Abstract

A review will be presented on the predominant factors leading to bacterial outbreaks and how to recognize them. The actual role that bacteria play in a disease outbreak may vary from pathogen to pathogen causing disease only when the fish has become weakened by other causes such as parasites, poor water quality and other stresses. The discussion will include methodology for the isolation of bacteria from fish and submission of specimens. Major aquarium fish pathogens such as Aeromonas complex, Flexibacter, sp., Pseudomonas sp., Vibrio sp. and Mycobacteria sp. will be briefly reviewed.

Bacterial diseases can be a cause of high mortality in aquarium fish. There are members of at least 25 bacterial genera which have been implicated as possible causative agents of bacterial disease in fish, (1) most of which are Gram-negative aerobes or facultative anaerobes (2).

Bacteria which produce disease in fish are usually present in the fish's environment, in the surrounding water, on the fish surface, or in their internal organs (2). The total number of bacteria per milliliter of water in an established aquarium varies from 10(4) to 10(6) organisms per milliliter (3). Collins et al. (4) demonstrated that in a tank undergoing the nitrification cycle the number of lactose fermentors per milliliter of water increases to approximately 10(4) organisms per milliliter on day 7 and then decreases to 10(2). Organisms per milliliter by day 28 while the counts of aeromonads initially are at very low levels and increase to approximately 10(4) organisms per milliliter by day 7. These counts are dependent upon the presence of fish in the tank. If fish are removed from the tank, counts decrease greatly but increase rapidly when fish are reintroduced.

Many investigators have demonstrated the presence of bacterial pathogens such as Aeromonas complex, Pseudomonas spp., and Vibrio spp., in the water, on the surface, and in the tissue of healthy fish (5,6,7,8,9). The question arises, 'what causes the fish to become bacterially infected?" Fish usually succumb to bacterial disease when they are submitted to a period of stress or a series of stresses such as poor water quality, parasitic infections, poor nutrition or temperature extremes (10,11). Bacteria are also frequently secondary invaders following disruption of the integument by parasites, rough netting, or rough handling. It has been established that stress causes the immune system to become compromised and can thus lead to decreased resistance to disease (12).

In establishing a diagnosis of bacterial etiology in a fish disease problem, it is necessary to first exclude factors such as water quality problems or environmental problems (toxicities, nutrition, or oxygen depletion). The exclusion of parasitic infections is also required. These factors frequently act as stressors and frequently precede bacteria or act concomitantly with bacterial infections (2,3).

Clinical signs and lesions for most bacterial disease are not pathognomonic and etiologic diagnosis based on clinical signs alone is ill advised if not impossible. The most frequently observed lesions are hemorrhages, ulcers, fin and tail rot, "mouth fungus", "saddle back lesions", ascites, exophthalmia, and color changes. Fish frequently may appear lethargic and inappetent (2).

If a fish is suspected of having a bacterial disease, identification of the etiological agent will require isolation and identification. This process verifies the presence of a bacterial infection and allows the diagnostician to perform an antibiotic sensitivity test to ensure that correct medication is being used. The possibility of culturing is dictated by the type of client or clinical situation. In commercial production where hundreds of dollars may be at stake, culture and sensitivity testing is mandatory; also in large aquariums it may be advisable to keep a record of the persistent bacteria and sensitivity patterns while in home aquaria, where the amount of money invested is less, it is not economically realistic to culture. If the situation warrants culture, the clinician should either attempt to culture the fish, take samples for culture, or send the fish to a qualified laboratory (2).

The preferred method of submitting specimens is to send both moribund and apparently healthy fish to a diagnostic laboratory. This allows the laboratory to culture the fish as well as keep a few fish in an aquaria for observation of disease development. when sending live specimens is not possible, an alternate method of submitting specimens would be to aseptically remove the kidney of a fish, place it in a sterile receptacle such as a syringe barrel or a petri dish, freeze it, and mail it frozen to the laboratory. A third possibility when dealing with large fish is to culture the kidney using a Culturette® (Charion Scientific, Kansas City, MO) swab and mail the swab to the laboratory. It is imperative to culture live or moribund fish and not dead fish because of rapid postmortem migration of normal bacteria into the tissues. Only as a last resort should fish packed in ice be sent to the laboratory (13,14).

The following are brief descriptions on how to obtain bacterial cultures from fish. For identification of the isolated bacteria the diagnostician is referred to laboratory procedures published by Shotts and Bullock or the Fish Health Blue Book (15)

For Large Fish (2):

A. To facilitate handling, wrap fish in a paper towel.

B. Examine the fish and make smears of gross external lesions and gram stain the smears.

C. Kill the animal.

D. Sear area immediately behind the dorsal fin using a glowing spatula. Make sure that both sides as well as the dorsal fin are seared.

E. Dip scissors in 95% alcohol and flame.

F. With the cooled scissors cut into the middle of the seared area and push downward until the spinal column is exposed and sever it.

G. Bend the tail and head together, keeping dorsal fin uppermost.

H. With the spinal column severed, the surrounding tissue will tear and the exposed dark red mass vertical to spinal column is the kidney.

I. Insert loop down into the kidney tissue and streak the culture plate.

For Small-Fish (2):

A. Kill the fish.

B. Place fish in a receptacle containing a surface disinfectant (chlorine, 1:5000 Roccal).

C. Transfer fish to a small flask of 95% alcohol. Use forceps which have been previously sterilized by flaming to transfer the fish.

D. Cut the fish behind the dorsal fin and proceed as described in (F) for large fish.

As previously discussed, there are many genera of bacterial fish pathogens. Five of the most commonly isolated are: Aeromonas complex, Flexibacter spp., Pseudomonas ssp., Vibrio ssp., and Mycrobacteria ssp., Edwardsiella ictaluri is another species which is gaining importance. The methods of transmission of these bacteria are similar; these include the oral route, skin abrasions, gill damage, and latent carriers. The following is a table containing some of the salient features of the diseases caused by the above mentioned bacteria.

Table 1. Common Bacterial Pathogens of Fish

Considerations when medicating fish include how the antibiotic is administered, legality of the antibiotic, effectiveness, dose, toxicity, and adsorbability.

Many antibiotics may be administered by inclusion in food; others as bath treatments or by injection. Antibiotics in food are used in the aquarium trade prior to a stress period such as transportation. Administration of antibiotics in water is popular in the aquarium industry. Problems encountered include adsorbability, effect on the biologic flora of the filter bed, and with indiscriminate use possible development of resistant strains (2,3,16,17,18).

Effectiveness of therapy is always a consideration. Most bacterial fish pathogens are gram negative. Thus the use of antimicrobials with a gram positive spectrum is questionable. Legal aspects are also a consideration in food fish therapy. Presently, only tetracycline, Romet (Hoffman-LaRoche, Nutley, NJ) and sulfas are allowable for food fish (2,15).

In the aquarium trade a variety of antibiotics are used as additives to the water. The success of this type of treatment depends on the adsorbability of the antibiotic, a sufficient dose, and lack of toxicity. In general, it is a good practice to use an adequate dose (see below) and limit a treatment to 5 hrs. Overtreatment with nitrofurans and sulfonamides can cause nephrotoxicity (2,19,20).

Antibiotics may be injected which assures a therapeutic blood level. This method is particularly useful in larger fish and can be used prior to or immediately after a stress period, or in the event of a suspected bacterial disease often associated with frank ulcerations. A preferred treatment is an intraperitoneal injection of chloramphenicol. Use of a tuberculin syringe with a 26 gauge needle works well. Fish can be netted with an injection being given through the mesh (16).

On the following page is a list of some antibiotics and dosages which are commonly used in aquaria therapy.

Table II. Common Antibiotics Used in Therapy of Aquarium Fish

Note: Most antibiotics will be adsorbed within 5 hours. During therapy: remove carbon, remove air stems from lift stacks, change water at 5-8 hours. Treatment can be repeated daily for 3-4 days or until signs of infection have disappeared.

Mycobacteria (13,16,19,21,22,)

Mycobacteria are ubiquitous in nature and piscine mycobacteriosis is common in fish in home aquaria. Signs of the disease may include wasting, external ulcerations, lifting of the scales (edema), fin rot, ascites and exophthalmia which may be unilateral or bilateral. In home aquaria, the aquarist notes a fish which will not eat and which is slowly wasting away. The fish eventually dies and is eaten by other fish thus perpetuating the disease.

In routine examination of fish, many have granulomas associated with acid fast bacteria. It is apparent from clinical observation that a mycobacterial infection in a tank need not be a catastrophe, eventually leading to widespread mortalities. This is especially true if the water quality is maintained, fish are not overcrowded, and the level of nutrition is kept high.

Once a diagnosis has been made in an aquarium (by acid-fast staining of impression smears of organs) the question arises whether to treat with antibiotics. Indications are that good water quality and diet is an adequate therapy. However, if treatment is requested or deemed necessary, feeding of food with antibiotics added (Kanamycin 3 mg/100 g food, Rifampin 6 mg/100 g food, Cycloserine 3 mg/100 g food, Minocycline 0.05 mg/100 g food) is recommended. Remissions should be expected.

Bacterial disease in aquaria can be minimized by maintaining good water quality, eliminating parasitic infections, providing good nutrition and minimizing stress.

References

1.  Austin, B. and Allen-Austin, D. A review. Bacterial pathogens of fish. J. Appl. Bact. 51,483-506 (1985).

2.  Brown, E. E. and Gratzek, J. B. Fish Farming Handbook. AVI Publishing Co., Westport, Connecticut, 391 pp. (1980).

3.  Gratzek, J. B. An overview of ornamental fish diseases and therapy. J. Small Anim. Pract. 22r 345-366 (1981).

4.  Collins, M. T., Gratzek, J. B., Shotts, E. B., Dawe, D. L., Campbell, C. M., and Senn, D. R. Nitrification in an aquatic recirculating system. J. Fish. Res. Ed. Can. 20 2 5-20 3 1 (197 5).

5.  Gratzek, J. B., Shotts, E. B., and Blue, J. L. A survey of parasites, bacteria and viruses associated with tropical fish imported from southeast asia. Aquatic Mammals 1, 1-5 (1976).

6.  Sugeta, H., Oshina, K., Tamura, M., and Dugudii, Y. Bacterial flora in the gastrointestine of freshwater fishes in the river. Bull. Jap. Soc. Sci. Fish. 49(9), 1387­1395 (1983).

7.  Shotts, E. B. and Gratzek, J. B. Bacteria, parasites and viruses of aquarium fish and their shipping waters. In: Courting and Stauffer (eds) Distribution, biology and management of exotic fish. John Hopkins Press, Baltimore, pp. 215-232 (1984).

8.  Shotts, E. B., Kleckner, A. L., Gratzek, J. B., and Blue. J. L. Bacterial flora of aquarium fishes and their shipping waters imported from Southeast Asia. J. Fish. Res. Ed. Can. al, 732-735 (1976).

9.  Trust, T. J. and Bartlett, K. H. Occurrence of potential fish pathogens in water containing ornamental fishes. Appl. microbiol. 28(l),35-40 (1974).

10. Gratzek, J. B. and Reinert, R. Physiological responses of experimental fish to stressful conditions. Natl. Cancer Inst. Monogram 65, 187-193 (1984).

11. Snieszko, S. F. The effect of environmental stress on outbreaks of infectious diseases of fish. J. Fish Biol. 197-208 (1974).

12. Wedemeyer, G. A., Meyer, F. P., and Smith, L. Environmental stress of fish diseases. In: S. F. Snieszko and H. R. Axelrod (eds) Diseases of Fishes. Book 5, TFH Publications, Neptune City, NJ (1976).

13. Shotts, E. B. and Bullock, G. L. Bacterial diseases of fishes: diagnostic procedures for gram-negative pathogens. J. Fish. Res. Rd. Can. 22, 1243-1247 (1975).

14. Shotts, E. B. and Bullock, G. L. Rapid diagnostic approach in the identification of Gram-negative bacterial diseases of fish. Fish Pathology 102, 187-190 (1976).

15. Amos, K. H. (Editor). Procedures for the detection and identification of certain fish pathogens. 3rd ed. Fish Health Section, American Fisheries Society, Corvalis, Oregon (1985).

16. Gratzek, J. B. Control and therapy of fish diseases. Advances in Vet. Sci. and Comp. Med. 21, 297-324 (1983).

17. Aoki, T. and Kitao, T. Detection of transferable R plasmids in strains of the fish pathogenic bacterium, Pasteurella piscida. J. Fish Dis. D., 345-350 (1985). Shotts, E. B., Vanderwork, V.L., and Campbell, L. M. Occurrence of R factors associated with Aeromonas hydrophila isolates from aquarium fish and waters. J. Fish. Res. Board Can. 31, 736-740 (1976).

18. Post, G. W. Textbook of Fish Health. TFH Publication, Neptune, NJ (1983).

19. Roberts, R. J. Fish Pathology. Bailliere Tindall, London (1978).

20. Snieszko, S. F. Mycobacteriosis (tuberculosis) of fish. Fish & Wildlife Service. Fish disease leaflet #55 (1978).

21. van Duijn, C. J. Tuberculosis in fishes. J. Small Anim. Pract. 21, 391-414, (1981).