Biotechnology offers revolution to fishhealth managementAlexandra Adams and Kim D. Thompson

Institute of Aquaculture, University of Stirling, Stirling FK9 4LA, UK

Biotechnology has many applications in fish health

management. The application of monoclonal antibodies

(mAbs) provides a rapid means of pathogen identifi-

cation; antibodies to immunoglobulins from different

fish species can be used to monitor the host response

following vaccination; and mAbs also have the potential

for screening broodstock for previous exposure to

pathogens. Luminex technology exemplifies a novel

antibody-based method that can be applied to both

pathogen detection and vaccine development. Mole-

cular technologies, such as the polymerase chain

reaction (PCR), real time PCR and nucleic acid

sequence-based amplification (NASBA), have enabled

detection, identification and quantification of extremely

low levels of aquatic pathogens, and microarray tech-

nologies offer a new dimension to multiplex screening

for pathogens and host response. Recombinant DNA

technology permits large-scale, low-cost vaccine pro-

duction, moreover DNA vaccination, proteomics, adju-

vant design and oral vaccine delivery will undoubtedly

foster the development of effective fish vaccines in the

future.

Introduction

Fish in culture are susceptible to a wide range of bacterial,viral, parasitic and fungal infections, and losses throughdisease currently make a significant impact on the qualityand volume of the fish produced in Europe and throughoutthe world [1]. An effective health management programmust cover all aspects of aquaculture activity including:up-to-date knowledge of the health status of the fish;identifying and managing risks to fish health; reducingexposure to or the spread of pathogens; and managing theuse of drugs and /or chemicals [1].

Biotechnology can have a direct positive impact onmany of the main elements of fish health management,with knock-on effects to other important issues. Rapiddetection and identification of pathogens, for example, iscrucial for effective disease management and this, in turn,leads to a reduction in the use of antibiotics and chemicalsin the environment; prevention of disease by vaccinationsignificantly reduces antibiotic use [2]. Moreover, themovement of infected fish needs to be restricted to preventthe spread of disease; therefore, regular screening for thepresence of pathogens is essential [1].

Corresponding author: Adams, A. (alexandra.adams@stir.ac.uk).Available online 29 March 2006

www.sciencedirect.com 0167-7799/$ - see front matter Q 2006 Elsevier Ltd. All rights reserved

Biotechnology has many applications in fish healthmanagement, and significant contributions have beenmade in the past decade in the development of vaccines forfish [3,4] in addition to diagnostic probes and tests [5,6].Consequently, many novel technologies are now availableto assist in the improvement of fish health, and becausescientific advances in aquatic health continue to close thegap with clinical and veterinary medicine, these arebecoming a reality that offers untold benefits to theaquaculture industry. This article will highlight some ofthe main areas where biotechnology has already made asignificant impact on fish healthmanagement; in addition,it will focus on new cutting-edge biotechnology that can beapplied to aquatic health.

The impact of biotechnology in fish health management

Vaccines

The stress and disease that accompany intensive fishculture have led to treatment with antibiotics andchemicals. Disease prevention by means of optimalhusbandry, the use of biological control methods such asvaccination and the use of immunostimulants should,however, be developed because concerns regarding thepollution associated with chemical treatments, and theemergence of multiple resistance to antibiotics, makes thecontrol of infections increasingly difficult [7].

The primary considerations for any successful vaccinefor aquaculture are cost-effectiveness and safety. Toaccomplish these, the vaccine must provide long-termprotection against the disease under the intensive rearingconditions found on commercial fish farms (Figure 1).Consideration must be given to all the serotype variants ofthe agent that causes the disease, the time and/or agewhen the animal is most susceptible to disease, the routeof administration and the method of vaccine preparation(e.g. killed, attenuated, sub-unit, recombinant).

Most of the commercial vaccines presently availablecomprise inactivated (killed) disease agents. When thisapproach fails in the development of vaccines, particularlyagainst viruses, then live attenuated vaccines aredeveloped; however, whenever a live vaccine is used,there is always concern that the attenuated strain(usually weakened as a result of gene deletion) mightback-mutate and revert to the virulent wild type [4,8].Many of the successful vaccines against viral diseases inhumans (e.g. rubella, measles, poliomyelitis) and indomestic animals (e.g. rabies, distemper) are live atte-nuated organisms. Licensing of such vaccines might,

Review TRENDS in Biotechnology Vol.24 No.5 May 2006

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Figure 1. Intensive rearing conditions are found on commercial fish farms. Here, seabream (Sparus aurata) are cultured in Malta using flexible rubber cage technology.

Review TRENDS in Biotechnology Vol.24 No.5 May 2006202

however, prove to be difficult in aquaculture. An alterna-tive approach is to prepare sub-unit vaccines in which thespecific components of the agent that causes a disease areisolated and then used in vaccines. To increase the amountof available antigens, the recent trend has been to clonethe genes encoding specific antigens and then to incorpor-ate them into bacterial DNA, from where they areexpressed. These are known as recombinant vaccines [9].

The majority of commercial vaccines are multivalentand target salmon and trout but there are expandingopportunities in Mediterranean marine fish species (e.g.European sea bass and sea bream) and the temperate andwarm-water species found in the Asia–Pacific region, forexample, grouper, Asian seabass, Japanese yellowtail,amberjack and tilapia. Presently, vaccines are availableagainst bacterial and some viral diseases and, currently,no vaccines against parasites of fish exist [10–12].Recombinant DNA technology is being used, increasingly,in vaccine development, and a commercial recombinantvaccine against infectious pancreatic necrosis virus(IPNV) is in use in Norway. A recent major developmentin fish vaccinology is DNA vaccination [13], enabling theeffective stimulation of cellular responses, and the firstcommercial DNA fish vaccine (against infectious hemato-poietic necrosis virus, IHNV) is now available in Canada.Proteomics [14] and epitope mapping (J. Costa, PhDthesis, University of Stirling, 2005) [15] offer an alterna-tive approach to the identification of vaccine antigens, andthe latter presents the potential to develop peptidevaccines. These methods have the capability to revolutio-nize fish vaccine development. Novel approaches invaccine adjuvants (e.g. using interleukin-1) [16] andvaccine delivery systems (e.g. PLGA micro-particles) are

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also being pursued, including the development of anantigen protection vehicle (APV) for oral vaccines [17].

Rapid detection methods

The rapid detection of pathogens in infected fish, bothclinically and sub-clinically, is desirable for effectivehealth management in aquaculture. If pathogens canalso be detected and identified in the environment, forexample, between harvesting and re-stocking or before adisease outbreak, then this can be extremely useful as anearly warning signal.

Traditional methods

Traditional bacteriology, virology, parasitology and my-cology are appropriate for the detection of common, easilycultured pathogens; however, for many pathogens thesemethods can be expensive, time-consuming and might notlead to a definitive diagnosis being made, even whencomplemented with histological evidence [18]. Forexample, routine bacteriological screening by plating outonto agar involves a lag phase dependent on the growthcycle of the particular bacterium, which might vary fromovernight to several weeks. In some instances wheregrowth is extremely slow, culture on agar medium mightnot be achieved at all owing to overgrowth withcontaminants. Therefore, a combination of methods isoften required for a definitive diagnosis of disease, andimmunodiagnostic and molecular methods have beendeveloped to detect many of the common pathogens.

Immunological methods for the detection of pathogens

Immunological methods, such as fluorescent antibodytechnique (FAT), indirect fluorescent antibody technique

Figure 3. Immunohistochemistry (IHC) is an extension of traditional histology, and

the sensitivity can be increased using biotin–streptavidin amplification. Mycobac-

teriummarinum (stained brown) in fixed tissue can be detected in Siamese fighting

fish spleen by amplified IHC using mAb 8F7. Counterstained with haematoxylin.

Review TRENDS in Biotechnology Vol.24 No.5 May 2006 203

(IFAT), immunohistochemistry (IHC), ELISA and dot blotor western blot, enable the rapid and specific detection ofpathogens without the need to isolate the pathogen first[18]. Monoclonal antibodies provide ideal standardizedreagents for such tests and many are now commerciallyavailable [19]. The antibody-based test selected for theidentification of pathogens depends on a variety of factors,and each method has its merits and disadvantages. TheFAT and IFAT are simple, sensitive methods that can beperformed within 2 hours [18]. There is, however, arequirement for specialized equipment (fluorescent micro-scope or confocal microscope) and a skilled operator toread results because there can be problems with non-specific background staining. Nevertheless, this method iswidely used for the detection of fish pathogens and isparticularly useful for the identification of viruses such asISAV (Figure 2). IHC, an extension of traditional histology,is an equally straightforward method, although it takesbetween 2 and 3 days to complete because samples aretreated with formalin, embedded in paraffin wax and thenincubated with a pathogen-specific antibody [20]. How-ever, it only requires the use of a simple light microscope, avariety of substrates are available and the assay can beamplified using biotin–streptavidin (Figure 3). AlthoughIHC is regarded as less sensitive than IFAT, it has theadvantage of being able to visualize the surrounding cellsand, therefore, the pathology associated with the infec-tion. The ELISA can be used in a variety of formats, bothfor the detection of the pathogen and for serology – thedetection of antibodies to the pathogen [19]. In general,the sandwich ELISA used for the detection of pathogens iscomplex to set up because standards are required forquantification; furthermore, although this assay is usefulfor the detection of pathogens during clinical disease, it is

Figure 2. Fluorescent staining is a simple and rapid method that is widely used for

the detection of fish pathogens and is particularly useful for the identification of fish

viruses, such as infectious salmon anemia virus (ISAV). Anti-ISAV monoclonal

antibodies (mAb 37) and anti-mouse–FITC conjugate (green staining) have also

been used in confocal microscopy to examine Salmon Head Kidney (SHK-1) cells

infected with ISAV. Nuclei stained blue with 4 0,6-Diamidino-2-phenylindole (DAPI).

Scale barZ50 mm.

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limited in its application to detect sub-clinical infections[21]. The advantages of the ELISA method are highthroughput, automated equipment is available and itis quantitative.

Immunological methods for the detection of fish

antibodies to specific pathogens

Serology is an essential screening tool in clinical medicineand in most programs for the control of the significantdiseases affecting domestic animals [22–24]; therefore, ithas potential as a means of disease surveillance inaquaculture. Detection of specific antibodies in theserum of animals is recognized as a useful indicator ofprevious exposure to pathogens and is regularly used inboth clinical and veterinary medicine. This type ofserology is often used when it is not possible to isolatethe pathogen by traditional methods or when there are norapid tests to identify the pathogen. Such methods arepresently underused in aquaculture, although theirpotential for disease management in other animals iswell proven. The tests for fish require anti-fish species IgMantibodies and many such probes are now commerciallyavailable [19]. The ELISA provides a convenient methodfor testing large numbers of samples and the testing isnon-destructive, requiring only a serum sample. Theprevalence and antibody titers in subgroups of fish withinfarms might reflect the infectious load before mortalitybecomes a problem and, because this sampling is carriedout independently of clinical signs of the disease, a muchlarger time frame is available for monitoring the spread ofthe disease. This ELISA method has many potentialapplications and can be used for the screening of fishpopulations suspected of having a particular disease (inculture or in the wild), for health testing in valuablebroodstock (Figure 4) or post vaccination to verifyantibody response.

Molecular methods

Sometimes, even with such sensitive immunologicalmethods, it is not possible to detect the pathogens ofinterest: perhaps the level in the environment (e.g. inwater samples), and the fish tissue, is below the sensitivity

Figure 4. The enzyme linked immunosorbent assay (ELISA) has many potential

applications and can be used for the screening of valuable broodstock such as

Tilapia (Oreochromis niloticus).Figure 5. 3D reconstruction of a spore of T. bryosalmonae from confocal

microscopy scans. Four central capsular cells (from one of which a polar filament

has extruded) are encompassed by nucleated valve cells. (polysaccharide stain).

Source: C. McGurk, PhD thesis, University of Stirling, 2005.

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threshold or the antigens on the pathogen have altered asit enters into a different life cycle stage (this is commonwith parasites). The possibility of amplifying smallamounts of defined sequences of DNA using PCR so thatthey can be detected by conventional methods hasincreased the potential of pathogen detection and identi-fication. PCR is exceptionally sensitive and is unaffectedby the altered expression of antigens when DNA isdetected (RNA can also be used as a template); however,false-positive and false-negative results because of con-tamination or inhibition can lead to problems in theanalysis of results [25]. Despite these considerations,molecular methods are now widely used for the detectionof pathogens in fish and shrimp [6].

The development of PCR technology has led toinnovations in methods for the detection of pathogens indiverse environments such as water, soil, and foodsamples. PCR, often in combination with other tech-niques, has opened up numerous possibilities in epide-miological studies for the identification of individualstrains and, in particular, the differentiation of closelyrelated strains. rRNA genes (5S, 16S and 23S rRNA) areoften used as the target DNA for detecting bacterialpathogens because they contain both highly conservedregions and variable species-specific regions. StandardPCR might be sufficient for the detection of pathogens,however if the pathogen needs to be identified to specieslevel [26], differentiated from closely related species, or iflive pathogens need to be distinguished from deadpathogens then modifications and extensions to themethod are required. Some of the most common PCRvariations used in diagnostics are: nested PCR; randomamplified polymorphic DNA (RAPD); reverse transcrip-tase-PCR (RT–PCR); reverse cross blot PCR (rcb-PCR);and RT–PCR enzyme hybridization assay [6,26,27], whichoffers the advantages of detecting live pathogens and alarge sample throughput. In-situ hybridization and in situhybridization–PCR are techniques for visualizing specificDNA and RNA sequences within cells. These are mainlyused for research purposes to identify and localizeinfections, map gene sequences to chromosomes, identify

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sites of gene expression and the portals of entry ofpathogens [28].

Powerful new technologies that can be applied to

aquatic health

Although immunodiagnostics have already made a sig-nificant impact on disease management in aquaculturethere is potential for rapid advancements in the future:novel methods of antibody production (e.g. recombinantantibody technology) will extend the existing range ofprobes available [29]. Multiplex testing systems (e.g. usingLuminex technology) [30,31], in which a variety ofpathogens or immune response molecules (e.g. cytokines,lysozyme, IgM) can be detected, simultaneously, offer anew dimension to fish health management and, in thefuture, such tests will make a significant impact ondisease screening programs in aquaculture. Luminextechnology also offers the potential for epitope mappingand therefore for vaccine development (J. Costa, PhDthesis, University of Stirling, 2005) [15], whereas confocalmicroscopy can provide novel basic information onparasite morphology (Figure 5) (C. McGurk, PhD thesis,University of Stirling, 2005) [32].

The rapid development of molecular technologies suchas real time PCR and nucleic acid-based sequenceamplification (NASBA) [33,34] provide methods thatreduce the possibility of contamination and offer highsample-throughput. In addition, microarray technology,although complex and in its infancy with regard toaquaculture, offers a new dimension to diagnostics andthe investigation of host–pathogen interactions, withpossibilities for the detection of DNA, RNA orproteins [35].

Future directions

During the past decade, the term ‘Omics’ has been coinedand applied to different areas such as the genome(genomics) and the proteome (proteomics). The merging

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of two existing technologies (2-dimensional gel electro-phoresis and mass spectrometry) led to the new field ofproteomics, which is a fast growing area for clinical andveterinary medicine [36,37]. Because this technology canassist both in the development of new vaccines and thediagnosis of disease it is of great interest to fish healthspecialists [38]. A major drawback is that the databasescurrently hold little information relating to aquaculture;datasets from diseased fish and from fish pathogens needto be collected on a large scale before this technology canbe fully used. In addition, although 2D electrophoresis isthe current gold standard for detecting variation in theexpression of proteins, this procedure is time-consuming,expensive and reproducibility is a problem. Even incombination with mass spectrometry, only the moreabundant proteins can be detected, thus indicating theneed for new technologies. Protein arrays and, inparticular, antibody micro-arrays might hold potentialfor the future [39,40].

References

1 Hill, B.J. (2005) The need for effective disease control in internationalaquaculture. Dev. Biol. (Basel) 121, 3–12

2 Engelstad, M. (2005) Vaccination and consumer perception of seaquality. Dev. Biol. (Basel) 121, 245–254

3 Gudding, R. et al. (1999) Recent developments in fish vaccinology. Vet.Immunol. Immunopathol. 72, 203–212

4 Thompson, K.D. and Adams, A. (2004) Current trends in immu-notherapy and vaccine development for bacterial diseases of fish. InMolecular Aspects of Fish and Marine Biology: Current Trends in theStudy of Bacterial and Viral Fish and Shrimp Diseases (Vol 3) (Leung,K-Y., ed.), pp. 313–362, World Scientific Publishing Co.

5 Adams, A. (1999) Application of antibody probes in the diagnosis andcontrol of fish diseases. In Aquaculture and Biotechnology(Karunasagar, I., Karunasagar, I. and Reilly, A., eds), pp. 1–12,Oxford & IHB Publishing Co. PVT. Ltd

6 Cunningham, C.O. (2004) Use of molecular diagnostic tests in diseasecontrol: making the leap from laboratory to field application. InMolecular Aspects of Fish and Marine Biology: Current Trends in theStudy of Bacterial and Viral Fish and ShrimpDiseases (Vol. 3) (Leung,K-Y., ed.), pp. 292–312, World Scientific Publishing Co.

7 Chinabut, S. and Puttinaowarat, S. (2005) The choice of diseasecontrol strategies to secure international market access for aqua-culture products. Dev. Biol. (Basel) 121, 255–261

8 Benmansour, A. and de Kinkelin, P. (1997) Live fish vaccines: historyand perspectives. Dev. Biol. Stand. 90, 279–289

9 Leong, J.C. et al. (1997) Fish vaccine antigens produced or delivered byrecombinant DNA technologies. Dev. Biol. Stand. 90, 267–277

10 H stein, T. et al. (2005) Bacterial vaccines for fish – an update of thecurrent situation worldwide. Dev. Biol. (Basel) 121, 55–74

11 Biering, E. et al. (2005) Update on viral vaccines for fish. Dev. Biol.121, 97–113

12 Sommerset et al. (2005) Vaccines for fish in aquaculture. Expert Rev.Vaccines 4, 89–101

13 Heppell, J. et al. (1998) Development of DNA vaccines for fish: vectordesign, intramuscular injection and antigen expression using viralhaemorrhagic septicaemia virus genes as model. Fish ShellfishImmunol. 8, 271–286

14 Lee, K.H. (2001) Proteomics: a technology-driven and technology-limited discovery science. Trends Biotechnol. 19, 217–222

15 Komatsu, N. et al. (2004) New multiplexed flow cytometric assay tomeasure anti-peptide antibody: a novel tool for monitoring immuneresponses to peptides used for immunization. Scand. J. Clin. Lab.Invest. 64, 535–546

www.sciencedirect.com

16 Staats, H.F. et al. (1999) IL-1 is an effective adjuvant for mucosal andsystemic immune responses when coadministered with proteinimmunogens. J. Immunol. 162, 6141–6147

17 Anderson, J.M. and Shive, M.S. (1997) Biodegradation and biocompat-ibility of PLA and PLGA microspheres. Adv. Drug Deliv. Rev. 28, 5–24

18 Adams, A. et al. (1995) Development and use of monoclonal antibodyprobes for immunohistochemistry, ELISA and IFAT to detect bacterialand parasitic fish pathogens. Fish Shellfish Immunol. 5, 537–547

19 Adams, A. and Thompson, K.D. (2005). Applications of monoclonalantibodies in fish health control. In Fish and Shellfish Immunology

(Swain, P., ed.), The Narendra Publisher20 Adams, A. and Marin de Mateo, M. (1994) Immunohistochemical

detection of fish pathogens. In Techniques in Fish Immunology (Vol 3)(Stolen, J.S., et al., eds), SOS Publications

21 Adams, A. (1992) Sandwich enzyme linked immunosorbent assay(ELISA) to detect and quantify bacterial pathogens in fish tissue. InTechniques in Fish Immunology (Vol 2) (Stolen, J.S. et al., eds), pp177–184, SOS Publications

22 Fournier, P-E. and Raoult, D. (2003) Comparison of PCR and serologyassays for early diagnosis of acute Q fever. J. Clin. Microbiol. 41,5094–5098

23 Palmer-Densmore, M.L. et al. (1998) Development and evaluation ofan ELISA to measure antibody responses to both the nucleocapsid andspike proteins of canine coronavirus. J. Immunoassay 19, 1–22

24 Yuce, A. et al. (2001) Serodiagnosis of tuberculosis by enzymeimmunoassay using A60 antigen. Clin. Microbiol. Infect. 7, 372–376

25 Morris, D.C. et al. (2002) Development of improved PCR to preventfalse positives and false negatives in the detection of Tetracapsula

bryosalmonae, the causative agent of proliferative kidney disease.J. Fish Dis. 25, 483–490

26 Puttinaowarat, S. et al. (2000) Mycobacteriosis: detection andidentification of aquatic Mycobacterium species. Fish Vet. J. 5, 6–21

27 Wilson, T. and Carson, J. (2003) Development of sensitive, high-throughput one-tube RT–PCR-enzyme hybridisation assay to detect

selected bacterial fish pathogens. Dis. Aquat. Organ. 54, 127–13428 Morris, D.J. et al. (2000) In situ hybridisation identifies the gill as a

portal of entry for PKX (Phylum: Myxozoa), the causative agent ofproliferative kidney disease. Parasit. Res. 86, 950–956

29 Breitling, F. and Dubel, S. (1999).Recombinant antibodies, JohnWileyand Sons, Inc.

30 Oliver, K.G. et al. (1998) Mulitplexed analysis of human cytokines byuse of the FlowMetrix system. Clin. Chem. 44, 2057–2060

31 Seideman, J. and Peritt, D. (2002) A novel monoclonal antibodyscreening method using the Luminex-100 microsphere system.J. Immunol. Methods 267, 165–171

32 McGurk, C. et al. (2005) Microscopic studies of the link betweensalmonid proliferative kidney disease (PKD) & bryozoans. Fish Vet. J.

8, 62–7133 Overturf, K. et al. (2001) Real-time PCR for the quantitative analysis

of IHNV in salmonids. J. Fish Dis. 24, 325–33334 Starkey, W. et al. (2004) Detection of piscine noda viruses using real

time nucleic acid based sequence amplification (NASBA). Dis. Aquat.

Org. 59, 93–10035 Kusnezow, W. and Hoheisel, J.D. (2002) Antibody microarrays:

promises and problems. Biotechniques 33(suppl), 14–2336 Arnon, R. and Ben-Yedidia, T. (2003) Old and new vaccine approaches.

Int. Immunopharmacol. 3, 1195–120437 Harry, J.L. et al. (2000) Proteomics: capacity versus utility. Electro-

phoresis 21, 1071–108138 Chen et al. (2004) Rapid screening of highly efficient vaccine

candidates by immunoproteomics. Proteomics 4, 3203–321339 MacBeath, G. (2002) Protein microarrays and proteomics. Nature

Genetics 32, 526–53240 Belov, L. et al. (2001) Immunophenotyping of leukemias using a

cluster of differentiation antibody microarray. Cancer Res. 61,4483–4489