas hydrophila Catla, rohu, mrigal Gopalakrishnan (1961)Karunasagar et al. (1986,1989)Catfish Djajadiredja et al. (1982)Crucian carp Miyazaki and Kage (1985)Ornamental fish Hettiarchi and Cheong (1994)Murrels, barbsKarunasagar andpearlspot Karunasagar (1994)Aeromonas salmonicida Common carp, tilapia Reddy et al. (1994)Common carp Bootsma et al. (1977)Citrobacter fruendii Common carp Karunasagar et al. (1992)Edwardsiella tarda Eels Miyazaki and Egusa (1976)Flexibacter columnaris Eels Lia-Po et al. (1982)Flexibacter maritimus Seabass Chong and Chao (1986)Red sea bream Wakabayashi et al. (1984)Flavobacterium spp. Salmonids Wakabayashi et al (1980)Mycobacterium spp. Snakehead Adams et al. (1996)Pseudomonas spp. Eels Lia-Po et al. (1982)Staphylococcus aureus Silver carp Shah and Tyagi (1986)Vibrio spp. Seabass Kasornchandra and Boonyaratpal<strong>in</strong>(1984)Chong and Chao (1986)Grouper Chua and Teng (1987)Chong and Chao (1986)Milkfish Muroga et al. (1986)Snapper Leong (1992)Eels Miyazaki et al. (1977)Yers<strong>in</strong>ia ruckeri Ra<strong>in</strong>bow trout Sharma et al. (1995)pathogens or opportunistic <strong>in</strong>vaders of stressed fish. The virulencemechanisms of some of the fish pathogens have been <strong>in</strong>tensively studied,though the mechanism by which these pathogens cross the epithelialbarrier and overcome the natural defences of the fish is not yetclearly understood.The virulence factors of motile aeromonads have been <strong>in</strong>vestigatedby a number of workers, and this <strong>in</strong>terest is perhaps driven by the factthat these organisms are emerg<strong>in</strong>g human pathogens (Carnahan 1993).Some of the suspected virulence-associated characteristics are serotype(Mittal et al. 1980), surface characters (Dooley and Trust 1988), andability to produce various extra-cellular products (Thune et al. 1993).
Downloaded by [Central Institute for Brackishwater Aquaculture-ICAR] at 05:12 17 August 2012236 SUSTAINABLE AQUACULTURE: GLOBAL PERSPECTIVESMittal et al. (1980) noted that stra<strong>in</strong>s of A. hydrophila virulent for fishshared a common O antigenic group, did not settle after boil<strong>in</strong>g, andwere resistant to the bactericidal action of mammalian serum. Studiesdone on tropical isolates suggested that pellet<strong>in</strong>g after boil<strong>in</strong>g and resistanceto serum may be associated with virulent stra<strong>in</strong>s (Karunasagar etal. 1988). Pai (1992) studied a number of factors such as hemolytic activity,surface hydrophobicity, pellet<strong>in</strong>g after boil<strong>in</strong>g, lack of agglut<strong>in</strong>ation<strong>in</strong> acriflav<strong>in</strong>e, congo red reaction, suicidal phenomenon, elastaseproduction, and resistance to the bactericidal action of serum of motileaeromonads associated with tropical fish ponds and did not f<strong>in</strong>d directcorrelation between many of these factors and virulence to fish. Mittalet al. (1980), Lallier et al. (1981), and Corral et al. (1990) noted that virulentstra<strong>in</strong>s of A. hydrophila were resistant to complement-mediatedlysis of fresh serum, and this was attributed to either lipopolysaccharideor surface prote<strong>in</strong> components of gram negative bacteria (Munn et al.1982; Buchanan and Pearce 1997). Pai (1992) noted that resistance tobactericidal action of serum was associated with tropical stra<strong>in</strong>s hav<strong>in</strong>ga low LD50, support<strong>in</strong>g the view that this activity constitutes an importantvirulence factor of motile aeromonads. It has also been suggestedthat the virulent stra<strong>in</strong>s show higher elastase and gelat<strong>in</strong>ase activities(Hsu et al. 1981; Wakabayashi et al. 1981; Pai 1992).Dur<strong>in</strong>g recent years, cell culture systems are be<strong>in</strong>g used to study howpathogens <strong>in</strong>teract with host cells, and <strong>in</strong> comb<strong>in</strong>ation with molecularbiological studies of gene <strong>in</strong>activation and complementation, such studieshave greatly contributed to the understand<strong>in</strong>g of the pathogenesis ofa number of diseases, particularly salmonellosis, listeriosis, <strong>in</strong>fectionscaused by pathogenic Escherichia coli, etc. (Henderson et al. 1999).Interaction of fish pathogenic A. hydrophila with fish cells has beenstudied by some <strong>in</strong>vestigators. Krovacek et al. (1987) noted that A. hydrophilaand V. anguillarum adhere to fish cells and to glass slides coatedwith fish mucus. Leung et al. (1995, 1996) suggested that A. hydrophilais capable of <strong>in</strong>vad<strong>in</strong>g and multiply<strong>in</strong>g <strong>in</strong>side tilapia phagocytes andcarp epithelial tissue cultured cells. They further noted that virulentstra<strong>in</strong>s <strong>in</strong>duced cytopathic changes <strong>in</strong> cells dur<strong>in</strong>g <strong>in</strong>vasion, and the <strong>in</strong>ternalizationwas accompanied by the rearrangement of micro-filaments(Low et al. 1998). However, studies with more virulent and avirulentstra<strong>in</strong>s are required to assess whether the cell culture assay could be usedto differentiate virulent from avirulent stra<strong>in</strong>s of A. hydrophila.Compared to motile aeromonads, fish pathogenic Vibrio sp. havebeen little <strong>in</strong>vestigated, with the possible exception of V. anguillarum.Crosa (1980) noted that highly virulent stra<strong>in</strong>s of V. anguillarum carry a
Downloaded by [Central Institute for Brackishwater Aquaculture-ICAR] at 05:12 17 August 2012Karunasagar et al. 23765 kilobase plasmid named pJM1, which encodes an efficient iron uptakesystem that allows the organism to grow under iron restricted conditions.On los<strong>in</strong>g the plasmid, the organism loses both the ability togrow under such conditions and also the virulence (Crosa and Hodges1981). In some virulent stra<strong>in</strong>s of this organism, the genes encod<strong>in</strong>g thehigh aff<strong>in</strong>ity iron transport system are located <strong>in</strong> the chromosome(Toranzo et al. 1983). Milton et al. (1996) noted that a 40.1 kDaflagell<strong>in</strong> A prote<strong>in</strong> encoded by fla A gene is essential for the virulence ofV. anguillarum, s<strong>in</strong>ce the virulence could be reduced 500-fold whenflagella was lost by transposon mutagenesis. Lee (1995) and Balebonaet al. (1995) suggested that extracellular products such as proteases are<strong>in</strong>volved <strong>in</strong> the pathogenicity of V. alg<strong>in</strong>olyticus to cultured grouper andseabream. In vitro, V. alg<strong>in</strong>olyticus has been shown to adhere to sk<strong>in</strong>mucus and sk<strong>in</strong> cells of seabream; however, the <strong>in</strong>fection experimentsus<strong>in</strong>g bath immersion suggested that V. alg<strong>in</strong>olyticus could be considereda pathogen for seabream, only when the mucus layer is removedand the sk<strong>in</strong> damaged (Balebona et al. 1998).Detailed studies on the pathogenicity of Flexibacter group are stilllack<strong>in</strong>g, though challenge experiments have been conducted with a fewstra<strong>in</strong>s (Wakabayashi et al. 1980, 1984). It has been suggested that thevirulence of this group is associated with extracellular prote<strong>in</strong> (ECP),subcellular components, hemolys<strong>in</strong>, and lipopolysaccharides (Aust<strong>in</strong>and Aust<strong>in</strong> 1999).VIRAL DISEASESThough a number of viral fish disease problems have been reportedfrom both freshwater as well as mar<strong>in</strong>e habitats <strong>in</strong> North America, Canada,Australia and Japan, reports of such <strong>in</strong>cidences <strong>in</strong> warmer parts ofAsia are very scanty (Table 2). This could also reflect the lack of facilitiesrequired to <strong>in</strong>vestigate viral diseases <strong>in</strong> this region. In the freshwatersystems, a few viral agents, particularly, rhabdoviruses have been isolatedfrom EUS-affected fish (Frerichs et al. 1986, 1989; Kasornchandraet al. 1991; Roberts et al. 1994). However, their precise role <strong>in</strong> the syndromeis not yet established. Inability to isolate viruses from many epizooticscould be due to various reasons. It is possible that the viruses arepresent early dur<strong>in</strong>g the syndrome (Kanchanakhan 1997). Other viralagents isolated <strong>in</strong>clude reo-like viruses (Roberts et al. 1994). In freshwatersystems, there have been two reports of detection of <strong>in</strong>fectious
Downloaded by [Central Institute for Brackishwater Aquaculture-ICAR] at 05:12 17 August 2012Karunasagar et al. 239ary pathogens. Interest<strong>in</strong>gly, Limsuwan and Ch<strong>in</strong>abut (1983) describedsevere chronic granulomatous mycoses <strong>in</strong> Thailand from which thesefungi were isolated, but later, they were considered secondary pathogens<strong>in</strong> EUS (Tonguthai 1985). Before EUS was a serious problem <strong>in</strong>Asia, Srivastava (1979) reported aphanomycosis due to Aphanomycespisci <strong>in</strong> mrigal and suggested that this disease could be a new threat tofish populations, but it was only after the report of Roberts et al. (1993)that the association between an <strong>in</strong>vasive fungus, Ap. <strong>in</strong>vaderis and EUShas been <strong>in</strong>creas<strong>in</strong>gly recognized and debated on its role as the causativeagent. They isolated a stra<strong>in</strong> of Aphanomyces from the muscle ofEUS-affected fish, and this fungus was shown to be slow-grow<strong>in</strong>g andthermolabile when compared to other saprophytic Aphanomyces. Whenplaced below the dermis of striped snakehead fish, Channa striatus, themycelium of slow-grow<strong>in</strong>g Aphanomyces succeeded <strong>in</strong> migrat<strong>in</strong>g <strong>in</strong>tothe tissues caus<strong>in</strong>g severe myonecrosis with chronic epithelial reactiontypical of EUS (Roberts et al. 1993). This fungus was named Ap.<strong>in</strong>vaderis by Willoughby et al. (1995). Interest<strong>in</strong>gly, Ap. <strong>in</strong>vaderis isnot detected on the surface of EUS-affected fish. Lilley and Roberts(1997) compared the <strong>in</strong>fectivity of several Ap. <strong>in</strong>vaderis isolates withthat of other Ap., Achlya, and Saprolegnia found on the surface ofEUS-affected fish. Only Ap. <strong>in</strong>vaderis produced <strong>in</strong>vasive lesion typicalof EUS. However, they po<strong>in</strong>t out that Ap. <strong>in</strong>vaderis cannot be consideredthe primary pathogen unless the <strong>in</strong>fective zoospore stage can beshown to breach the sk<strong>in</strong> unaided. Therefore, many <strong>in</strong>vestigators stillconsider EUS to be a syndrome of complex aetiology. Under experimentalconditions, this fungus could <strong>in</strong>fect juvenile snakehead fish,only when it was <strong>in</strong>jected or <strong>in</strong>serted <strong>in</strong>to the body of the fish. Severalworkers have shown that it is unable to <strong>in</strong>fect the fish when challengedby bath<strong>in</strong>g <strong>in</strong> the fungus (Kanchanakhan 1997). It has been suggested,therefore, that the fungus may need special environmental conditionsand/or other fish pathogens to <strong>in</strong>duce an <strong>in</strong>fection (Roberts et al. 1993;Ch<strong>in</strong>abut et al. 1995). The ecology of Ap. <strong>in</strong>vaderis is still a mystery,s<strong>in</strong>ce no isolates have been reported from natural waters. Moreover, thefungus has not been shown to sporulate <strong>in</strong> fish tissue, and therefore thequestion rema<strong>in</strong>s as to where the fungus sporulates and how it spreadsfrom fish to fish to cause an epizootic.An epizootic <strong>in</strong>fection due to A. debaryana <strong>in</strong> the Mastacembelusarmantus, has been reported from an artificial reservoir <strong>in</strong> India (Khulbeet al. 1994). In the same reservoir, when water temperatures were moderate,mycotic <strong>in</strong>fections were observed to be severe, with fungi like A.flagellata and S. parasitica be<strong>in</strong>g more virulent (Khulbe et al. 1995).
Downloaded by [Central Institute for Brackishwater Aquaculture-ICAR] at 05:12 17 August 2012240 SUSTAINABLE AQUACULTURE: GLOBAL PERSPECTIVESMETHODS FOR DIAGNOSIS OF MICROBIAL DISEASESSo far, diagnosis of microbial diseases of tropical fish has been basedon classical methods such as cl<strong>in</strong>ical signs, histopathology, and isolationand identification of pathogens by conventional methods. However,dur<strong>in</strong>g recent years, application of nucleic-acid based and antibody-baseddiagnostic methods are be<strong>in</strong>g <strong>in</strong>creas<strong>in</strong>gly applied <strong>in</strong> medical and veter<strong>in</strong>aryfields and have been widely used for a number of fish and shellfishdiseases (Karunasagar and Karunasagar 1999). Even <strong>in</strong> tropical countriessuch as Taiwan, India, Thailand, and Malaysia, polymerase cha<strong>in</strong>reaction (PCR)-based detection methods are be<strong>in</strong>g rout<strong>in</strong>ely used forwhitespot syndrome virus affect<strong>in</strong>g shrimp (Lo et al. 1996; Kasornchandraet al. 1998; Otta et al. 1999). However, such methods are not yet usedfor diseases of f<strong>in</strong>fishes. There may be several reasons for this. Shrimpis a high-value product, and the farmers have a high f<strong>in</strong>ancial stake.They are therefore will<strong>in</strong>g to spend on expensive diagnostic methods,but the economics of a fish farm is on a different scale. Nevertheless,these methods will be extremely useful, particularly <strong>in</strong> cases where traditionalmethods are also expensive and time consum<strong>in</strong>g, e.g., <strong>in</strong> viraldiseases.DNA-based methods, such as hybridization with an oligonucleotideprobe or PCR/reverse transcriptase PCR, has been reported to be usefulfor detection of fish viral pathogens such as Infectious Pancreatic NecrosisVirus (Rimstaad et al. 1990; Rodriguez et al. 1995), fish rhabdovirus(Estepa et al. 1995), fish iridovirus (Tamai et al. 1997), and redseabream iridovirus (Oshima et al. 1996), and for bacteria that are difficultto culture or slow grow<strong>in</strong>g, such as mycobacteria (Talaat et al.1997). Therefore, adopt<strong>in</strong>g these methods for viruses or bacteria affect<strong>in</strong>gtropical fishes should not be difficult.Similarly, antibody-based methods such as fluorescent antibody test(FAT) and enzyme-l<strong>in</strong>ked immunosorbant assay (ELISA) have been reportedfor detection of a number of fish pathogens <strong>in</strong>clud<strong>in</strong>g bacteria,viruses, and parasites (Karunasagar and Karunasagar 1999). However,these are mostly experimental studies, and commercial kits for diagnosisof fish diseases are still not available for rout<strong>in</strong>e use. There are alsoproblems <strong>in</strong> application of antibody-based methods for bacterial pathogens,such as A. hydrophila, which are serologically heterogenous. Theserological structure of Vibrio spp., such as V. alg<strong>in</strong>olyticus, affect<strong>in</strong>gtropical fishes has not been studied <strong>in</strong> great detail. Such studies wouldbe important for development of diagnostic tests suitable for pathogensof tropical fishes.
Downloaded by [Central Institute for Brackishwater Aquaculture-ICAR] at 05:12 17 August 2012Karunasagar et al. 241IMMUNOPROPHYLAXIS OF FISH DISEASESPrevention of diseases would be the major goal of scientific aquaculture.The possibility of vacc<strong>in</strong>at<strong>in</strong>g fishes aga<strong>in</strong>st diseases has been<strong>in</strong>vestigated for nearly half a century, but successful vacc<strong>in</strong>es have beendeveloped to protect aga<strong>in</strong>st only a few disease conditions (Karunasagarand Karunasagar 1999). One of the most successful vacc<strong>in</strong>es has beenaga<strong>in</strong>st vibriosis, and this generally conta<strong>in</strong>s V. anguillarum serotype 1and 2, but polyvalent vacc<strong>in</strong>es conta<strong>in</strong><strong>in</strong>g other antigens such as V.salmonicida, A. salmonicida or V. ordalii are also available (Toranzo etal. 1997). Whether they are effective aga<strong>in</strong>st vibriosis of tropical mar<strong>in</strong>efishes is not known. Leong et al. (1998) tested the effect of an importedvacc<strong>in</strong>e (not specified) and a locally produced vibrio vacc<strong>in</strong>e on the survivalof grouper, Ep<strong>in</strong>ephelus cocciodes, cultured <strong>in</strong> float<strong>in</strong>g net cages<strong>in</strong> Malaysia. After 12 weeks, the overall survival for the control grouperranged between 20.5% to 35%, while grouper vacc<strong>in</strong>ated with locallyproduced vacc<strong>in</strong>e showed a survival range of 8.5% to 43.5% and thegroup vacc<strong>in</strong>ated with imported vacc<strong>in</strong>e showed a survival range of30% to 80%. However, high mortalities occurred <strong>in</strong> the first 4 weeks,regardless of whether groupers were vacc<strong>in</strong>ated or not (Leong et al.1998). These results suggest that more appropriate vacc<strong>in</strong>es need to bedeveloped for vibriosis affect<strong>in</strong>g tropical fish.Among diseases of freshwater fish, motile aeromonad septicaemia isvery important and there have been a number of attempts to developvacc<strong>in</strong>es aga<strong>in</strong>st motile aeromonads. Vacc<strong>in</strong>ation aga<strong>in</strong>st the non-motileaeromonad A. salmonicida affect<strong>in</strong>g temperate fish has been possiblebecause of the serological homogeneity <strong>in</strong> this species. Motile aeromonadsaffect<strong>in</strong>g tropical fish are, on the other hand, serologically veryheterogenous. However, experimental studies have shown that bacter<strong>in</strong>sdelivered by <strong>in</strong>jection or immersion can <strong>in</strong>duce antibody productionand protection aga<strong>in</strong>st homologous challenge <strong>in</strong> a number of fish, <strong>in</strong>clud<strong>in</strong>gcommon carp, Cypr<strong>in</strong>us carpio (Baba et al. 1988), catla, rohu,mrigal (Karunasagar et al. 1991, 1997), and tilapia, Aureochromismosambicus (Ruangpan et al. 1986). There was good correlation betweenthe levels of agglut<strong>in</strong>at<strong>in</strong>g antibody and extent of protectionaga<strong>in</strong>st challenge; cross-react<strong>in</strong>g antibodies <strong>in</strong>duced by monovalentvacc<strong>in</strong>es showed vary<strong>in</strong>g degrees of protection aga<strong>in</strong>st heterologouschallenge (Karunasagar et al. 1997). When polyvalent vacc<strong>in</strong>es wereused, antibody response aga<strong>in</strong>st all the component stra<strong>in</strong>s was observed;however, the level of antibody was less compared to that <strong>in</strong>fishes immunized with monovalent vacc<strong>in</strong>es (Karunasagar et al. 1997).
Downloaded by [Central Institute for Brackishwater Aquaculture-ICAR] at 05:12 17 August 2012242 SUSTAINABLE AQUACULTURE: GLOBAL PERSPECTIVESS<strong>in</strong>ce <strong>in</strong>clusion of all serotypes of the organism <strong>in</strong> polyvalent vacc<strong>in</strong>eswould not be possible, it is necessary to understand whether there areany common protective antigens <strong>in</strong> the fish pathogenic stra<strong>in</strong>s of motileaeromonads. Therefore, many basic studies are necessary before a successfulvacc<strong>in</strong>e for this organism can be developed. The possibility ofus<strong>in</strong>g live attenuated vacc<strong>in</strong>e has been proposed by some <strong>in</strong>vestigators.Karunasagar et al. (1991) noted that an aerolys<strong>in</strong> deletion mutant <strong>in</strong>ducedsignificant protection, as evidenced by challenge experiments.Protection aga<strong>in</strong>st heterologous stra<strong>in</strong> was only partial. Azad et al.(1997) reported that A. hydrophila grown as a biofilm on substratessuch as chit<strong>in</strong> and bagasse could be used as an oral vacc<strong>in</strong>e; however,they did not address the question of protection aga<strong>in</strong>st heterologousstra<strong>in</strong>s or attenuation of the vacc<strong>in</strong>e stra<strong>in</strong>. Hernanz Moral et al. (1998)developed an aromatic-dependent mutant of A. hydrophila as a candidatefor live vacc<strong>in</strong>e and noted that the stra<strong>in</strong> conferred significant protectionaga<strong>in</strong>st the wild-type stra<strong>in</strong> <strong>in</strong> ra<strong>in</strong>bow trout, Oncorhynchusmykiss. However, the extent of protection aga<strong>in</strong>st heterologous stra<strong>in</strong>swas not evaluated.REFERENCESAdams, A., K.D. Thompson, H. McEvan, S.C. Chen, and R.H. Richards. 1996. Developmentof monoclonal antibodies to Mycobacterium spp. isolated from Chevronsnakehead and Siamese fight<strong>in</strong>g fish. Journal of Aquatic Animal Health 8: 208-215.Aust<strong>in</strong>, B., and D.A. Aust<strong>in</strong>. 1999. Bacterial <strong>Fish</strong> Pathogens: <strong>Disease</strong> of Farmed andWild <strong>Fish</strong>. Spr<strong>in</strong>ger Praxis Publish<strong>in</strong>g, Chichester, United K<strong>in</strong>gdom.Azad, I.S., K.M. Shankar, and C.V. Mohan. 1997. Evaluation of an Aeromonashydrophila biofilm for oral vacc<strong>in</strong>ation of carp. Pages 181-185 <strong>in</strong> T.W. Flegel, andMacRae, Eds., <strong>Disease</strong>s <strong>in</strong> Asian Aquaculture III, Asian <strong>Fish</strong>eries Society, Manila,Philipp<strong>in</strong>es.Baba, T., J. Imamura, K. Izawa, and K. Ikeda. 1988. Cell mediated protection <strong>in</strong> carp,Cypr<strong>in</strong>us carpio L., aga<strong>in</strong>st Aeromonas hydrophila. Journal of <strong>Fish</strong> <strong>Disease</strong>s11:171-178.Balebona, M.C., M.A. Mor<strong>in</strong>igo, and J.J. Borrego. 1995. Role of extracellular products<strong>in</strong> the pathogenicity of Vibrio stra<strong>in</strong>s on cultured gilthead seabream (Sparus aurata).Microbiologia 11: 439-446.Balebona, M.C., M.J. Andreu, M.A. Bordas, I. Zorrilla, M.A. Mor<strong>in</strong>igo, and J.J.Borrego. 1998. Pathogenicity of Vibrio alg<strong>in</strong>olyticus for cultured gilt-head seabream(Sparus aurata L.). Applied and Environmental Microbiology 64: 4269-4275.Bootsma, R., N. Fijan, and J. Blommaert. 1977. Isolation and identification of the causativeagents of carp-erythrodermatitis. Veter<strong>in</strong>arski Archiv 47: 291-302.Boonyaratpal<strong>in</strong>, S., K. Supamattaya, J. Kasornchandra, and R.W. Hoffman. 1996.Picorna-like virus associated with mortality and a spongious encephalopathy <strong>in</strong>grouper Ep<strong>in</strong>ephelus malabaricus. <strong>Disease</strong>s of Aquatic Organisms 26: 75-80.
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