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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 effi- cient 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 identi- fied 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 in its infancy, is highly adapt- able and, depending on the ingenuity of the screening protocol, might be used for identifying novel enzymes, bioactive peptides or even the products of linked multi-gene complexes.

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 assay and the polymerase chain reaction have detected

A. salmonicida in the absence of colony-forming 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 (b.austin@hw.ac.uk) is ut the Department oj’ Biological Sciences, Heriot- Watt University, Riccurton, Edinbugh, UK EH14 4AS.

septicaemia, termed vibriosisl. The fungal species Zchthyophonlrs hoferi affects many marine species, caus- ing 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).

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Box 1. Characteristics of Aeromonas salmonicidal

Gram-negative, non-motile, fermentative rods, which typically produce brown diffusible pigment on protein- containing medium such as brain heart infusion agar (BHIAI. Incapable of growth at 37°C. On BHIA it may develop any of three different colony types, termed ‘rough’, ‘smooth’ and ‘G-phase’ because of the presence or absence of an external proteinaceous layer (A-layer). Cell-wall-defective/deficient forms (L-forms) have been recognized. Atypical isolates may be slow in pigment- ing and nutritionally fastidious, requiring blood-products for growth. By definition, cells do not occur in surface water, and are pathogenic to fish.

A. salmor~icida is the causal agent of furunculosis, and is one of the oldest known fish pathogens1,3. The pathogen has a worldwide distribution, causing infec- tions in representatives of many families of fish, includ- ing Anoplopomidae, Cyprinidae, Salmonidae and Serranidae. Classical furunculosis involves a haemor- rhagic septicaemia, including the presence of furuncles (boils) on the flanks. The seriousness of furunculosis to aquaculture (the rearing of aquatic species in con- trolled conditions) is illustrated by the epidemic in 1991-1992, which led to the loss of -10000 tonnes (roughly equivalent to 25% of the total production) of Atlantic salmon (S&no sular) in Scotland. Although troublesome initially in freshwater, A. salmorlicida has emerged as a major pathogen of salmonids and other fish, such as dabs and flounder, in the seaJ.“. In these fish, the disease may be of a different form from classical furunculosis, 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 precise location 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 effect- ive disease control measures, especially for fish other than salmonids.

Detection A. salmorzicida is difficult to recover from fish that do

not show clinical signs of disease, and from the aquatic environment, even during the epizootics of furuncu- losis. Routinely, cultures may be grown on tryptone soya agar or brain heart infusion agar following incu- bation at 15-25°C. Atypical isolates often need the inclusion of blood or serum in the isolation medium’. Pre-incubation of pathological material for 24-48 h in tryptone soya broth6 followed by the use of Coomassie Brilliant agar improves recovery of the pathogen”,‘. However, an effective selective medium remains to be described. Alternative approaches to culturing for the detection of A. sulmonicida and diagnosis of furuncu- losis have included serology and molecular biolo,T techniques.

Serology has been used successfully on pure and mixed cultures and with pathological material to

confirm the presence ofA. sulmonicidu. Methods range from slide agglutination8 to the latex agglutination test9 and enzyme-linked immunosorbent assay (ELISA) lo. Latex agglutination, of which one system has been commercialized, may result in diagnoses within 2 min to 2 h for pure/mixed cultures or heavily infected fish tissues, respectively. ELISA enables reliable diagnoses in 3G60 min, and appears to be effective for use with asymptomatic carrier fishl.10. The ongoing debate concerning the relative merits of monoclonal and polyclonal antibodies in serological techniques remains to be resolved.

DNA probes have the ability to detect A. salmoni- cidu in clinical and environmental samples. Moreover, a DNA fragment specific to A. sulmonicidu was incor- porated into a polymerase chain reaction (PCR) tech- nique and used to detect approximately two celIsll. By means of PCR and a specific DNA probe, the pres- ence of A. satmonicidu has been reported in effluent, water, faeces and sediment from a commercial fresh- water Atlantic salmon farm in Ireland12*13. In contrast, culturing only recovered colony-forming units from clinically diseased fish. 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 molecu- lar 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 sensi- tivity of modern detection methods may not be too helpful with the management of disease caused by A. salmorzicida.

The possible survival of A. sulmonicida in water has been extensively researched’. The data suggest that the pathogen can survive through varying periods and temperatures in fresh, brackish and seawater, and the underlying sediment. A consensus view is that there is a concomitant reduction in pathogenicity when the organism leaves the fish and enters the aquatic envi- ronment’j. Sakai has suggested that the long-term sur- vival ofA. sulmonicida in the aquatic environment may reflect electrostatic charge differences on individual cells, with positive and negative charges on avirulent and virulent cells, respectivelylj. Virulent cells may be able to survive under starvation conditions in river sediments. A decline in numbers might be caused by the spontaneous occurrence of avirulent free-living cells, which enter a dormant phase due to a lack of nutrientsls. Furthermore, these tiee-living cells could represent a transitional form, leading eventually to a loss of viability. Interestingly, the onset of the dor- mant/non-culturable phase may be delayed by the presence of the amino acids arginine and methion- inel”. Although the possible presence of dormant/ non-culturable cells in the aquatic environment has been vigorously debated, there is accumulating evi- dence to suggest that intact cells of A. sulmonicidu remain in aquatic habitats after the number of colony- forming units has declined to zeroIT. Attempts to revive these cells, principally by the addition of

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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 fil- ters; 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 conven- tional plate counts had reached zero. These findings suggest that specialized forms of A. salmonicida, such as L-forms, may contribute to the difficulty in recov- ering 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”. I f A. salmonicida cells that are dormant, non-culturable, altered, senes- cent 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 lim- it.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 infec- tion’. Carriers harbour the pathogen in the external mucus, gills and spleenb, enabling release when clini- cal 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 cor- ticosteroids is often employed to induce the develop- ment of clinical disease from carriers’,

Antibiotic therapy, which is often used to combat outbreaks of furunculosisr, does not necessarily elimi- nate the carrier state. Indeed, it is possible that inap- propriate 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 sub-

ject 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 dam- aged areas on the surface of the fish. Further work demonstrated that uptake was enhanced by the pres- ence 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 develop- ing effective control strategies for furunculosis. Meth- ods 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, non- specific 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 immunostimu- latory 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. salmoni- cida when administered by injection25. B-1,3-Glucans are now being routinely incorporated into fish diets but this possibly reflects a misrepresentation of scien- titic data for commercial gain; specifically, the scien- tific 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 encom- passes use of subcellular components (namely inacti- vated extracellular products and lipopolysaccharides), genetic manipulation of cells and live (avirulent) vac- cines with or without adjuvants. Iron-regulated outer- membrane proteins, the so-called IROMPs, are es- pecially immunogenic to Atlantic salmona7, and have been developed into a commercial vaccine. This prod- uct 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 outer- membrane porin has also shown potential in vaccine trials”‘. A significant development is an in viuo growth model for A. salmonicida, i.e. a specialized intraperi- toneal chamber implanted in rainbow trout, which has allowed the prospect of developing gene products that are only synthesized in the host”“J1. Genetic manipu- lation 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 extra- cellular A-layer (this is involved in pathogenicity) have

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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 anti- body 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 high- est antibody titre and level of protectionss. Within 16 weeks of administration by injection, A. salmonicida lipopolysaccharide was located mostly in the abdomi- nal granulomas, head kidney and spleerG5. The ben- efit of adjuvants, especially those comprising mineral oil, for stimulating a protective immune response following intraperitoneal vaccination has been con- firmeds6, 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 algino- lyticus, which was recovered from penaeid shrimps, has been found to aid the control of furunculosis in salmonid.G7. Other probiotics that may control furun- culosis will undoubtedly be identified in the future.

Conclusion Biotechnology is involved in the detection and con-

trol of infections caused by A. salmonicida. Clearly, there is an urgent need for rapid and reliable diagnos- tic 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 poss- ible ‘natural’ population levels of A. salmonicida. In addition, disease control measures will continue to be researched, particularly concerning probiotics, non- specific 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 dis- ease 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., AItwegg, M., Goshng, P. J. and Joseph, S., eds), pp. 197-243, Wiley

4 WikIund, T. (1995) Dir. Aquat. Ox. 21, 145-150 5 Wikhmd, T. 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/~ Shel@.rh

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. 6. 335-35U

37 Austm, B.. Stuckey. L. F.. Robertson, P. A. W., Effendi, I. and Gnffith, D. R. W. (1995) 1. Fisll DIS. 18, 93-96

In the next issue: Bioremediation of marine oil spills

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