Vol. 57, No. 6
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1991, p. 1813-1816 0099-2240/91/061813-04$02.00/0
Effect of Sublethal Concentrations of Four Chemicals on Susceptibility of Juvenile Rainbow Trout (Oncorhynchus mykiss) to Saprolegniosis M. CARBALLO* AND M. J. MUNOZ
Department of Animal Health, CIT-I.N.I.A., Embajadores 68, 28012 Madrid, Spain Received 28 December 1990/Accepted
5
April 1991
The effects of sublethal concentrations of a variety of chemicals on the susceptibility of rainbow trout (Oncorhynchus mykiss) juveniles to Saprolegnia parasitica infection was examined. Sublethal concentrations of un-ionized ammonia (0.05 mg/liter) and nitrite (0.12 mg/liter) increased fish susceptibility after 10 days of exposure to the toxin, this increase being higher for ammonia (75% and 20% morbidity) than for nitrite (20 % and 0% morbidity, respectively) with inoculum doses of 1.4 x 106 and 9.5 x 105 zoospores per liter, respectively. Sublethal concentrations of copper (0.05 mg/liter) or cyanide (0.05 mg/liter) did not show enhancement of infection by S. parasitica, even though the toxin exposure was for 21 days and the inoculum 106 and 3.2 x 106 doses were higher than those for the experiments with the nitrogen compounds (4 zoospores per liter. However, infections began to appear in control animals.
sublethal concentrations of different toxicants on the susceptibility of juvenile Oncorhynchus mykiss to S. parasitica infection.
Mycotic infections with members of the family Saprolegniaceae are widely reported in freshwater fish. Although salmonid fish come into contact with many fungal spores, only Saprolegnia parasitica (sin. S. diclina type 1) and other Saprolegnia spp., characterized by groups of long hookered hairs on the secondary zoospore cyst, have been isolated from fungal lesions on live salmonid fish (23). Pathogenic Saprolegnia spp. produce an easily recognizable cottonywhite growth on the epidermis of the affected animal. Infection is normally restricted to the superficial tissues, resulting in a breakdown of the fish osmoregulatory mechanism, and unless fish can be treated, the condition is usually lethal. Pickering and Willoughby (22) described some predisposing factors that increased the susceptibility of salmonids to Saprolegnia infection. These factors included sexual maturation, stress, integumentary damage, and evidence of other pathogenic agents. Virtually nothing is known about the process of infection and the characteristics that enable this pathogen alone to successfully colonize live fish. The presence of sublethal concentrations of toxicants in water have been found to be important factors in the emergence and development of infectious diseases. Many studies exist regarding changes in susceptibility of fish to viral (13), bacterial (24), and parasitic (8) diseases after exposure to sublethal concentrations of different toxins. The presence of noxious chemicals at sublethal concentrations in freshwater environments causes physiological alterations which could increase the disease incidence in fish. These alterations depend on the chemical, its concentration, and the length of exposure. Toor et al. (31) found that high levels
MATERIALS AND METHODS Fish. Juvenile rainbow trout, 0. mykiss (17 + 8 g) were obtained from a fish farm. Acclimatization to laboratory conditions was conducted for 1 week before transferring the fish to a 115-liter glass aquarium with flowing dechlorinated tap water. Fungi and zoospore inocula. Isolates of S. parasitica CM2101a from infected trout were used in this study. The isolates had the typical secondary cyst coat ornamentation of pathogens (23). Stock cultures were maintained on slants of weak glucose-peptone agar and were kept at 8 to 12°C and subcultured every 3 to 6 months. Secondary zoospore suspensions, for the experimental infection of fish, were obtained by the method of Willoughby and Pickering (37) and Willoughby et al. (36). Hemp seeds colonized by the fungi were incubated in autoclave-sterilized tap water at 20°C for 2 days. The water surrounding the seeds contained numerous motile secondary zoospores. Zoospore suspensions were then obtained by filtering this water through double layers of Whatman 541 filter paper, and the concentrations were estimated by inoculating 0.1 ml of different dilutions of spore suspensions on glucose-peptone agar plates and incubating at 20°C for 8 to 12 h. For each dilution, an average count was made from four plates. The different zoospore suspensions in which the fish were maintained for infection were estimated to be 5 x 105, 9.75 x 105, 1.45 x 106, 3.2 x 106, and 4 x 106 zoospores per liter. Experimental design and characteristics of toxic exposure. A group of 18 fish were exposed for 10 days to a sublethal concentration of un-ionized ammonia in static conditions in an 80-liter glass aquarium, with water renewal every 3 days. The experimental concentration of ammonia was 0.05 mg of N per liter as un-ionized ammonia, obtained by the addition of reagent grade ammonium chloride (NH4Cl) solution (Merck, Darmstadt, Germany). Total ammonia-nitrogen (NH3-N) was measured with an ammonia-selective electrode
of organic compounds from different wastes are predisposing environmental factors to outbreaks of saprolegniosis, but, until now, nothing was known about the specific effects of physicochemical parameters on saprolegniosis. Ammonia, nitrite, copper, and cyanide are common pollutants in aquatic environments, and their lethal and sublethal toxicities for salmonids are well documented (1, 6, 10, 17, 29). The present study was undertaken to observe the effects of *
Corresponding author. 1813
1814
APPL. ENVIRON. MICROBIOL.
CARBALLO AND MUNOZ
(95-12; Orion Research, Cambridge, Mass.) calibrated with ammonium chloride solution. Un-ionized ammonia (NH3) concentrations were calculated from total concentrations (1, 7). Three groups of six fish that had been exposed to ammonia were immersed in the following doses of fungi: 5 x 105, 9.75 x 105, and 1.4 x 106 zoospores per liter. For each fungal dose, six animals without toxin exposure were used as controls. Similar conditions, including the same numbers of fish, the same exposure time, and the same zoospore concentrations were used to test the effects of a sublethal concentration of nitrite. Sublethal levels of nitrite (0.12 mg/liter) were maintained by the addition of potassium nitrite (KNO2) solutions (Merck) determined by a standard method (25). The infections was obtained with the same fungal doses as in the ammonia infection. A group of 20 fish were exposed for 21 days to a sublethal concentration of copper in static conditions in an 80-liter glass aquarium. The experimental copper concentration was 0.05 mg/liter. Copper was added as a copper nitrate [Cu(NO3)2] (Merck reagent grade) solution, and the copper concentration was determined with a Perkin-Elmer atomic absorption spectrophotometer (3030 B) equipped with a graphite furnace (HG-300). Two groups of 10 fish were then infected with two different doses of fungal inocula. A group of 10 fish unexposed to this toxin was used as controls. Cyanide tests were made with the same numbers of fish and the same exposure time as the copper tests. Cyanide levels (0.05 mg/liter) were maintained throughout the experimental period by the addition of a potassium cyanide (KCN) solution (Merck) and were measured with a cyanide-selective electrode (94-06000; Orion Research) calibrated with a potassium cyanide solution. Infections were obtained by using the same doses of fungal inocula used in the copper tests. Dissolved oxygen, temperature, pH, ammonia, nitrite, copper, and cyanide levels were analyzed daily. Water alkalinity, hardness, carbon dioxide, sodium, potassium, chloride, and permanganate-oxidizable matter levels were analyzed before and after water renewal. Selective electrodes (Orion Research) or standard procedures (2, 25) were used. Toxicant concentrations were measured daily. Infection of fish. After exposure, fish were anesthetized in a 0.03% solution of 2-phenoxy ethanol and wounded by removing 5- to 7-mm2 patches of scales from each side immediately in front of the dorsal fin above the lateral line. Animals were then immersed for 10 min in an experimental fungal inoculum (19, 26). Pathogen-exposed fish were transferred to an 80-liter glass aquarium with flow-through dechlorinated tap water and maintained 15 days or until the appearance of saprolegniosis. Infections were estimated by the presence of cottony-white patches on the body of the fish. Mucus samples were plated on glucose-peptone-penicillin-streptomycin agar plates, incubated at 20°C for 8 to 12 h (37), and observed for fungal growth. an
RESULTS The water characteristics used in the experiments (mean values + standard deviations) were (i) alkalinity (milligrams of CaCO3 per liter), 36.7 + 14; (ii) chloride concentration 5; (iii) hardness (milligrams of NaCl per liter), 13.1 5; (iv) sodium (milligrams of CaCO3 per liter), 33.7 concentration (milligrams of Na per liter), 5.6 + 1.3; (v) potassium concentration (milligrams of K per liter), 0.14 + 0.05; and (vi) organic matter concentration (milligrams of 02
5.7 + 1.6. Throughout the study the temperature in the fish aquaria was 15 + 3°C, and the pH was 7.3 + 0.2. The
per liter),
TABLE 1. Percentage of fish with fungal infections after being exposed for 10 days to un-ionized ammonia (0.05 mg of N per liter) or nitrite (0.12 mg of N per liter) at three inoculum concentrations Toxicant or control
Ammonia Nitrite Control
% of infected fish at inoculum concn (zoospores per liter) of: 1.4 x106
9.75 x105
5 x 105
75 50 0
20 0 0
0 0 0
concentration of carbon dioxide was 5.6 + 0.6 mg/liter, and the dissolved oxygen concentration was 7.3 + 1 mg/liter. Un-ionized ammonia and nitrite in aquaria without exposure to these chemicals did not exceed the recommendations of the U.S. Environmental Protection Agency (32). The mean toxicant concentrations + the standard deviations were 0.057 0.02 mg of N per liter for un-ionized ammonia, 0.117 0.02 mg of N per liter for nitrite, 0.045 0.01 mg of Cu per liter for total copper, and 0.052 + 0.06 mg of cyanide per liter for cyanide. The results of infection in fish exposed to ammonia and nitrite are in Table 1 and the results for copper and cyanide exposure are in Table 2. Infection occurred in the group of fish exposed to ammonia and nitrite, with the percentage of infected fish being higher when exposed to ammonia (75%) than to nitrite (50%) at the highest fungi dose used (1.45 x 106 zoospores per liter). At the medium fungal dose (9.75 x 105 zoospores per liter), infection was found only in ammonia-exposed fish (20%), and no infection was found in any group of tested fish at the lowest fungal dose used (5 x 105 zoospores per liter). No infection was found in fish exposed to copper and cyanide. The control fish had a very low percentage of infections at the highest fungal dose used. Infection was clearly evident as cottony-white mycelial masses on the fish epithelium in the descaled areas of the fish, appearing at 2 to 4 days. Cultures of the fungi isolated from the fish were analyzed, and typical S. parasitica characteristics were recorded. No mortalities occurred among infected fish. ±
±
±
DISCUSSION Nolard-Tintigner (19) found that the level of parasitism by members of the Saprolegniaceae depends on the number of secondary zoospores in the water. The experience of one of the authors with Lebistes reticulatus and Xiphophorus belleri, small tropical and very sensitive fish, indicated that a 100% infective dose was approximately 1.5 x 105 zoospores per liter or more; no infection was observed even at levels as high as 2.4 x 104 zoospores per liter. The concentration of TABLE 2. Percentage of fish with fungal infections after being exposed for 21 days to copper (0.05 ppm) or cyanide (0.05 ppm) at two inoculum concentrations Toxicant or control
Copper Nitrite Control
% of infected fish at inoculum concn (zoospores per liter) of:
4 x 106
3.2 x 106
0 0 1
0 0 0
VOL. 57, 1991
the fungal inoculum used in our experiments was higher, and infection was found at levels 6 and 10 times higher, but only in ammonia- and nitrite-exposed fish. Furthermore, the animals used in our study were immature fish, which have been described as being more resistant than sexually mature fish (22). The general susceptibility to a disease in animals has been described in terms of mortality rates, but it has been suggested (8) that changes in rates of infection and/or changes in morbidity are more subtle changes and are more important in evaluating the susceptibility to sublethal toxic effects than are mortality rates. Therefore, we studied the morbidity, as the percentage of infected fish, and evaluated the effects of different levels of exposure to sublethal concentrations of four chemicals. In addition, exposure to S. parasitica by scale removal was used to obtain a high percentage of infection (26). Our findings revealed higher percentages of infection when fishes were exposed to ammonia. Increases of susceptibility to bacterial infection after exposure to NH3 have been observed in Ictalurus punctatus (24, 33), which increases of ammonia were associated with bacterial gill disease and other bacterial infections. I. punctatus exposed to sublethal concentrations of ammonia (0.02 to 0.04 mg/ liter) were more susceptible to invasion by Aeromonas hydrophila (9). The exposure times at these concentrations were correlated with lowered host resistance. Smart (27) found that rainbow trout exposed to ammonia (0.05 to 0.4 mg/liter) for 1 week to 3 months had diminished numbers of erythrocytes, irreversible blood damage, inflammatory and degenerative lesions in the gills, and increased susceptibility to bacterial disease. Several alterations and toxic effects related to sublethal concentrations of ammonia include gill damage (16, 29), alterations of the mucous cells (16, 21), and an enhancement of corticosteroid levels (30). Infection by Saprolegnia spp. indicated that external mucus renewal was employed as a defense mechanism against the fungus (35). Mucus production can be influenced by the presence of environmental factors such as stress and pollutants (21). The effects of ammonia on mucus production are unknown. Possibly, the number of mucous cells in the epidermis remains constant, but the time required for the formation and discharge of mucus by the mucous cells varies under the influence of ammonia (16). When fish have been infected by a Saprolegnia sp., the fungus destroys the essential waterproofing properties of the skin, and the results are massive osmoregulatory problems (22). In fish exposed to ammonia, increases in ventilation frequency in an effort to fight against the osmoregulatory problems and gill damage generated by this chemical, are continually observed (16). Therefore, additional changes in osmotic balance in these fish might also be observed. Once initiated, a fungal infection is also a stress factor (18). Ammonia exposure increased corticosteroid levels (3, 30) in fish exposed to sublethal concentrations. Several of these factors could be involved in the high percentages of infection observed in ammonia-exposed fishes. Some well-documented effects of nitrite exposure are changes in electrolyte concentrations in plasma as well as the production of methemoglobinemia, hematological consequences of short-term nitrite exposure (34). In addition, corticosteroids released into the circulation after nitrite exposure (3), or after fungal infection has been initiated (18), can lead to an electrolyte imbalance (as a secondary effect of
Cu AND CN- EFFECTS ON SAPROLEGNIOSIS
1815
stress) that could be additive to the toxic effects of nitrite exposure. Saprolegnia infection of fish exposed to copper and cyanide was not observed, although the exposure times and fungal doses were twice those of the experiments with nitrogen compounds. The effects of sublethal copper concentrations on the increased susceptibility of fish to infection appear to be dependent on the concentration of copper as well as on the length of time the fish are exposed to the metal. In addition, it has been found that 0. mykiss exposed for 24 h to 0.01 mg of total copper per liter showed an increased susceptibility to Yersinia ruckeri infection (14). Infections increased after 48 h of copper exposure, but by 96 h of copper exposure the fish had begun to lose their susceptibility. Similar results were obtained (13) when rainbow trout were exposed to sublethal concentrations of copper (0.01 mg/liter), whereupon a 24-h exposure had a bigger effect than a longer exposure (9 days) on the increased susceptibility of rainbow trout to the infectious hematopoietic necrosis virus. Our findings were in agreement with these, and no infection was found after 21 days of exposure at 0.05 mg of copper per liter. Increases and decreases of corticosteroids were registered (5) when Oncorhynchus nerka was exposed for 24 h to concentrations of copper and water quality similar to those used in our experiments, indicating that the fish adapted to these conditions. Thus, changes in copper levels could affect, depending on the exposure time, the immunosuppressive effect of corticosteroids (20). The result could be either an increase or decrease in susceptibility to the disease, in spite of the direct toxic effect of copper on osmoregulation, as has been described (11, 12). The effects of cyanide on changes of fish susceptibility to infectious diseases have not been well documented. Cyanide is essentially an inhibitor of oxygen metabolism, rendering the tissues incapable of exchanging oxygen (6). At sublethal concentrations (0.01 to 0.042 mg/liter), it produced physiological changes such as reduced growth, altered respiration, and some degree of hepatic necrosis in juvenile rainbow trout (4, 15). A progressive acclimatization to this pollutant has been observed; after exposure of juvenile rainbow trout to 0.02 mg of HCN per liter for 21 days, an early depression of growth occurred, followed by an increase in growth rate by the end of the experiment (28). When juvenile rainbow trout were exposed to 0.01, 0.02, or 0.03 mg of HCN per liter, increased respiration rates were observed at 1 to 4 days after exposure, which gradually decreased at 5 to 6 days (4). Possibly, acclimatization occurred, and this toxic concentration did not affect the susceptibility to the disease. Our experiments confirm that specific toxicants (ammonia and nitrite) predispose fish to saprolegniosis. These effects might explain the results of Toor et al. (31) that a high organic load was a predisposing factor to an outbreak of saprolegniosis. Considering the relationship of organic matter to the accumulation of ammonia and nitrite in the water by the biological degradation of nitrogen-rich products, we might conclude that high ammonia and nitrite levels found in fish farms due to inadequate water renovation, insufficient cleaning, or very high fish density increase the risk of saprolegniosis. ACKNOWLEDGMENTS This work was supported in part by a grant from I.N.I.A. (research project no. I.N.I.A. 8171). The technical assistance of M. Cuellar and F. Plasencia is gratefully acknowledged.
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REFERENCES 1. Alabaster, J. S., and T. Lloyd. 1980. Water quality criteria for freshwater fish. Butterworth and Co. Ltd., London. 2. American Public Health Association. 1981. Standard methods for the examination of water and wastewater, 15th ed. American Public Health Association, Washington, D.C. 3. Carballo, M., M. J. Munoz, and J. V. Tarazona. 1990. Induccion de estres y aumento de susceptibilidad a Saprolegnia parasitica por compuestos toxicos en Oncorhynchus mykiss, p. 733-737. Actas III Concreso Nacional Acuicultura. Santiago de Compostela, Spain. 4. Dixon, D. G., and G. Leduc. 1981. Chronic cyanide poisoning of rainbow trout and its effects on growth, respiration and liver histopathology. Arch. Environ. Contam. Toxicol. 10:117-131. 5. Donaldson, E. M., and H. M. Dye. 1975. Corticosteroid concentrations in sockeye salmon (Oncorhynchus nerka) exposed to low concentrations of copper. J. Fish. Res. Board Can. 32:533539. 6. Doudoroff, P. 1976. Toxicity to fish of cyanide and related compounds: a review. U.S. Environ. Prot. Agency Off. Res. Dev. Res. Rep. Ecol. Res. Ser. EPA-600 3-76-038:1-154. 7. Emerson, K., R. C. Russo, R. E. Lund, and R. V. Thurson. 1975. Aqueous ammonia equilibrium calculations: effect of pH and temperature. J. Fish Biol. 32:2379-2383. 8. Ewing, M. S., S. A. Ewing, and M. A. Zimmer. 1982. Sublethal copper stress and susceptibility of channel catfish to experimental infections with Ichthyophthirius multifiliis. Bull. Environ. Contam. Toxicol. 28:674-681. 9. Flagg, R. H., and L. W. Hinck. 1979. Influence of ammonia on Aeromonas susceptibility in channel catfish. Proc. Annu. Conf. Southeast. Assoc. Fish Wildl. Agencies 32:415-419. 10. Harrison, L. F. 1986. The impact of increased copper concentrations on freshwater ecosystems. Rev. Environ. Toxicol. 2:134-141. 11. Heath, A. G. 1984. Changes in tissue adenylates and water content of bluegill, Lepomis macrochirus, exposed to copper. J. Fish Biol. 24:299-309. 12. Heath, A. G. 1987. Effects of waterborne copper or zinc on the osmoregulatory response of bluegill to a hypertonic NaCl challenge. Comp. Biochem. Physiol. 88:307-311. 13. Hetrick, F. M., M. D. Knittel, and J. L. Fryer. 1979. Increased susceptibility of rainbow trout to infectious hematopoietic necrosis virus after exposure to copper. Appl. Environ. Microbiol. 37:198-201. 14. Knittel, M. D. 1981. Susceptibility of steelhead trout, Salmo gairdneri R., to redmouth infection by Yersinia ruckeri following exposure to copper. J. Fish Dis. 4:33-40. 15. Kovaccs, T. G. 1979. The effect of temperature on cyanide toxicity to rainbow trout (Salmo gairdneri). I. Acute effects. II. Chronic effects. M.S. thesis. Concordia University, Montreal, Canada. 16. Lang, T., G. Peters, R. Hoffman, and E. Meyer. 1987. Experimental investigations on the toxicity of ammonia: effects on ventilation frequency, growth, epidermal mucous cells, and gill structure of rainbow trout Salmo gairdneri. Dis. Aquat. Org. 3:159-165. 17. Lewis, W. M., and D. P. Morris. 1986. Toxicity of nitrite to fish: a review. Trans. Am. Fish. Soc. 115:183-195. 18. Neish, G. A., and G. C. Hughes. 1980. Fungal diseases in fish. T.F.H. Publications, Inc. Ltd., Neptune, New Jersey.
19. Nolard-Tintigner, R. N. 1978. Ability of zoospores versus oospores to cause saprolegniasis in fish. Acta Zool. Pathol. Antverp. 57:1-27. 20. Pickering, A. D. 1984. Cortisol-induced lymphocytopenia in brown trout Salmo trutta. Gen. Comp. Endocrinol. 53:252-259. 21. Pickering, A. D., and D. J. Macey. 1977. Structure, histochemistry and the effects of handling on the mucous cells of the epidermis of the char Salvelinus alpinus. J. Fish Biol. 10:505512. 22. Pickering, A. D., and L. G. Willoughby. 1982. Saprolegnia infections of salmonid fish, p. 271-297. In R. J. Roberts (ed.), Microbial diseases of fish. Academic Press, Inc. (London), Ltd., London. 23. Pickering, A. D., L. G. Willoughby, and C. B. McGrory. 1979. Fine structure of secondary zoospore cyst cases of Saprolegnia isolates from infected fish. Trans. Br. Mycol. Soc. 72:427-436. 24. Plumb, J. A. 1984. Relationship of water quality and infectious diseases in cultured channel catfish. Symp. Biol. Hung. 23:189198. 25. Rodier, J. 1981. Analisis de las aguas. Omega Ed. Barcelona, Spain. 26. Singhal, R. N., S. Jeet, and R. W. Davies. 1987. Experimental transmission of Saprolegnia and Achlya to fish. Aquaculture 64:1-7. 27. Smart, G. 1976. The effect of ammonia exposure on gill structure of the rainbow trout, Salmo gairdneri. J. Fish Biol. 8:471-475. 28. Speyer, M. R. 1975. Some effects of chronic combined arsenic and cyanide poisoning on the physiology of rainbow trout. M.S. thesis. Sir George Williams Campus, Concordia University, Montreal, Canada. 29. Tarazona, J. V., M. J. Munoz, J. A. Ortiz, 0. Nuniez, and J. Camargo. 1987. Fish mortality due to acute ammonia exposure. Aquacult. Fish. Manage. 18:167-172. 30. Tomasso, J. R., K. B. Davis, and B. A. Simco. 1981. Plasma corticosteroid dynamics in channel catfish, Ictalurus punctatus, exposed to ammonia and nitrite. Can. J. Fish. Aquat. Sci. 38:1106-1112. 31. Toor, H. S., H. Sehgal, and R. S. Sehdev. 1983. A case study of acute fish diseases in tanks loaded with high levels of organic manures. Aquaculture 35:277-282. 32. U.S. Environmental Protection Agency. 1976. Quality criteria for water. Office of Water and Hazardous Materials, U.S. Environmental Protection Agency, Washington, D.C. 33. Walters, G. R., and J. A. Plumb. 1980. Environmental stress and bacterial infection in channel catfish Ictalurus punctatus R. J. Fish Biol. 17:177-185. 34. Williams, E. M., and F. B. Eddy. 1988. Regulation of blood haemoglobin and electrolytes in rainbow trout Salmo gairdneri exposed to nitrite. Aquat. Toxicol. 13:13-28. 35. Willoughby, L. G. 1989. Continued defence of salmonid fish against Saprolegnia fungus, after its establishment. J. Fish Dis.
12:63-67. 36. Willoughby, L. G., C. B. Mcgrory, and A. D. Pickering. 1983. Zoospore germination of Saprolegnia pathogenic to fish. Trans. Br. Mycol. Soc. 80:421-435. 37. Willoughby, L. G., and A. D. Pickering. 1977. Viable Saprolegniaceae spores on the epidermis of the salmonid fish Salmo trutta and Salvelinus alpinus. Trans. Br. Mycol. Soc. 68:91-95.
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1991, p. 1813-1816 0099-2240/91/061813-04$02.00/0
Effect of Sublethal Concentrations of Four Chemicals on Susceptibility of Juvenile Rainbow Trout (Oncorhynchus mykiss) to Saprolegniosis M. CARBALLO* AND M. J. MUNOZ
Department of Animal Health, CIT-I.N.I.A., Embajadores 68, 28012 Madrid, Spain Received 28 December 1990/Accepted
5
April 1991
The effects of sublethal concentrations of a variety of chemicals on the susceptibility of rainbow trout (Oncorhynchus mykiss) juveniles to Saprolegnia parasitica infection was examined. Sublethal concentrations of un-ionized ammonia (0.05 mg/liter) and nitrite (0.12 mg/liter) increased fish susceptibility after 10 days of exposure to the toxin, this increase being higher for ammonia (75% and 20% morbidity) than for nitrite (20 % and 0% morbidity, respectively) with inoculum doses of 1.4 x 106 and 9.5 x 105 zoospores per liter, respectively. Sublethal concentrations of copper (0.05 mg/liter) or cyanide (0.05 mg/liter) did not show enhancement of infection by S. parasitica, even though the toxin exposure was for 21 days and the inoculum 106 and 3.2 x 106 doses were higher than those for the experiments with the nitrogen compounds (4 zoospores per liter. However, infections began to appear in control animals.
sublethal concentrations of different toxicants on the susceptibility of juvenile Oncorhynchus mykiss to S. parasitica infection.
Mycotic infections with members of the family Saprolegniaceae are widely reported in freshwater fish. Although salmonid fish come into contact with many fungal spores, only Saprolegnia parasitica (sin. S. diclina type 1) and other Saprolegnia spp., characterized by groups of long hookered hairs on the secondary zoospore cyst, have been isolated from fungal lesions on live salmonid fish (23). Pathogenic Saprolegnia spp. produce an easily recognizable cottonywhite growth on the epidermis of the affected animal. Infection is normally restricted to the superficial tissues, resulting in a breakdown of the fish osmoregulatory mechanism, and unless fish can be treated, the condition is usually lethal. Pickering and Willoughby (22) described some predisposing factors that increased the susceptibility of salmonids to Saprolegnia infection. These factors included sexual maturation, stress, integumentary damage, and evidence of other pathogenic agents. Virtually nothing is known about the process of infection and the characteristics that enable this pathogen alone to successfully colonize live fish. The presence of sublethal concentrations of toxicants in water have been found to be important factors in the emergence and development of infectious diseases. Many studies exist regarding changes in susceptibility of fish to viral (13), bacterial (24), and parasitic (8) diseases after exposure to sublethal concentrations of different toxins. The presence of noxious chemicals at sublethal concentrations in freshwater environments causes physiological alterations which could increase the disease incidence in fish. These alterations depend on the chemical, its concentration, and the length of exposure. Toor et al. (31) found that high levels
MATERIALS AND METHODS Fish. Juvenile rainbow trout, 0. mykiss (17 + 8 g) were obtained from a fish farm. Acclimatization to laboratory conditions was conducted for 1 week before transferring the fish to a 115-liter glass aquarium with flowing dechlorinated tap water. Fungi and zoospore inocula. Isolates of S. parasitica CM2101a from infected trout were used in this study. The isolates had the typical secondary cyst coat ornamentation of pathogens (23). Stock cultures were maintained on slants of weak glucose-peptone agar and were kept at 8 to 12°C and subcultured every 3 to 6 months. Secondary zoospore suspensions, for the experimental infection of fish, were obtained by the method of Willoughby and Pickering (37) and Willoughby et al. (36). Hemp seeds colonized by the fungi were incubated in autoclave-sterilized tap water at 20°C for 2 days. The water surrounding the seeds contained numerous motile secondary zoospores. Zoospore suspensions were then obtained by filtering this water through double layers of Whatman 541 filter paper, and the concentrations were estimated by inoculating 0.1 ml of different dilutions of spore suspensions on glucose-peptone agar plates and incubating at 20°C for 8 to 12 h. For each dilution, an average count was made from four plates. The different zoospore suspensions in which the fish were maintained for infection were estimated to be 5 x 105, 9.75 x 105, 1.45 x 106, 3.2 x 106, and 4 x 106 zoospores per liter. Experimental design and characteristics of toxic exposure. A group of 18 fish were exposed for 10 days to a sublethal concentration of un-ionized ammonia in static conditions in an 80-liter glass aquarium, with water renewal every 3 days. The experimental concentration of ammonia was 0.05 mg of N per liter as un-ionized ammonia, obtained by the addition of reagent grade ammonium chloride (NH4Cl) solution (Merck, Darmstadt, Germany). Total ammonia-nitrogen (NH3-N) was measured with an ammonia-selective electrode
of organic compounds from different wastes are predisposing environmental factors to outbreaks of saprolegniosis, but, until now, nothing was known about the specific effects of physicochemical parameters on saprolegniosis. Ammonia, nitrite, copper, and cyanide are common pollutants in aquatic environments, and their lethal and sublethal toxicities for salmonids are well documented (1, 6, 10, 17, 29). The present study was undertaken to observe the effects of *
Corresponding author. 1813
1814
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CARBALLO AND MUNOZ
(95-12; Orion Research, Cambridge, Mass.) calibrated with ammonium chloride solution. Un-ionized ammonia (NH3) concentrations were calculated from total concentrations (1, 7). Three groups of six fish that had been exposed to ammonia were immersed in the following doses of fungi: 5 x 105, 9.75 x 105, and 1.4 x 106 zoospores per liter. For each fungal dose, six animals without toxin exposure were used as controls. Similar conditions, including the same numbers of fish, the same exposure time, and the same zoospore concentrations were used to test the effects of a sublethal concentration of nitrite. Sublethal levels of nitrite (0.12 mg/liter) were maintained by the addition of potassium nitrite (KNO2) solutions (Merck) determined by a standard method (25). The infections was obtained with the same fungal doses as in the ammonia infection. A group of 20 fish were exposed for 21 days to a sublethal concentration of copper in static conditions in an 80-liter glass aquarium. The experimental copper concentration was 0.05 mg/liter. Copper was added as a copper nitrate [Cu(NO3)2] (Merck reagent grade) solution, and the copper concentration was determined with a Perkin-Elmer atomic absorption spectrophotometer (3030 B) equipped with a graphite furnace (HG-300). Two groups of 10 fish were then infected with two different doses of fungal inocula. A group of 10 fish unexposed to this toxin was used as controls. Cyanide tests were made with the same numbers of fish and the same exposure time as the copper tests. Cyanide levels (0.05 mg/liter) were maintained throughout the experimental period by the addition of a potassium cyanide (KCN) solution (Merck) and were measured with a cyanide-selective electrode (94-06000; Orion Research) calibrated with a potassium cyanide solution. Infections were obtained by using the same doses of fungal inocula used in the copper tests. Dissolved oxygen, temperature, pH, ammonia, nitrite, copper, and cyanide levels were analyzed daily. Water alkalinity, hardness, carbon dioxide, sodium, potassium, chloride, and permanganate-oxidizable matter levels were analyzed before and after water renewal. Selective electrodes (Orion Research) or standard procedures (2, 25) were used. Toxicant concentrations were measured daily. Infection of fish. After exposure, fish were anesthetized in a 0.03% solution of 2-phenoxy ethanol and wounded by removing 5- to 7-mm2 patches of scales from each side immediately in front of the dorsal fin above the lateral line. Animals were then immersed for 10 min in an experimental fungal inoculum (19, 26). Pathogen-exposed fish were transferred to an 80-liter glass aquarium with flow-through dechlorinated tap water and maintained 15 days or until the appearance of saprolegniosis. Infections were estimated by the presence of cottony-white patches on the body of the fish. Mucus samples were plated on glucose-peptone-penicillin-streptomycin agar plates, incubated at 20°C for 8 to 12 h (37), and observed for fungal growth. an
RESULTS The water characteristics used in the experiments (mean values + standard deviations) were (i) alkalinity (milligrams of CaCO3 per liter), 36.7 + 14; (ii) chloride concentration 5; (iii) hardness (milligrams of NaCl per liter), 13.1 5; (iv) sodium (milligrams of CaCO3 per liter), 33.7 concentration (milligrams of Na per liter), 5.6 + 1.3; (v) potassium concentration (milligrams of K per liter), 0.14 + 0.05; and (vi) organic matter concentration (milligrams of 02
5.7 + 1.6. Throughout the study the temperature in the fish aquaria was 15 + 3°C, and the pH was 7.3 + 0.2. The
per liter),
TABLE 1. Percentage of fish with fungal infections after being exposed for 10 days to un-ionized ammonia (0.05 mg of N per liter) or nitrite (0.12 mg of N per liter) at three inoculum concentrations Toxicant or control
Ammonia Nitrite Control
% of infected fish at inoculum concn (zoospores per liter) of: 1.4 x106
9.75 x105
5 x 105
75 50 0
20 0 0
0 0 0
concentration of carbon dioxide was 5.6 + 0.6 mg/liter, and the dissolved oxygen concentration was 7.3 + 1 mg/liter. Un-ionized ammonia and nitrite in aquaria without exposure to these chemicals did not exceed the recommendations of the U.S. Environmental Protection Agency (32). The mean toxicant concentrations + the standard deviations were 0.057 0.02 mg of N per liter for un-ionized ammonia, 0.117 0.02 mg of N per liter for nitrite, 0.045 0.01 mg of Cu per liter for total copper, and 0.052 + 0.06 mg of cyanide per liter for cyanide. The results of infection in fish exposed to ammonia and nitrite are in Table 1 and the results for copper and cyanide exposure are in Table 2. Infection occurred in the group of fish exposed to ammonia and nitrite, with the percentage of infected fish being higher when exposed to ammonia (75%) than to nitrite (50%) at the highest fungi dose used (1.45 x 106 zoospores per liter). At the medium fungal dose (9.75 x 105 zoospores per liter), infection was found only in ammonia-exposed fish (20%), and no infection was found in any group of tested fish at the lowest fungal dose used (5 x 105 zoospores per liter). No infection was found in fish exposed to copper and cyanide. The control fish had a very low percentage of infections at the highest fungal dose used. Infection was clearly evident as cottony-white mycelial masses on the fish epithelium in the descaled areas of the fish, appearing at 2 to 4 days. Cultures of the fungi isolated from the fish were analyzed, and typical S. parasitica characteristics were recorded. No mortalities occurred among infected fish. ±
±
±
DISCUSSION Nolard-Tintigner (19) found that the level of parasitism by members of the Saprolegniaceae depends on the number of secondary zoospores in the water. The experience of one of the authors with Lebistes reticulatus and Xiphophorus belleri, small tropical and very sensitive fish, indicated that a 100% infective dose was approximately 1.5 x 105 zoospores per liter or more; no infection was observed even at levels as high as 2.4 x 104 zoospores per liter. The concentration of TABLE 2. Percentage of fish with fungal infections after being exposed for 21 days to copper (0.05 ppm) or cyanide (0.05 ppm) at two inoculum concentrations Toxicant or control
Copper Nitrite Control
% of infected fish at inoculum concn (zoospores per liter) of:
4 x 106
3.2 x 106
0 0 1
0 0 0
VOL. 57, 1991
the fungal inoculum used in our experiments was higher, and infection was found at levels 6 and 10 times higher, but only in ammonia- and nitrite-exposed fish. Furthermore, the animals used in our study were immature fish, which have been described as being more resistant than sexually mature fish (22). The general susceptibility to a disease in animals has been described in terms of mortality rates, but it has been suggested (8) that changes in rates of infection and/or changes in morbidity are more subtle changes and are more important in evaluating the susceptibility to sublethal toxic effects than are mortality rates. Therefore, we studied the morbidity, as the percentage of infected fish, and evaluated the effects of different levels of exposure to sublethal concentrations of four chemicals. In addition, exposure to S. parasitica by scale removal was used to obtain a high percentage of infection (26). Our findings revealed higher percentages of infection when fishes were exposed to ammonia. Increases of susceptibility to bacterial infection after exposure to NH3 have been observed in Ictalurus punctatus (24, 33), which increases of ammonia were associated with bacterial gill disease and other bacterial infections. I. punctatus exposed to sublethal concentrations of ammonia (0.02 to 0.04 mg/ liter) were more susceptible to invasion by Aeromonas hydrophila (9). The exposure times at these concentrations were correlated with lowered host resistance. Smart (27) found that rainbow trout exposed to ammonia (0.05 to 0.4 mg/liter) for 1 week to 3 months had diminished numbers of erythrocytes, irreversible blood damage, inflammatory and degenerative lesions in the gills, and increased susceptibility to bacterial disease. Several alterations and toxic effects related to sublethal concentrations of ammonia include gill damage (16, 29), alterations of the mucous cells (16, 21), and an enhancement of corticosteroid levels (30). Infection by Saprolegnia spp. indicated that external mucus renewal was employed as a defense mechanism against the fungus (35). Mucus production can be influenced by the presence of environmental factors such as stress and pollutants (21). The effects of ammonia on mucus production are unknown. Possibly, the number of mucous cells in the epidermis remains constant, but the time required for the formation and discharge of mucus by the mucous cells varies under the influence of ammonia (16). When fish have been infected by a Saprolegnia sp., the fungus destroys the essential waterproofing properties of the skin, and the results are massive osmoregulatory problems (22). In fish exposed to ammonia, increases in ventilation frequency in an effort to fight against the osmoregulatory problems and gill damage generated by this chemical, are continually observed (16). Therefore, additional changes in osmotic balance in these fish might also be observed. Once initiated, a fungal infection is also a stress factor (18). Ammonia exposure increased corticosteroid levels (3, 30) in fish exposed to sublethal concentrations. Several of these factors could be involved in the high percentages of infection observed in ammonia-exposed fishes. Some well-documented effects of nitrite exposure are changes in electrolyte concentrations in plasma as well as the production of methemoglobinemia, hematological consequences of short-term nitrite exposure (34). In addition, corticosteroids released into the circulation after nitrite exposure (3), or after fungal infection has been initiated (18), can lead to an electrolyte imbalance (as a secondary effect of
Cu AND CN- EFFECTS ON SAPROLEGNIOSIS
1815
stress) that could be additive to the toxic effects of nitrite exposure. Saprolegnia infection of fish exposed to copper and cyanide was not observed, although the exposure times and fungal doses were twice those of the experiments with nitrogen compounds. The effects of sublethal copper concentrations on the increased susceptibility of fish to infection appear to be dependent on the concentration of copper as well as on the length of time the fish are exposed to the metal. In addition, it has been found that 0. mykiss exposed for 24 h to 0.01 mg of total copper per liter showed an increased susceptibility to Yersinia ruckeri infection (14). Infections increased after 48 h of copper exposure, but by 96 h of copper exposure the fish had begun to lose their susceptibility. Similar results were obtained (13) when rainbow trout were exposed to sublethal concentrations of copper (0.01 mg/liter), whereupon a 24-h exposure had a bigger effect than a longer exposure (9 days) on the increased susceptibility of rainbow trout to the infectious hematopoietic necrosis virus. Our findings were in agreement with these, and no infection was found after 21 days of exposure at 0.05 mg of copper per liter. Increases and decreases of corticosteroids were registered (5) when Oncorhynchus nerka was exposed for 24 h to concentrations of copper and water quality similar to those used in our experiments, indicating that the fish adapted to these conditions. Thus, changes in copper levels could affect, depending on the exposure time, the immunosuppressive effect of corticosteroids (20). The result could be either an increase or decrease in susceptibility to the disease, in spite of the direct toxic effect of copper on osmoregulation, as has been described (11, 12). The effects of cyanide on changes of fish susceptibility to infectious diseases have not been well documented. Cyanide is essentially an inhibitor of oxygen metabolism, rendering the tissues incapable of exchanging oxygen (6). At sublethal concentrations (0.01 to 0.042 mg/liter), it produced physiological changes such as reduced growth, altered respiration, and some degree of hepatic necrosis in juvenile rainbow trout (4, 15). A progressive acclimatization to this pollutant has been observed; after exposure of juvenile rainbow trout to 0.02 mg of HCN per liter for 21 days, an early depression of growth occurred, followed by an increase in growth rate by the end of the experiment (28). When juvenile rainbow trout were exposed to 0.01, 0.02, or 0.03 mg of HCN per liter, increased respiration rates were observed at 1 to 4 days after exposure, which gradually decreased at 5 to 6 days (4). Possibly, acclimatization occurred, and this toxic concentration did not affect the susceptibility to the disease. Our experiments confirm that specific toxicants (ammonia and nitrite) predispose fish to saprolegniosis. These effects might explain the results of Toor et al. (31) that a high organic load was a predisposing factor to an outbreak of saprolegniosis. Considering the relationship of organic matter to the accumulation of ammonia and nitrite in the water by the biological degradation of nitrogen-rich products, we might conclude that high ammonia and nitrite levels found in fish farms due to inadequate water renovation, insufficient cleaning, or very high fish density increase the risk of saprolegniosis. ACKNOWLEDGMENTS This work was supported in part by a grant from I.N.I.A. (research project no. I.N.I.A. 8171). The technical assistance of M. Cuellar and F. Plasencia is gratefully acknowledged.
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