- Email: [email protected]
Progress in understanding the fish pathogen aeromonas salmonicida
131
marine biotechnology At least in the microbial sector, this requirement for species separation has been a major impediment to the comprehensive analysis of biotechnological potential. The current belief that less than 10% (and possibly less than 0.1% in certain environments) of the microbial diversity in any environmental sample is culturable” is evidence of the gulf between the potential of the resource and our present ability to access it. Recent developments in molecular technology offer a route to the analysis of natural microbial biodiversity which circumvents the limitations imposed by the need to isolate and culture individual species. The efficient extraction of total DNA &om an environmental sample provides a mechanism for the simultaneous analysis of multiple genomes, potentially representing the total microbial diversity within that sample. Homologues of known gene sequences can be identified and isolated by hybridization probes or PCR amplification, while gene products of any type may be identified by screening of expression libraries. The lat-
ter approach, currently able and, depending on protocol, might be used bioactive peptides or multi-gene complexes.
in its infancy, is highly adaptthe ingenuity of the screening for identifying novel enzymes, even the products of linked
References 1 Van Dover, C. L. (1995) m Hydr~rhemral lienrs and Processes (Parsons, L. M., Walker, C. L. and DIXON, D. R., eds), pp. 257-294, Geological Socxq 2 Bull, A. T., Goodfellow, M. and Slacer, J. H. (1992) Annu. Rev. Microbid.
46, 219-252
3 DeLong, E. F., Wu, K. Y., Prkzelin, B. B. andJovine, V. M. (1994) Mature 371, 695-697 4 Car&, B. K. (1992) Cur. Opin. Biotechnol. 4, 275-279 5 Ireland, C. M. et al. (1992) in Marine BioterhnoloXy (Vol. 1) (Attaway, D. H. and Zabonky, A., eds), pp. l-43, Plenum Press 6 Gnffith, M. and Ewart, K. V. (1995) Biotechnol. Ado. 13, 375-402 7 Warren, G. J. (1987) Biotechnol. Genet. Erg. Rev. 5, 107-135 8 Yamamoto, H. (1996) Biotechnol. Gtwer. Eng. Rev. 13, 133-165 9 Bruce, K. D., Hioms, W. D., Hobman, J. L., Osbom, A. M., Strike, P. and Rx&e, D. A. (1992) Appl. Environ. Minobiol. 58, 3413-3416
Progress in understanding the fish pathogen Aeromonas salmonicida Brian Austin Aeromonas salmonicida is the causal agent of furunculosis in various fish species. Detection methods include culturing, serology and molecular biology techniques. Controversy surrounds its possible independent existence in water; enzyme-linked immunosorbent A. salmonicida
assay and the polymerase in the absence of colony-forming
chain reaction
have detected
units, but cells that are non-
culturable may be significant to fish pathology. Furunculosis is probably transmitted by the pathogen’s entry into gills, mouth, anus and/or surface injury of fish through contact with infected fish or contaminated
water. Disease-control is possible by
good husbandry practices, disease-resistant stock, improved diets, nonspecific immunostimulants,
antimicrobial
compounds and vaccines.
A diverse range ofbacteria, fungi, protozoa and viruses is associated with diseases of marine animals. Among the bacteria, Vibrio spp., including K anguillanrm, L! (Photobacterium) damsela, K ordalii and V salmonicida, are commonly associated with a haemorrhagic B. Austin ([email protected]) is ut the Department oj’ Biological Sciences, Heriot- Watt University, Riccurton, Edinbugh, UK EH14 4AS. Copyright
0 1997, Elsevler
Scmce
Ltd. All rghts reserved.
0167
- 7799/97/$17.00.
septicaemia, termed vibriosisl. The fungal species Zchthyophonlrs hoferi affects many marine species, causing a disease known as ‘ich’*. One of the most serious virus diseases, affecting many fish species in the sea, is lymphocystis2. AU these pathogens cause losses in wild and farmed fish stocks. In terms of the application of biotechnology to the control of fish disease, emphasis has been placed on one bacterial fish pathogen, namely Aeromonas salmonicida (Box 1). PII: SO167-7799(97)01026-3
TIBTECH APRIL 1997 NOL 15)
132
marine biotechnology Box 1. Characteristics of Aeromonas salmonicidal Gram-negative,non-motile,fermentative rods, which typically producebrown diffusiblepigmenton proteincontainingmediumsuch as brain heart infusionagar (BHIAI.Incapableof growth at 37°C. On BHIAit may develop any of three different colony types, termed ‘rough’,‘smooth’and‘G-phase’ becauseof the presence or absenceof anexternalproteinaceouslayer (A-layer). Cell-wall-defective/deficient forms (L-forms)have been recognized.Atypical isolatesmay be slow in pigmentingandnutritionallyfastidious,requiringblood-products for growth. By definition,cellsdo not occur in surface water, and are pathogenicto fish. A. salmor~icida is the causalagent of furunculosis, and is one of the oldest known fish pathogens1,3.The pathogen hasa worldwide distribution, causinginfections in representativesof many families of fish, including Anoplopomidae, Cyprinidae, Salmonidae and Serranidae. Classicalfurunculosis involves a haemorrhagic septicaemia,including the presenceof furuncles (boils) on the flanks. The seriousnessof furunculosis to aquaculture (the rearing of aquatic speciesin controlled conditions) is illustrated by the epidemic in 1991-1992, which led to the lossof -10000 tonnes (roughly equivalent to 25% of the total production) of Atlantic salmon (S&no sular) in Scotland. Although troublesome initially in freshwater, A. salmorlicidahas emerged asa major pathogen of salmonidsand other fish, such asdabs and flounder, in the seaJ.“.In these fish, the diseasemay be of a different form from classicalfurunculosis, as it results in ulcers, and the aetiological agent is deemed to be ‘atypical’A,j. Three outstanding problems in A, salmonicida biology remain to be resolved:the determination of the preciselocation and form of the organism in asymptomatic/carrier fish, the location of the reservoir of the pathogen in the aquatic environment, and the availability of effective diseasecontrol measures,especially for fish other than salmonids.
confirm the presenceofA. sulmonicidu. Methods range from slideagglutination8 to the latex agglutination test9 and enzyme-linked immunosorbent assay(ELISA) lo. Latex agglutination, of which one system has been commercialized, may result in diagnoseswithin 2 min to 2 h for pure/mixed cultures or heavily infected fish tissues,respectively. ELISA enablesreliable diagnoses in 3G60 min, and appearsto be effective for usewith asymptomatic carrier fishl.10. The ongoing debate concerning the relative merits of monoclonal and polyclonal antibodiesin serologicaltechniques remains to be resolved. DNA probes have the ability to detect A. salmonicidu in clinical and environmental samples.Moreover, a DNA fragment specific to A. sulmonicidu was incorporated into a polymerasechain reaction (PCR) technique and usedto detect approximately two celIsll. By meansof PCR and a specific DNA probe, the presence of A. satmonicidu has been reported in effluent, water, faecesand sediment from a commercial freshwater Atlantic salmon farm in Ireland12*13. In contrast, culturing only recovered colony-forming units from clinically diseasedfish. PCR technology has enabled the demonstration of DNA, four months after the use of furunculosis vaccines, in the kidney and spleen of Atlantic salmon (S. sulur)‘-‘. Both serology and molecular methods suffer f?om the disadvantage that they may not distinguish living (pathogenic) from dead cells, such as those incorporated in many vaccines. Therefore, in the long term, the increasing sensitivity of modern detection methods may not be too helpful with the management of diseasecaused by A. salmorzicida.
The possiblesurvival of A. sulmonicida in water has been extensively researched’.The datasuggestthat the pathogen can survive through varying periods and temperatures in fresh, brackish and seawater,and the underlying sediment. A consensusview is that there is a concomitant reduction in pathogenicity when the organism leavesthe fish and enters the aquatic environment’j. Sakaihassuggestedthat the long-term survival ofA. sulmonicida in the aquatic environment may reflect electrostatic charge differences on individual cells, with positive and negative chargeson avirulent Detection A. salmorzicida is difficult to recover from fish that do and virulent cells, respectivelylj. Virulent cells may be not show clinical signsof disease,and from the aquatic able to survive under starvation conditions in river environment, even during the epizootics of furuncusediments.A decline in numbers might be causedby the spontaneous occurrence of avirulent free-living losis. Routinely, cultures may be grown on tryptone soya agar or brain heart infusion agar following incucells, which enter a dormant phase due to a lack of bation at 15-25°C. Atypical isolates often need the nutrientsls. Furthermore, these tiee-living cells could inclusion of blood or serum in the isolation medium’. represent a transitional form, leading eventually to a loss of viability. Interestingly, the onset of the dorPre-incubation of pathological material for 24-48 h in tryptone soyabroth6 followed by the useof Coomassie mant/non-culturable phase may be delayed by the Brilliant agar improves recovery of the pathogen”,‘. presence of the amino acids arginine and methionHowever, an effective selective medium remainsto be inel”. Although the possible presence of dormant/ described. Alternative approachesto culturing for the non-culturable cells in the aquatic environment has been vigorously debated, there is accumulating evidetection of A. sulmonicidaand diagnosisof furunculosis have included serology and molecular biolo,T dence to suggest that intact cells of A. sulmonicidu techniques. remain in aquatic habitats after the number of colonySerology has been used successfully on pure and forming units has declined to zeroIT. Attempts to mixed cultures and with pathological material to revive these cells, principally by the addition of TIBTECH APRIL 1997 WOL 151
133
marine biotechnology nutrients, have been mostly unsuccessful. Whether the cells are damaged or dying has not been resolved but it is possible that they are present in an altered condition, necessitating specialized growth conditions. This is supported by studies by Effendi and Austin’s ofmarine samples considered to be devoid of culturable A. salmonicida but that contained cells capable of passing through the pores of 0.22 and 0.45 p,rn porosity filters; some of these cells grew on specialized media developed for the growth of cell-wall-defective/ deficient forms of A. salmonicida, i.e. L-formsrg. Thus, populations of A. salmonicida of -10” cells ml-l were recorded in experimental microcosms after conventional plate counts had reached zero. These findings suggest that specialized forms of A. salmonicida, such as L-forms, may contribute to the difficulty in recovering the pathogen from environmental samples. Yet, the role of such modified cells in disease outbreaks is unclear. Certainly, natural L-forms have been observed in salmonids but conclusive proof of an association with disease outbreaks is lacking”. If A. salmonicida cells that are dormant, non-culturable, altered, senescent or dying are unable to reproduce disease then their relevance to fish pathology is questionable. It has been realized that nucleic acids from A. salmonicida may be shed into the aquatic environment. In particular, DNA has been found in aquatic sediments > 13 weeks after colony counts declined to below detectable limit.s20. In future, molecular biology techniques may well be used to address the issue of the presence of living versus dead cells, principally by the detection of specific RNAs that should be present in truly viable cells. Transmission Fish are undoubtedly important in the transmission of furunculosis. Indeed, clinically diseased and carrier fish have long been associated with the spread of infection’. Carriers harbour the pathogen in the external mucus, gills and spleenb, enabling release when clinical disease occurs, such as after immunosuppressionr. Therefore, effective methods are essential for the detection of carriers. A combination of increasing the water temperature to 18°C and the injection of corticosteroids is often employed to induce the development of clinical disease from carriers’, Antibiotic therapy, which is often used to combat outbreaks of furunculosisr, does not necessarily eliminate the carrier state. Indeed, it is possible that inappropriate antibiotic treatment regimes might lead to the establishment of altered forms of A. salmonicida, such as L-forms, in fish. Certainly, contact with infected fish or contaminated water must be regarded as the most likely means for the transmission of furunculosisl. The site(s) of uptake of the pathogen into fish, although remaining the subject of conjecture, seems likely to include gills, mouth, anus and/or surface injury. By immunofluorescence, Klontz*r determined that the intestine is the primary site of infection, leading to the development of the asymptomatic carrier state. McCarthy2a showed that
rainbow trout (Oncorhynchus mykiss) resisted infection with A. salmonicida unless the flanks were abraded. This suggests that A. salmonicida may enter through damaged areas on the surface of the fish. Further work demonstrated that uptake was enhanced by the presence of particulates, leading to the presence of the pathogen in the blood within a few minutes2s. Control Because of the economic importance of salmonid farming, much emphasis has been placed on developing effective control strategies for furunculosis. Methods have involved use of good husbandry practices (including ‘good’ water quality, adequate disinfection of equipment and eggs, and lower stocking densities), and disease-resistant fish stock, improved diets, nonspecific immunostimulants, antimicrobial compounds, probiotics (microorganisms that exert a beneficial effect on the host) and vaccines. Recent research has led to the recognition of the value of immunostimulatory compounds such as B-1,3glucans, synthetic peptides and killed mycobacterial cells, the latter of which enhanced disease resistance in coho salmon to A. salmonicida24. Commercial interest has centred on B-1,3-glucans for control of infections by A. salmonicida when administered by injection25. B-1,3-Glucans are now being routinely incorporated into fish diets but this possibly reflects a misrepresentation of scientitic data for commercial gain; specifically, the scientific evidence pointed to the benefit of glucans as nonspecific immunostimulants when administered by injection, not orally. It remains for further work to establish the effect of B-1,3-glucans on fish when administered orally. Vaccine research has developed since the work in 1942 of Duff2h, who used a chloroform-inactivated whole-cell suspension of A. salmonicida, and encompasses use of subcellular components (namely inactivated extracellular products and lipopolysaccharides), genetic manipulation of cells and live (avirulent) vaccines with or without adjuvants. Iron-regulated outermembrane proteins, the so-called IROMPs, are especially immunogenic to Atlantic salmona7, and have been developed into a commercial vaccine. This product is now extensively used in the UK, and may be responsible for the marked reduction in the incidence of fiu-unculosis in farmed Atlantic salmon since 1992. Yet, in laboratory-based studies, an IROMP-based vaccine was less successful than a product containing inactivated L-forms of A. salmonicida2”. An outermembrane porin has also shown potential in vaccine trials”‘. A significant development is an in viuo growth model for A. salmonicida, i.e. a specialized intraperitoneal chamber implanted in rainbow trout, which has allowed the prospect of developing gene products that are only synthesized in the host”“J1. Genetic manipulation of A. salmonicidu has led to the development of a subunit vaccine, through expression of a 587 kbp fragment of the 70 kDa serine protease gene in Escherichia coli3’. Avirulent cells lacking the extracellular A-layer (this is involved in pathogenicity) have TIETECH APRIL 1997
NOL 15)
134
marine biotechnology been proposed as suitable vaccines32. An attenuated live vaccine (DELTA-aroA) has been developed, and preferentially stimulated T-cell responses in rainbow trout33. Here, an aroA mutant (BRIVAX 1) of A. salmonicida was constructed, and found to elicit antibody response. In additi on, a live attenuated vaccine has been used as a carrier for heterologous antigen expression3”. Certainly, the administration of such vaccines to fish led to the development of antibodies specific for A. ralmonicida3s. Following a comparison of intraperitoneal, immersion and oral vaccination methods in Atlantic salmon, the benefit of the first method was clearly demonstrated in terms of the highest antibody titre and level of protectionss. Within 16 weeks of administration by injection, A. salmonicida lipopolysaccharide was located mostly in the abdominal granulomas, head kidney and spleerG5. The benefit of adjuvants, especially those comprising mineral oil, for stimulating a protective immune response following intraperitoneal vaccination has been confirmeds6, despite the potential harmful side-effects to fish. Studies are now being directed at developing improved, non-oily adjuvants, which have negligible harmful effects on fish. In addition, an effective oral vaccine is sorely needed by the aquaculture industry. Members of the normal microflora of fish may be useful as probiotics. Already, a strain of Vibrio alginolyticus, which was recovered from penaeid shrimps, has been found to aid the control of furunculosis in salmonid.G7. Other probiotics that may control furunculosis will undoubtedly be identified in the future.
Conclusion Biotechnology is involved in the detection and control of infections caused by A. salmonicida. Clearly, there is an urgent need for rapid and reliable diagnostic systems suitable for field use. Such systems should provide information about the presence or likely onset of a disease condition, and not generate data on possible ‘natural’ population levels of A. salmonicida. In addition, disease control measures will continue to be researched, particularly concerning probiotics, nonspecific immunostimulants and vaccines. A pessimistic view is that infections caused by A. salmonicida will not disappear from farmed fish populations, necessitating the continual development of ‘new’ or improved disease control measures.
References 1 Austin, B. and Austin, D. A. (1993) Bacterial Fi& P&qen~: Disease in Farmed and Wild Fish (2nd edn), EIIis Horwood 2 Austin, B. (1988) Marine Microbiology, Cambridge Umversity Press 3 Austin, B. and Adams, C. (1996) in 77~ Genus Aeromonas
(Austm, B., pp. 197-243, 4 WikIund, T. 5 Wikhmd, T.
AItwegg, M., Goshng, P. J. and Joseph, S., eds), Wiley (1995) Dir. Aquat. Ox. 21, 145-150 and Dalsgaard, I. (1995) j, Aquat. A&. Health 7,
218-224
6 Clpnano, R. C., Bullock, G. L. and Noble, A. (1996)J. Aquat. Anim. Healtl1 8, 47-5 1 7 Daly, J. G. and Stevenson. R. M. W. (1985) Trans. Am. Fish Sm. 114, 909-910 8 Rabb, L., Comlck, J. W. and MacDermott, L. A. (1964) Prof. Fir/~ Cult. 26, 118-120 9 McCarthy, D. H. (1975)J. Gw. &ficrubiol. 88, 384-386 10 Austin, B., Bishop, 1.. Gray. C., Watt, B. and Dawes, J. (1986)]. Ficlt Dis. 9, 469-474 11 Hiney, M., Dawson, M. T., Heery, D. M., Smith, P. R., Gannon, F. and Powell, R. (1992) ilppl. Environ. Microbial. 58, 103(rlO42 12 O’Brien, F. et al. (1994) Appl. Et~vmn. Mimbiol. 60, 387+3877 13 Mooney, J., Powell, E., Clabby, C. and Powell, R. (1995) Dir. Aquar. Orf. 21,131-135 14 Hoie, S., Hewn, M. and Thoreren. 0. F. (1996) Fir/~ [email protected] Zmmrrnol. 6, 19’%206 15 Sakal, D. K. (1986) ,;lppl. 6wrrun. Mim>biof. 51, 1343-1349 16 Plckup, R. W., Rhodes. C., Cobban, R. J. and Clarke, K. J. (1996) 1. Fix/l Die. 19, 65-74 17 Morgan, J. A. W., Cramwell, P. A. and Plckup. R. W. (1991) Appl. Environ. Miuobrol. 57, 1777-1782 18 Effendi, I. and Aurnn. 8. (1994)]. FIS/I Die. 17, 375-385 19 McIntosh, D. and Austm, B. (1990) Syst. Appl. Microbial. 13,378-381 20 Deere, D., Porter, J.. Plckup, R. W and Edwards, C. (1996)j. Appl. Bacferiol. 81. 309-3 18 21 KIonu, G. W. (1968) fit%. Spvrf Fich. Ret 39, 81-82 22 McCarthy, D. H. (1980) .4qrcar. .2licrobiul. 6, 299-324 23 Hodgkmson, J. J.. Bucke. I>. and Austm. B. (1987) FE1!4S Microbial. Left. 40,207-210 24 Ohvler, G., Evelyn, T. P. T. and Lallirr, R. (1985)j. Fisk Dis. 8, 43-55 25 NlkI, L.. AIbnght, L. J. and Evelyn. T. P. T. (1991) Dts. Aquaf. 0~. 12,7-12 26 Duff, D. B. (1942)]. Iwtwzsl. 41, 87-94 27 Lutwyche, P., Exner. M. M., Hancock, R. E. W. and Trust, T. J. (1995) Infcit. Im,nun. 63, 3137-3142 28 McIntosh, D. and Austin, B. (1993)J. Aqzmt. A&. Health 5,254-258 29 Bennett, A. J.. Whtby, P. W. and Coleman, G. (1992)J. Fish Dir. 15, 473-484 30 Garduno, R. A., Thornton, J. C. and Day, W. W. (1993) It@?. Zmmtrnol. 61, 485+4862 31 Garduno, K. A.. Thornton. J. C. and Day, W. W. (1993) Can. 1. &ficrobiol. 39, 1051-1058 32 Thornton, J. C , Garduno, R. A. and Day, W. W. (1994)j. Fi.ch Dis. 17, 195-204 33 Marsden, M. J., Vaughan, L. M.. Foster, T. J. and Secombes, C. J. (1996) @ct. 1mmuwl. 64, 3863-3869 34 Noonan, B., Enzmann, P. J. and Trust, T. J. (1996) Appl. Envirorz. ,2ficrobiol. 6 1, 3586-359 1 35 Press, C. M., Evencen, 0.. Reltan. L. J. and Landsverk, T. (1996) 1. Fish Dis. 19, 215-224 36 Mldtlyng, P. J.. Rexan, L. J. and Speilberg, L. (1996) Fisk Shel@h Immunol.
37 Austm, Gnffith,
6. 335-35U
B.. Stuckey. L. F.. Robertson, P. A. W., Effendi, D. R. W. (1995) 1. Fisll DIS. 18, 93-96
In the next issue: Bioremediationof marine oil spills
TIBTECH APRIL 1997 (VOL 15)
I. and
marine biotechnology At least in the microbial sector, this requirement for species separation has been a major impediment to the comprehensive analysis of biotechnological potential. The current belief that less than 10% (and possibly less than 0.1% in certain environments) of the microbial diversity in any environmental sample is culturable” is evidence of the gulf between the potential of the resource and our present ability to access it. Recent developments in molecular technology offer a route to the analysis of natural microbial biodiversity which circumvents the limitations imposed by the need to isolate and culture individual species. The efficient extraction of total DNA &om an environmental sample provides a mechanism for the simultaneous analysis of multiple genomes, potentially representing the total microbial diversity within that sample. Homologues of known gene sequences can be identified and isolated by hybridization probes or PCR amplification, while gene products of any type may be identified by screening of expression libraries. The lat-
ter approach, currently able and, depending on protocol, might be used bioactive peptides or multi-gene complexes.
in its infancy, is highly adaptthe ingenuity of the screening for identifying novel enzymes, even the products of linked
References 1 Van Dover, C. L. (1995) m Hydr~rhemral lienrs and Processes (Parsons, L. M., Walker, C. L. and DIXON, D. R., eds), pp. 257-294, Geological Socxq 2 Bull, A. T., Goodfellow, M. and Slacer, J. H. (1992) Annu. Rev. Microbid.
46, 219-252
3 DeLong, E. F., Wu, K. Y., Prkzelin, B. B. andJovine, V. M. (1994) Mature 371, 695-697 4 Car&, B. K. (1992) Cur. Opin. Biotechnol. 4, 275-279 5 Ireland, C. M. et al. (1992) in Marine BioterhnoloXy (Vol. 1) (Attaway, D. H. and Zabonky, A., eds), pp. l-43, Plenum Press 6 Gnffith, M. and Ewart, K. V. (1995) Biotechnol. Ado. 13, 375-402 7 Warren, G. J. (1987) Biotechnol. Genet. Erg. Rev. 5, 107-135 8 Yamamoto, H. (1996) Biotechnol. Gtwer. Eng. Rev. 13, 133-165 9 Bruce, K. D., Hioms, W. D., Hobman, J. L., Osbom, A. M., Strike, P. and Rx&e, D. A. (1992) Appl. Environ. Minobiol. 58, 3413-3416
Progress in understanding the fish pathogen Aeromonas salmonicida Brian Austin Aeromonas salmonicida is the causal agent of furunculosis in various fish species. Detection methods include culturing, serology and molecular biology techniques. Controversy surrounds its possible independent existence in water; enzyme-linked immunosorbent A. salmonicida
assay and the polymerase in the absence of colony-forming
chain reaction
have detected
units, but cells that are non-
culturable may be significant to fish pathology. Furunculosis is probably transmitted by the pathogen’s entry into gills, mouth, anus and/or surface injury of fish through contact with infected fish or contaminated
water. Disease-control is possible by
good husbandry practices, disease-resistant stock, improved diets, nonspecific immunostimulants,
antimicrobial
compounds and vaccines.
A diverse range ofbacteria, fungi, protozoa and viruses is associated with diseases of marine animals. Among the bacteria, Vibrio spp., including K anguillanrm, L! (Photobacterium) damsela, K ordalii and V salmonicida, are commonly associated with a haemorrhagic B. Austin ([email protected]) is ut the Department oj’ Biological Sciences, Heriot- Watt University, Riccurton, Edinbugh, UK EH14 4AS. Copyright
0 1997, Elsevler
Scmce
Ltd. All rghts reserved.
0167
- 7799/97/$17.00.
septicaemia, termed vibriosisl. The fungal species Zchthyophonlrs hoferi affects many marine species, causing a disease known as ‘ich’*. One of the most serious virus diseases, affecting many fish species in the sea, is lymphocystis2. AU these pathogens cause losses in wild and farmed fish stocks. In terms of the application of biotechnology to the control of fish disease, emphasis has been placed on one bacterial fish pathogen, namely Aeromonas salmonicida (Box 1). PII: SO167-7799(97)01026-3
TIBTECH APRIL 1997 NOL 15)
132
marine biotechnology Box 1. Characteristics of Aeromonas salmonicidal Gram-negative,non-motile,fermentative rods, which typically producebrown diffusiblepigmenton proteincontainingmediumsuch as brain heart infusionagar (BHIAI.Incapableof growth at 37°C. On BHIAit may develop any of three different colony types, termed ‘rough’,‘smooth’and‘G-phase’ becauseof the presence or absenceof anexternalproteinaceouslayer (A-layer). Cell-wall-defective/deficient forms (L-forms)have been recognized.Atypical isolatesmay be slow in pigmentingandnutritionallyfastidious,requiringblood-products for growth. By definition,cellsdo not occur in surface water, and are pathogenicto fish. A. salmor~icida is the causalagent of furunculosis, and is one of the oldest known fish pathogens1,3.The pathogen hasa worldwide distribution, causinginfections in representativesof many families of fish, including Anoplopomidae, Cyprinidae, Salmonidae and Serranidae. Classicalfurunculosis involves a haemorrhagic septicaemia,including the presenceof furuncles (boils) on the flanks. The seriousnessof furunculosis to aquaculture (the rearing of aquatic speciesin controlled conditions) is illustrated by the epidemic in 1991-1992, which led to the lossof -10000 tonnes (roughly equivalent to 25% of the total production) of Atlantic salmon (S&no sular) in Scotland. Although troublesome initially in freshwater, A. salmorlicidahas emerged asa major pathogen of salmonidsand other fish, such asdabs and flounder, in the seaJ.“.In these fish, the diseasemay be of a different form from classicalfurunculosis, as it results in ulcers, and the aetiological agent is deemed to be ‘atypical’A,j. Three outstanding problems in A, salmonicida biology remain to be resolved:the determination of the preciselocation and form of the organism in asymptomatic/carrier fish, the location of the reservoir of the pathogen in the aquatic environment, and the availability of effective diseasecontrol measures,especially for fish other than salmonids.
confirm the presenceofA. sulmonicidu. Methods range from slideagglutination8 to the latex agglutination test9 and enzyme-linked immunosorbent assay(ELISA) lo. Latex agglutination, of which one system has been commercialized, may result in diagnoseswithin 2 min to 2 h for pure/mixed cultures or heavily infected fish tissues,respectively. ELISA enablesreliable diagnoses in 3G60 min, and appearsto be effective for usewith asymptomatic carrier fishl.10. The ongoing debate concerning the relative merits of monoclonal and polyclonal antibodiesin serologicaltechniques remains to be resolved. DNA probes have the ability to detect A. salmonicidu in clinical and environmental samples.Moreover, a DNA fragment specific to A. sulmonicidu was incorporated into a polymerasechain reaction (PCR) technique and usedto detect approximately two celIsll. By meansof PCR and a specific DNA probe, the presence of A. satmonicidu has been reported in effluent, water, faecesand sediment from a commercial freshwater Atlantic salmon farm in Ireland12*13. In contrast, culturing only recovered colony-forming units from clinically diseasedfish. PCR technology has enabled the demonstration of DNA, four months after the use of furunculosis vaccines, in the kidney and spleen of Atlantic salmon (S. sulur)‘-‘. Both serology and molecular methods suffer f?om the disadvantage that they may not distinguish living (pathogenic) from dead cells, such as those incorporated in many vaccines. Therefore, in the long term, the increasing sensitivity of modern detection methods may not be too helpful with the management of diseasecaused by A. salmorzicida.
The possiblesurvival of A. sulmonicida in water has been extensively researched’.The datasuggestthat the pathogen can survive through varying periods and temperatures in fresh, brackish and seawater,and the underlying sediment. A consensusview is that there is a concomitant reduction in pathogenicity when the organism leavesthe fish and enters the aquatic environment’j. Sakaihassuggestedthat the long-term survival ofA. sulmonicida in the aquatic environment may reflect electrostatic charge differences on individual cells, with positive and negative chargeson avirulent Detection A. salmorzicida is difficult to recover from fish that do and virulent cells, respectivelylj. Virulent cells may be not show clinical signsof disease,and from the aquatic able to survive under starvation conditions in river environment, even during the epizootics of furuncusediments.A decline in numbers might be causedby the spontaneous occurrence of avirulent free-living losis. Routinely, cultures may be grown on tryptone soya agar or brain heart infusion agar following incucells, which enter a dormant phase due to a lack of bation at 15-25°C. Atypical isolates often need the nutrientsls. Furthermore, these tiee-living cells could inclusion of blood or serum in the isolation medium’. represent a transitional form, leading eventually to a loss of viability. Interestingly, the onset of the dorPre-incubation of pathological material for 24-48 h in tryptone soyabroth6 followed by the useof Coomassie mant/non-culturable phase may be delayed by the Brilliant agar improves recovery of the pathogen”,‘. presence of the amino acids arginine and methionHowever, an effective selective medium remainsto be inel”. Although the possible presence of dormant/ described. Alternative approachesto culturing for the non-culturable cells in the aquatic environment has been vigorously debated, there is accumulating evidetection of A. sulmonicidaand diagnosisof furunculosis have included serology and molecular biolo,T dence to suggest that intact cells of A. sulmonicidu techniques. remain in aquatic habitats after the number of colonySerology has been used successfully on pure and forming units has declined to zeroIT. Attempts to mixed cultures and with pathological material to revive these cells, principally by the addition of TIBTECH APRIL 1997 WOL 151
133
marine biotechnology nutrients, have been mostly unsuccessful. Whether the cells are damaged or dying has not been resolved but it is possible that they are present in an altered condition, necessitating specialized growth conditions. This is supported by studies by Effendi and Austin’s ofmarine samples considered to be devoid of culturable A. salmonicida but that contained cells capable of passing through the pores of 0.22 and 0.45 p,rn porosity filters; some of these cells grew on specialized media developed for the growth of cell-wall-defective/ deficient forms of A. salmonicida, i.e. L-formsrg. Thus, populations of A. salmonicida of -10” cells ml-l were recorded in experimental microcosms after conventional plate counts had reached zero. These findings suggest that specialized forms of A. salmonicida, such as L-forms, may contribute to the difficulty in recovering the pathogen from environmental samples. Yet, the role of such modified cells in disease outbreaks is unclear. Certainly, natural L-forms have been observed in salmonids but conclusive proof of an association with disease outbreaks is lacking”. If A. salmonicida cells that are dormant, non-culturable, altered, senescent or dying are unable to reproduce disease then their relevance to fish pathology is questionable. It has been realized that nucleic acids from A. salmonicida may be shed into the aquatic environment. In particular, DNA has been found in aquatic sediments > 13 weeks after colony counts declined to below detectable limit.s20. In future, molecular biology techniques may well be used to address the issue of the presence of living versus dead cells, principally by the detection of specific RNAs that should be present in truly viable cells. Transmission Fish are undoubtedly important in the transmission of furunculosis. Indeed, clinically diseased and carrier fish have long been associated with the spread of infection’. Carriers harbour the pathogen in the external mucus, gills and spleenb, enabling release when clinical disease occurs, such as after immunosuppressionr. Therefore, effective methods are essential for the detection of carriers. A combination of increasing the water temperature to 18°C and the injection of corticosteroids is often employed to induce the development of clinical disease from carriers’, Antibiotic therapy, which is often used to combat outbreaks of furunculosisr, does not necessarily eliminate the carrier state. Indeed, it is possible that inappropriate antibiotic treatment regimes might lead to the establishment of altered forms of A. salmonicida, such as L-forms, in fish. Certainly, contact with infected fish or contaminated water must be regarded as the most likely means for the transmission of furunculosisl. The site(s) of uptake of the pathogen into fish, although remaining the subject of conjecture, seems likely to include gills, mouth, anus and/or surface injury. By immunofluorescence, Klontz*r determined that the intestine is the primary site of infection, leading to the development of the asymptomatic carrier state. McCarthy2a showed that
rainbow trout (Oncorhynchus mykiss) resisted infection with A. salmonicida unless the flanks were abraded. This suggests that A. salmonicida may enter through damaged areas on the surface of the fish. Further work demonstrated that uptake was enhanced by the presence of particulates, leading to the presence of the pathogen in the blood within a few minutes2s. Control Because of the economic importance of salmonid farming, much emphasis has been placed on developing effective control strategies for furunculosis. Methods have involved use of good husbandry practices (including ‘good’ water quality, adequate disinfection of equipment and eggs, and lower stocking densities), and disease-resistant fish stock, improved diets, nonspecific immunostimulants, antimicrobial compounds, probiotics (microorganisms that exert a beneficial effect on the host) and vaccines. Recent research has led to the recognition of the value of immunostimulatory compounds such as B-1,3glucans, synthetic peptides and killed mycobacterial cells, the latter of which enhanced disease resistance in coho salmon to A. salmonicida24. Commercial interest has centred on B-1,3-glucans for control of infections by A. salmonicida when administered by injection25. B-1,3-Glucans are now being routinely incorporated into fish diets but this possibly reflects a misrepresentation of scientitic data for commercial gain; specifically, the scientific evidence pointed to the benefit of glucans as nonspecific immunostimulants when administered by injection, not orally. It remains for further work to establish the effect of B-1,3-glucans on fish when administered orally. Vaccine research has developed since the work in 1942 of Duff2h, who used a chloroform-inactivated whole-cell suspension of A. salmonicida, and encompasses use of subcellular components (namely inactivated extracellular products and lipopolysaccharides), genetic manipulation of cells and live (avirulent) vaccines with or without adjuvants. Iron-regulated outermembrane proteins, the so-called IROMPs, are especially immunogenic to Atlantic salmona7, and have been developed into a commercial vaccine. This product is now extensively used in the UK, and may be responsible for the marked reduction in the incidence of fiu-unculosis in farmed Atlantic salmon since 1992. Yet, in laboratory-based studies, an IROMP-based vaccine was less successful than a product containing inactivated L-forms of A. salmonicida2”. An outermembrane porin has also shown potential in vaccine trials”‘. A significant development is an in viuo growth model for A. salmonicida, i.e. a specialized intraperitoneal chamber implanted in rainbow trout, which has allowed the prospect of developing gene products that are only synthesized in the host”“J1. Genetic manipulation of A. salmonicidu has led to the development of a subunit vaccine, through expression of a 587 kbp fragment of the 70 kDa serine protease gene in Escherichia coli3’. Avirulent cells lacking the extracellular A-layer (this is involved in pathogenicity) have TIETECH APRIL 1997
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marine biotechnology been proposed as suitable vaccines32. An attenuated live vaccine (DELTA-aroA) has been developed, and preferentially stimulated T-cell responses in rainbow trout33. Here, an aroA mutant (BRIVAX 1) of A. salmonicida was constructed, and found to elicit antibody response. In additi on, a live attenuated vaccine has been used as a carrier for heterologous antigen expression3”. Certainly, the administration of such vaccines to fish led to the development of antibodies specific for A. ralmonicida3s. Following a comparison of intraperitoneal, immersion and oral vaccination methods in Atlantic salmon, the benefit of the first method was clearly demonstrated in terms of the highest antibody titre and level of protectionss. Within 16 weeks of administration by injection, A. salmonicida lipopolysaccharide was located mostly in the abdominal granulomas, head kidney and spleerG5. The benefit of adjuvants, especially those comprising mineral oil, for stimulating a protective immune response following intraperitoneal vaccination has been confirmeds6, despite the potential harmful side-effects to fish. Studies are now being directed at developing improved, non-oily adjuvants, which have negligible harmful effects on fish. In addition, an effective oral vaccine is sorely needed by the aquaculture industry. Members of the normal microflora of fish may be useful as probiotics. Already, a strain of Vibrio alginolyticus, which was recovered from penaeid shrimps, has been found to aid the control of furunculosis in salmonid.G7. Other probiotics that may control furunculosis will undoubtedly be identified in the future.
Conclusion Biotechnology is involved in the detection and control of infections caused by A. salmonicida. Clearly, there is an urgent need for rapid and reliable diagnostic systems suitable for field use. Such systems should provide information about the presence or likely onset of a disease condition, and not generate data on possible ‘natural’ population levels of A. salmonicida. In addition, disease control measures will continue to be researched, particularly concerning probiotics, nonspecific immunostimulants and vaccines. A pessimistic view is that infections caused by A. salmonicida will not disappear from farmed fish populations, necessitating the continual development of ‘new’ or improved disease control measures.
References 1 Austin, B. and Austin, D. A. (1993) Bacterial Fi& P&qen~: Disease in Farmed and Wild Fish (2nd edn), EIIis Horwood 2 Austin, B. (1988) Marine Microbiology, Cambridge Umversity Press 3 Austin, B. and Adams, C. (1996) in 77~ Genus Aeromonas
(Austm, B., pp. 197-243, 4 WikIund, T. 5 Wikhmd, T.
AItwegg, M., Goshng, P. J. and Joseph, S., eds), Wiley (1995) Dir. Aquat. Ox. 21, 145-150 and Dalsgaard, I. (1995) j, Aquat. A&. Health 7,
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6 Clpnano, R. C., Bullock, G. L. and Noble, A. (1996)J. Aquat. Anim. Healtl1 8, 47-5 1 7 Daly, J. G. and Stevenson. R. M. W. (1985) Trans. Am. Fish Sm. 114, 909-910 8 Rabb, L., Comlck, J. W. and MacDermott, L. A. (1964) Prof. Fir/~ Cult. 26, 118-120 9 McCarthy, D. H. (1975)J. Gw. &ficrubiol. 88, 384-386 10 Austin, B., Bishop, 1.. Gray. C., Watt, B. and Dawes, J. (1986)]. Ficlt Dis. 9, 469-474 11 Hiney, M., Dawson, M. T., Heery, D. M., Smith, P. R., Gannon, F. and Powell, R. (1992) ilppl. Environ. Microbial. 58, 103(rlO42 12 O’Brien, F. et al. (1994) Appl. Et~vmn. Mimbiol. 60, 387+3877 13 Mooney, J., Powell, E., Clabby, C. and Powell, R. (1995) Dir. Aquar. Orf. 21,131-135 14 Hoie, S., Hewn, M. and Thoreren. 0. F. (1996) Fir/~ [email protected] Zmmrrnol. 6, 19’%206 15 Sakal, D. K. (1986) ,;lppl. 6wrrun. Mim>biof. 51, 1343-1349 16 Plckup, R. W., Rhodes. C., Cobban, R. J. and Clarke, K. J. (1996) 1. Fix/l Die. 19, 65-74 17 Morgan, J. A. W., Cramwell, P. A. and Plckup. R. W. (1991) Appl. Environ. Miuobrol. 57, 1777-1782 18 Effendi, I. and Aurnn. 8. (1994)]. FIS/I Die. 17, 375-385 19 McIntosh, D. and Austm, B. (1990) Syst. Appl. Microbial. 13,378-381 20 Deere, D., Porter, J.. Plckup, R. W and Edwards, C. (1996)j. Appl. Bacferiol. 81. 309-3 18 21 KIonu, G. W. (1968) fit%. Spvrf Fich. Ret 39, 81-82 22 McCarthy, D. H. (1980) .4qrcar. .2licrobiul. 6, 299-324 23 Hodgkmson, J. J.. Bucke. I>. and Austm. B. (1987) FE1!4S Microbial. Left. 40,207-210 24 Ohvler, G., Evelyn, T. P. T. and Lallirr, R. (1985)j. Fisk Dis. 8, 43-55 25 NlkI, L.. AIbnght, L. J. and Evelyn. T. P. T. (1991) Dts. Aquaf. 0~. 12,7-12 26 Duff, D. B. (1942)]. Iwtwzsl. 41, 87-94 27 Lutwyche, P., Exner. M. M., Hancock, R. E. W. and Trust, T. J. (1995) Infcit. Im,nun. 63, 3137-3142 28 McIntosh, D. and Austin, B. (1993)J. Aqzmt. A&. Health 5,254-258 29 Bennett, A. J.. Whtby, P. W. and Coleman, G. (1992)J. Fish Dir. 15, 473-484 30 Garduno, R. A., Thornton, J. C. and Day, W. W. (1993) It@?. Zmmtrnol. 61, 485+4862 31 Garduno, K. A.. Thornton. J. C. and Day, W. W. (1993) Can. 1. &ficrobiol. 39, 1051-1058 32 Thornton, J. C , Garduno, R. A. and Day, W. W. (1994)j. Fi.ch Dis. 17, 195-204 33 Marsden, M. J., Vaughan, L. M.. Foster, T. J. and Secombes, C. J. (1996) @ct. 1mmuwl. 64, 3863-3869 34 Noonan, B., Enzmann, P. J. and Trust, T. J. (1996) Appl. Envirorz. ,2ficrobiol. 6 1, 3586-359 1 35 Press, C. M., Evencen, 0.. Reltan. L. J. and Landsverk, T. (1996) 1. Fish Dis. 19, 215-224 36 Mldtlyng, P. J.. Rexan, L. J. and Speilberg, L. (1996) Fisk Shel@h Immunol.
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