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Effect of heavy metal stressors and salinity shock on the susceptibility of grouper (Epinephelus sp.) to infectious pancreatic necrosis virus
Virus Research 63 (1999) 121 – 129 www.elsevier.com/locate/virusres
Effect of heavy metal stressors and salinity shock on the susceptibility of grouper (Epinephelus sp.) to infectious pancreatic necrosis virus Hsin-Yiu Chou a,*, Tsui-Yi Peng a, Su-Jung Chang a, Ya-Li Hsu b, Jen-Leih Wu b b
a Department of Aquaculture, National Taiwan Ocean Uni6ersity, Keelung 202, Taiwan, ROC Laboratory of Marine Molecular Biology and Biotechnology, Institute of Zoology, Academia Sinica, Nankang 115, Taiwan, ROC
Abstract In the present study, attempts were made to clarify the effect of heavy metal stressors and salinity shock on the disease susceptibility of grouper fry (Epinephelus sp.) to infetious pancreatic necrosis virus (IPNV) infection. Zinc, cadmium and copper (5 ppm ZnCl2, 3 ppm CdCl2 and 1 ppm CuCl2) were used to treat groupers before and after virus infection. Cumulative mortalities in the experimental groups were 96 – 100% within 42 days. Only 5 – 15% mortalities were observed in most of the groups that were exposed to either heavy metals or virus infection alone. Subsequently, virus was re-isolated from the experimentally infected groupers, and copper concentration was measured in fish that had been exposed to CuCl2. We also investigated the effect of salinity shock (i.e. an abrupt change of salinity level from 33 ppt to either 40 ppt or 20 ppt) on susceptibility of grouper to IPNV. Similar results were obtained, mortalities of groupers in the experimental groups reached 80 – 100%. The results of the present study suggest that an IPN virus with only low pathogenicity could cause high mortality in groupers when combined with environmental stress. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Birnavirus infection; Cadmium; Copper; Grouper; Salinity; Shock; Zinc
1. Introduction In Taiwan, fisheries production is important to both the food supply and the country’s economy, and this is particularly true of aquaculture (Taiwan Fisheries Yearbook, 1996, 1997). With the rapid industrialization and corresponding eco* Corresponding author. Tel: + 886-2-2462-2192; fax: + 886-2-2463-4176. E-mail address: [email protected] (H.-Y. Chou)
nomic improvement that have occurred over the last two decades, consumer preferences have changed from quantity to quality. Even at relatively high prices, gourmet marine species such as sea bass, grouper and sea bream have become popular and this has led to the development of a profitable local marine aquaculture industry (Chen, 1990; Liao, 1991). Groupers (Epinephelus spp.) are especially prized and they are among the most highly priced fish in Taiwan.
0168-1702/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 7 0 2 ( 9 9 ) 0 0 0 6 5 - 9
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H.-Y. Chou et al. / Virus Research 63 (1999) 121–129
Unfortunately, the coastal environment continues to deteriorate as industrial pollutants are dumped directly into the sea (Hung and Tsai, 1991). Like other environmental stressors such as adverse water temperature and overcrowding, as well as biological factors such as age or co-infection by other pathogens, chemical pollutants are capable of having significant effects on the susceptibility of aquatic animals to certain viruses. In previous studies, we demonstrated that either an increase in temperature or heavy metal stress has a marked effect on the susceptibility of hard clam to a low pathogenic infectious pancreatic necrosis virus (IPNV)-like aquatic birnavirus infection (Chou et al., 1994, 1998). Similar positive correlations between increased environmental stress (transport, crowding, temperature and catching stress, and exposure to heavy metal pollutants) and increased mortality have also been documented in cases involving infectious haematopoietic necrosis virus (IHNV) (Hetrick et al., 1979), infectious pancreatic necrosis virus (IPNV) (Stangeland et al., 1996) and white sturgeon iridovirus (WSIV) (LaPatra et al., 1996). IPNV has a worldwide distribution. Dorson (1983) remarked that the virus has been found in every country where it has been sought. In Taiwan, many species of fish and shellfish have been found to be infected with IPNV (Hedrick et al., 1983; Wu et al., 1983; Ueno et al., 1984; Lo et al., 1988; Hsu et al., 1989; Chou et al., 1993). In combination, IPNV and environmental stressors may constitute a threat for the continued expansion of the marine aquaculture sector on this island. In the present study, therefore, attempts were made to clarify the effect of heavy metal stressors on the susceptibility of grouper to IPNV infection. For comparison, the effect of another environmental stressor, salinity shock, was also investigated. As a preliminary step, we also tested the ability of the virus itself to survive at relatively high concentrations of Zn2 + , Cd2 + and Cu2 + . This was to ensure that any increase in mortality rates in the presence of heavy metal ions could be attributed to the combination of an infection by a still viable virus plus an environmental stressor. In an earlier study, we found that the protocol ‘stressor followed by virus’ produced different mortal-
ity patterns compared stressor’. Accordingly, groupers were treated salinity shock either infection.
to ‘virus followed by in the present study, with heavy metals and before or after virus
2. Materials and methods
2.1. Virus IPNV T42G virus was used for experimental infection in this study. This virus was isolated from rainbow trout (Oncorhynchus mykiss) at Lu Gu trout farm in Taiwan and has been shown to be closely related to the IPNV VR-299 fingerprint group (Hsu et al., 1989, 1995). The virus was propagated in CHSE-214 cells at 20°C.
2.2. Groupers The healthy grouper fry (Epinephelus sp.) used in this study were obtained from a grouper hatchery in southern Taiwan. They were verified to be virus-free by virus isolation tests on tissue homogenates using the four established fish cell lines (CHSE-214, EPC, FHM and RTG-2) before being subjected to the pathogenicity tests. The groupers were kept in aerated tanks where water temperature and salinity were 2591°C and 33 ppt, respectively. They were fed commercial feed twice daily.
2.3. Virus sur6i6al studies The ability of the T42G virus itself to survive in solutions containing different concentrations of Zn2 + , Cd2 + and Cu2 + was investigated. Survival studies of T42G were conducted for a period of more than 28 days. At specific time intervals, infectious virus titres were determined by microtiter plate techniques using CHSE-214 cells and expressed as TCID50/ml (50% tissue culture infective dose) (Rovozzo and Burke, 1973). Zinc, cadmium and copper stock solutions (1 M ZnCl2, 0.1 M CdCl2 and 1 M CuCl2) were prepared from reagent-grade ZnCl2, CdCl2·212H2O and CuCl2·2H2O (Hayashi Pure Chemical Indus-
H.-Y. Chou et al. / Virus Research 63 (1999) 121–129
tries Ltd., Japan). Stock solutions were prepared by dissolving CdCl2·212H2O and CuCl2·2H2O in sterilized double-distilled water (DDW) and ZnCl2 in sterilized HCl-acidified DDW. The stock solutions were filter sterilized through a 0.45-mm membrane and stored at room temperature. Heavy metal stock solutions were diluted with sterilized DDW into the appropriate concentrations for testing. After mixing with an equal volume of virus solution (108 TCID50/ml), the final mixtures contained either 20 or 2 mM of ZnCl2, CdCl2 and 20, 2, 0.2 or 0.02 mM of CuCl2. These mixtures were held at 20°C until virus titration. (For reference, 20 mM ZnCl2 =2729 ppm; 20 mM CdCl2 =4387 ppm; 20 mM CuCl2 =5420 ppm.)
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groupers were then caught for a third time and returned to their original 33 ppt seawater holding tanks; in the salinity shock experiments, the original 33 ppt environment was restored by flushing with seawater for 6 h. A simplified flow diagram and details of the experimental conditions are given in the scheme below.
2.4. Effect of three hea6y metal cations on susceptibility of grouper to T42G 6irus The metal concentrations used reflect government specified concentrations for heavy metal contaminants in water discharges from factories or mines in Taiwan. Based on the preliminary results of short-term exposure (48 h followed by observation for 7 days) of grouper to zinc, cadmium and copper solutions which had no influence on grouper survival, 5 ppm ZnCl2, 3 ppm CdCl2 and 1 ppm CuCl2 concentrations were selected and used in the subsequent experiments. In these infection trials, which were carried out by means of waterborne inoculation, the effect of exposure of groupers to seawater containing zinc, cadmium and copper before or after T42G virus infection was investigated. Two replicates of 20 groupers were used in every treatment. Water temperature and salinity were respectively 259 1°C and 33 ppt throughout the experiments unless otherwise stated. The basic protocols for all four of the studies described here were similar. Grouper fry (1 g average weight, 3.3 cm average length) were netted and transferred from seawater holding tanks to the first experimental environment (i.e. virus supernatant, environmental stressor or control). After 24 or 48 h, the groupers were recaptured and transferred to the second experimental environment. In the heavy metal experiment,
In experiment I, groupers were immersed in 105.0 TCID50/ml T42G virus suspension for 24 h. Control groups were exposed to supernatant from a CHSE-214 cell culture which did not contain the virus. Afterwards, the experimental groups were transferred to tanks of seawater which contained ZnCl2, CdCl2 and CuCl2, respectively. Mortality was observed daily for 42 days. In experiment II, the experimental groups (40 groupers each) were exposed to seawater which contained ZnCl2, CdCl2 and CuCl2, respectively, for 48 h. Half of each group was then infected with virus by immersion (105.0 TCID50/ml for 24 h), while half served as controls for the heavy metal treatment. Mortality was recorded daily for 42 days. Dead and moribund fish were collected and attempts were made to re-isolate the virus. Some of the fish collected had already been partially eaten (groupers are cannibalistic), but only complete fish were used for virus re-isolation. The viscera of each fish were removed, weighed and homogenized. HBSS (Hank’s buffered saline solu-
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tion) was added to homogenates in the ratio of 4:1. After being centrifuged at 1300×g (Sigma 2K15 rotor 12145) for 20 min, the supernatant fluid was removed to a new vial and mixed with antibiotics (1000 IU/ml penicillin, 1000 mg/ml streptomycin and 50 mg/ml fungizone). After 4 h of continuous shaking at 4°C, the mixture was inoculated into 24-well plates (50 ml/well) containing confluent monolayers of CHSE-214 cells prepared 24 h previously. The plates were then incubated at 20°C until a cytopathic effect (CPE) was observed.
2.5. Copper analysis At 2 days and 7 days after a 33 ppt seawater environment had been restored, the gill, viscera and muscle were removed from three groupers (5.3 g average weight, 7.5 cm average length) from each experimental group and from each corresponding control group. After being allowed to dry out at 60°C overnight, the tissues were then digested in concentrated nitric acid (15 ml per 0.5 – 1 g dry weight) and superoxide (5 ml per 0.5 – 1 g dry weight) for 2 h at 80°C and then for a further 48 h at room temperature. DDW was added to the mixtures to make a total volume of 10 ml, and the fluids were filtered through a 0.45-mm membrane. Flame atomic absorption spectrophotometry (HITACHI, Z-8200, Polarized Zeeman Atomic Absorption Spectrophotometer) was then used to analyze the copper content.
2.6. Effect of salinity shock on susceptibility of grouper to T42G 6irus The effect of salinity shock (33 ppt to 20 ppt, or 33 ppt to 40 ppt) before or after virus infection on the susceptibility of groupers to the T42G virus was investigated. Two replicates of 20 groupers (mean weight 1 g) were used in every treatment. In experiment III, groupers were immersed in 105.0 TCID50/ml T42G virus solution for 24 h. Control groups were exposed to supernatant from a CHSE-214 cell culture which did not contain the virus. Afterwards, the groupers were transferred to tanks of seawater in which salinity was 20 ppt, 33 ppt or 40 ppt for 48 h. A 33 ppt seawater
environment was restored by flushing for 6 h. Mortality was recorded daily for 28 days. In experiment IV, groups of 40 groupers were kept in seawater with salinity of 20 ppt, 33 ppt and 40 ppt, respectively, for 48 h. Half of each group were then transferred to tanks of virus solution (105.0 TCID50/ml), prepared in 20 ppt, 33 ppt and 40 ppt, respectively. The other half were transferred to tanks of seawater with the same three salinities and served as controls for the salinity shock treatment. After 24 h, the original 33 ppt seawater environment was restored by flushing for 6 h. Mortality was recorded daily for 28 days.
3. Results
3.1. Virus sur6i6al studies Fig. 1 shows the results of T42G virus survival in several concentrations of ZnCl2, CdCl2 and CuCl2 solutions. In ZnCl2/DDW and CdCl2/ DDW solutions, T42G virus was stable, with infectivity being sustained at 85–90% after 28 days. T42G was not stable in CuCl2 solution: virus infectivity declined rapidly in the 20 mM, 2 mM and 0.2 mM CuCl2 solutions, and more slowly in the 0.02 mM CuCl2 solution, although infectivity survived for 28 days in the 0.02 mM CuCl2 solution.
3.2. Effect of three hea6y metal cations on susceptibility of groupers to T42G 6irus In experiment I, mortalities increased sharply in the fifth or sixth week, and after 42 days cumulative mortalities had reached 96–100% in the experimental groups (Fig. 2). In experiment II, in which the groupers which had been stressed with the heavy metals first, onset of mortality was earlier than experiment I, and cumulative mortalities reached 100% in 42 days (Fig. 3). In both experiments I and II, only 10–15% mortalities were observed in the groups that were exposed to either heavy metals or virus infection, but not both. No groupers died in the control groups during the experimental period. Mortality
H.-Y. Chou et al. / Virus Research 63 (1999) 121–129
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among the experimentally infected groupers under stress was higher than that of groupers which were infected with virus only or exposed to heavy metals only. As Table 1 shows, virus was successfully re-isolated from almost all of the tested samples.
Fig. 2. Cumulative mortality (%) of grouper (1 g average weight, 3.3 cm average length) experimentally infected by immersion in T42G virus suspension (105.0 TCID50/ml) for 24 h, subsequently transferred to tanks of seawater containing either 5 ppm ZnCl2, 3 ppm CdCl2 or 1 ppm CuCl2, and reared in seawater at 25°C. Data shown are the mean of two replicates.
3.3. Copper analysis Fig. 1. Survival of T42G virus (107.5 TCID50/ml) in different concentrations of ZnCl2, CdCl2 and CuCl2 solutions.
Low copper contents (B5 mg/g) were measured in gill and viscera, and almost no copper was
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detected in muscle. The results of the 2-day and 7-day copper content analysis were very similar in the groupers from experimental and control groups (Fig. 4).
3.4. Effect of salinity shock on susceptibility of grouper to T42G 6irus After 28 days, cumulative mortalities of groupers exposed to the virus first (experiment III) were about 80% (Fig. 5). In experiment IV, survival time shortened and the mortalities reached 90–100% within 2 weeks (Fig. 6). Although mortality was also quite high (45%) in fish subjected to increased salinity shock, only 10% mortalities were observed in the other groups that were treated either with salinity shock or virus infection, but not both. No fish died in the control groups.
4. Discussion
Fig. 3. Cumulative mortality (%) of grouper (1 g average weight, 3.3 cm average length) experimentally exposed to either 5 ppm ZnCl2, 3 ppmCdCl2 or 1 ppm CuCl2 for 48 h, subsequently infected by immersion in T42G virus suspension (105.0 TCID50/ml) for 24 h, and then reared in seawater at 25°C . Data shown are the mean of two replicates.
Heavy metal poisoning alone cannot account for the results seen in experiments I and II (Fig. 2). Firstly, since milkfish fry (Chanos chanos) can survive copper levels of 250 ppm (Lai, 1987) and carp (Cyprinus carpio) can survive similar conditions (Lin and Tang, 1990), the much lower levels found in the present study ( B 5 mg/g; Fig. 3) appear to be well below lethal concentrations. The high mortalities found in the present study (see Figs. 2, 3, 5 and 6), however, can be accounted for if the virus and the environmental stressor— whether heavy metal or salinity shock—are having a combined effect. In fact, fishery biologists have long recognized that stresses such as rapid temperature changes, handling, crowding and deterioration of water quality adversely affect fish health and increase susceptibility to disease (Wedemeyer, 1970; Snieszko, 1974). A number of mechanisms by which pollutants may act to increase disease incidence in fish have been speculated, many suggesting immunosuppression as a factor in the impact of infectious disease among fishes in highly contaminated areas (Anderson et al., 1984; Moller, 1985; Sindermann, 1993; Zelikoff, 1993). Ross et al. (1996) have defined immunotoxicity as a toxicant-induced injury to part of the immune system that may lead to a diminished capacity of an organism to defend itself against infection by pathogens. They also demonstrated
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Table 1 Positive detection of re-isolated virus from dead or moribund groupers exposed to heavy metals before or after virus infection
Samplesa a
V/Zn
Zn/V
V/Cd
Cd/V
V/Cu
Cu/V
V
7/7 (100)
14/14 (100)
12/12 (100)
22/24 (92)
14/14 (100)
7/7 (100)
2/2 (100)
Number positive/total number tested; % in parentheses.
that environmental contaminants such as polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDDs), and polychlorinated dibenzofurans (PCDFs) can be immunotoxic at low doses in marine mammals (Ross et al., 1996). Although the immune system of grouper bears little similarity to that of a marine mammal, an immunotoxic effect (albeit via different pathways) may well account for the results of the present study. Some corroboration is provided by the virus titres that were taken at the end of experiments I and II (data not shown); although virus was not found in surviving fish that were exposed to virus only, a low virus titre was found in the dead fish of these same virus-only groups. Furthermore, virus titres of almost exactly the same low levels were found in all tested fish in the heavy metal/virus and virus/heavy metal groups. This suggests: (1) that the virus did not replicate in any of the fish; (2) that the fish’s immune system effectively eliminated the virus in most of the unstressed fish; (3) that, when the virus was not eliminated, even these low virus levels were sufficiently high to cause death in most cases; and (4) that the increased susceptibility of the fish exposed to heavy metals was due to their inability to eliminate the virus. These considerations support the conclusion that the high mortalities were the result of an immunotoxic effect. Other explanations are nonetheless possible, and indeed in the salinity stress studies (experiments III and IV) immunotoxicity seems an unlikely explanation. The earlier onset of mortality in experiments III and IV (cf. Figs. 2, 3, 5 and 6) also needs to be accounted for. In the absence of any specific mechanism for this immune suppression in this study, other explanations cannot be ruled out. More work to establish a link between environmental stressors, IPNV infection and immune responses of grouper is required. Since so
Fig. 4. Copper contents found in gill, viscera and muscle of groupers from experimental groups and their corresponding control groups at 2 days and 7 days. Data shown are the mean of three pooled samples from each group.
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Fig. 5. Cumulative mortality (%) of grouper (1 g average weight, 3.3 cm average length) experimentally infected by immersion in T42G virus suspension (105.0 TCID50/ml) for 24 h, subsequently transferred to lower (from 33 ppt to 20 ppt) or higher (33 ppt to 40 ppt) salinity for 48 h, and flushed with seawater until salinity returned to 33 ppt in 6 h. Water temperature was 25 91°C during the experiment. Data shown are the mean of two replicates.
many different environmental stressors in combination with a virus produce such similar effects, it might also prove worthwhile to continue the search for stress proteins analogous to those that are already well documented in humans.
Acknowledgements This work was supported by the National Science Council under Grant No. NSC86-2311-B019-003-B23. We also thank N.-H. Yu for providing the fish, and to P. Barlow for his helpful comments on the manuscript.
References
Fig. 6. Cumulative mortality (%) of grouper (1 g average weight, 3.3 cm average length) experimentally subjected to lower (33 ppt to 20 ppt) or higher (33 ppt to 40 ppt) salinity for 48 h and then infected by immersion in T42G virus suspension (105.0 TCID50/ml) for 24 h before being returned to 33 ppt seawater at 2591°C. Data shown are the mean of two replicates.
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H.-Y. Chou et al. / Virus Research 63 (1999) 121–129 Dorson, M., 1983. Infectious pancreatic necrosis of salmonids: overview of current problems. In: Anderson, D.P., Dorson, M., Dubourget, P. (Eds.), Antigens of Fish Pathogens— Development and Production Standards for Vaccines and Serodiagnostics. Collection Foundation Marcel Merieux, pp. 7 – 32. Hedrick, R.P., Fryer, J.L., Chen, S.N., Kou, G.H., 1983. Characteristics of four birnaviruses isolated from fish in Taiwan. Fish Pathol. 10, 91–97. Hetrick, F.M., Knuttek, M.D., Fryer, J.L., 1979. Increased susceptibility of rainbow trout to infectious hematopoietic necrosis virus after exposure to copper. Appl. Envir. Microbiol. 37, 198 – 201. Hsu, Y.L., Chen, B.S., Wu, J.L., 1989. Comparison of RNAs and polypeptides of infectious pancreatic necrosis viruses from eel and rainbow trout. J. Gen. Virol. 70, 2233–2239. Hsu, Y.L., Chen, C.C., Wu, J.L., 1995. Molecular relationships in infectious pancreatic necrosis virus. Virus Res. 37, 239 – 252. Hung, T.C., Tsai, C.C.H., 1991. Study of heavy metals on the western coast of Taiwan. J. Environ. Protect. Soc. ROC 14, 11 – 34. Lai, C.M., 1987. The residue and the influence of growth in Chanos chanos and Meretrix lusoria with different low concentrations of heavy metals. Bull. Taiwan Fish. Res. Inst. 42, 305 – 310 (in Chinese). LaPatra, S.E., Groff, J.M., Patterson, T.L., Shewmaker, W.D., Casten, M., Siple, J., Hauck, A.K., 1996. Preliminary evidence of sturgeon density and other stressors on manifestation of white sturgeon iridovirus disease. J. Appl. Aquacult. 6, 51 – 58. Liao, I.C., 1991. Aquaculture: the Taiwanese experience. Bull. Inst. Zool. Acad. Sinica Monogr. 16, 1–36 (in Chinese). Lin, T.S., Tang, H.C., 1990. Accumulation and elimination of copper and cadmium in common carp. Bull. Taiwan Fish. Res. Inst. 48, 127 – 136 (in Chinese). Lo, C.F., Hong, Y.W., Huang, S.Y., Wang, C.H., 1988. The
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characteristics of the virus isolated from the gill of clam, Meretrix lusoria. Fish Pathol. 23, 147 – 154. Moller, H., 1985. A critical review on the role of pollution as a cause of fish diseases. In: Ellis, A.E. (Ed.), Fish and Shellfish Pathology. Academic Press, London, pp. 169 – 182. Ross, P.S., De Swart, R.L., Van Loveren, H., Osterhaus, A.D.M.E., Vos, J.G., 1996. The immunotoxicity of environmental contaminants to marine wildlife: a review. Annu. Rev. Fish Dis. 6, 151 – 165. Rovozzo, G.C., Burke, C.N., 1973. A Manual of Basic Virological Technique. Prentice-Hall, Englewood Cliffs, NJ. Snieszko, S.F., 1974. The effects of environmental stress on outbreaks of infectious diseases of fishes. J. Fish Biol. 6, 197 – 208. Sindermann, C.J., 1993. Interactions of pollutants and disease in marine fish and shellfish. In: Couch, J.A., Fournie, J.W. (Eds.), Advances in Fisheries Science: Pathobiology of Marine and Estuarine Organisms. CRC Press, Boca Raton, pp. 451 – 482. Stangeland, K., Hoie, S., Taksdal, T., 1996. Experimental induction of infectious pancreatic necrosis in Atlantic salmon, Salmo salar L., post-smolts. J. Fish Dis. 19, 323 – 327. Ueno, Y., Chen, S.N., Kou, G.H., Hedrick, R.P., Fryer, J.L., 1984. Characterization of virus isolated from Japanese eel (Anguilla japonica) with nephroblastoma. Bull. Inst. Zool. Acad. Sinica 23, 47 – 55. Wedemeyer, G.A., 1970. The role of stress in the disease resistance of fishes. Am. Fish. Soc. Special Publ. 5, 30 – 35. Wu, J.L., Lin, H.M., Tang, F.J., Kou, G.H., Liu, K.C., 1983. Isolation and characterization of IPNV from rainbow trout in Taiwan. Proceedings of ROC – Japan Cooperative Science Seminar on Fish Diseases, pp. 59 – 67. Zelikoff, J.T., 1993. Metal pollution-induced immunomodulation in fish. Annu. Rev. Fish Dis. 3, 305 – 325.
Effect of heavy metal stressors and salinity shock on the susceptibility of grouper (Epinephelus sp.) to infectious pancreatic necrosis virus Hsin-Yiu Chou a,*, Tsui-Yi Peng a, Su-Jung Chang a, Ya-Li Hsu b, Jen-Leih Wu b b
a Department of Aquaculture, National Taiwan Ocean Uni6ersity, Keelung 202, Taiwan, ROC Laboratory of Marine Molecular Biology and Biotechnology, Institute of Zoology, Academia Sinica, Nankang 115, Taiwan, ROC
Abstract In the present study, attempts were made to clarify the effect of heavy metal stressors and salinity shock on the disease susceptibility of grouper fry (Epinephelus sp.) to infetious pancreatic necrosis virus (IPNV) infection. Zinc, cadmium and copper (5 ppm ZnCl2, 3 ppm CdCl2 and 1 ppm CuCl2) were used to treat groupers before and after virus infection. Cumulative mortalities in the experimental groups were 96 – 100% within 42 days. Only 5 – 15% mortalities were observed in most of the groups that were exposed to either heavy metals or virus infection alone. Subsequently, virus was re-isolated from the experimentally infected groupers, and copper concentration was measured in fish that had been exposed to CuCl2. We also investigated the effect of salinity shock (i.e. an abrupt change of salinity level from 33 ppt to either 40 ppt or 20 ppt) on susceptibility of grouper to IPNV. Similar results were obtained, mortalities of groupers in the experimental groups reached 80 – 100%. The results of the present study suggest that an IPN virus with only low pathogenicity could cause high mortality in groupers when combined with environmental stress. © 1999 Elsevier Science B.V. All rights reserved. Keywords: Birnavirus infection; Cadmium; Copper; Grouper; Salinity; Shock; Zinc
1. Introduction In Taiwan, fisheries production is important to both the food supply and the country’s economy, and this is particularly true of aquaculture (Taiwan Fisheries Yearbook, 1996, 1997). With the rapid industrialization and corresponding eco* Corresponding author. Tel: + 886-2-2462-2192; fax: + 886-2-2463-4176. E-mail address: [email protected] (H.-Y. Chou)
nomic improvement that have occurred over the last two decades, consumer preferences have changed from quantity to quality. Even at relatively high prices, gourmet marine species such as sea bass, grouper and sea bream have become popular and this has led to the development of a profitable local marine aquaculture industry (Chen, 1990; Liao, 1991). Groupers (Epinephelus spp.) are especially prized and they are among the most highly priced fish in Taiwan.
0168-1702/99/$ - see front matter © 1999 Elsevier Science B.V. All rights reserved. PII: S 0 1 6 8 - 1 7 0 2 ( 9 9 ) 0 0 0 6 5 - 9
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H.-Y. Chou et al. / Virus Research 63 (1999) 121–129
Unfortunately, the coastal environment continues to deteriorate as industrial pollutants are dumped directly into the sea (Hung and Tsai, 1991). Like other environmental stressors such as adverse water temperature and overcrowding, as well as biological factors such as age or co-infection by other pathogens, chemical pollutants are capable of having significant effects on the susceptibility of aquatic animals to certain viruses. In previous studies, we demonstrated that either an increase in temperature or heavy metal stress has a marked effect on the susceptibility of hard clam to a low pathogenic infectious pancreatic necrosis virus (IPNV)-like aquatic birnavirus infection (Chou et al., 1994, 1998). Similar positive correlations between increased environmental stress (transport, crowding, temperature and catching stress, and exposure to heavy metal pollutants) and increased mortality have also been documented in cases involving infectious haematopoietic necrosis virus (IHNV) (Hetrick et al., 1979), infectious pancreatic necrosis virus (IPNV) (Stangeland et al., 1996) and white sturgeon iridovirus (WSIV) (LaPatra et al., 1996). IPNV has a worldwide distribution. Dorson (1983) remarked that the virus has been found in every country where it has been sought. In Taiwan, many species of fish and shellfish have been found to be infected with IPNV (Hedrick et al., 1983; Wu et al., 1983; Ueno et al., 1984; Lo et al., 1988; Hsu et al., 1989; Chou et al., 1993). In combination, IPNV and environmental stressors may constitute a threat for the continued expansion of the marine aquaculture sector on this island. In the present study, therefore, attempts were made to clarify the effect of heavy metal stressors on the susceptibility of grouper to IPNV infection. For comparison, the effect of another environmental stressor, salinity shock, was also investigated. As a preliminary step, we also tested the ability of the virus itself to survive at relatively high concentrations of Zn2 + , Cd2 + and Cu2 + . This was to ensure that any increase in mortality rates in the presence of heavy metal ions could be attributed to the combination of an infection by a still viable virus plus an environmental stressor. In an earlier study, we found that the protocol ‘stressor followed by virus’ produced different mortal-
ity patterns compared stressor’. Accordingly, groupers were treated salinity shock either infection.
to ‘virus followed by in the present study, with heavy metals and before or after virus
2. Materials and methods
2.1. Virus IPNV T42G virus was used for experimental infection in this study. This virus was isolated from rainbow trout (Oncorhynchus mykiss) at Lu Gu trout farm in Taiwan and has been shown to be closely related to the IPNV VR-299 fingerprint group (Hsu et al., 1989, 1995). The virus was propagated in CHSE-214 cells at 20°C.
2.2. Groupers The healthy grouper fry (Epinephelus sp.) used in this study were obtained from a grouper hatchery in southern Taiwan. They were verified to be virus-free by virus isolation tests on tissue homogenates using the four established fish cell lines (CHSE-214, EPC, FHM and RTG-2) before being subjected to the pathogenicity tests. The groupers were kept in aerated tanks where water temperature and salinity were 2591°C and 33 ppt, respectively. They were fed commercial feed twice daily.
2.3. Virus sur6i6al studies The ability of the T42G virus itself to survive in solutions containing different concentrations of Zn2 + , Cd2 + and Cu2 + was investigated. Survival studies of T42G were conducted for a period of more than 28 days. At specific time intervals, infectious virus titres were determined by microtiter plate techniques using CHSE-214 cells and expressed as TCID50/ml (50% tissue culture infective dose) (Rovozzo and Burke, 1973). Zinc, cadmium and copper stock solutions (1 M ZnCl2, 0.1 M CdCl2 and 1 M CuCl2) were prepared from reagent-grade ZnCl2, CdCl2·212H2O and CuCl2·2H2O (Hayashi Pure Chemical Indus-
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tries Ltd., Japan). Stock solutions were prepared by dissolving CdCl2·212H2O and CuCl2·2H2O in sterilized double-distilled water (DDW) and ZnCl2 in sterilized HCl-acidified DDW. The stock solutions were filter sterilized through a 0.45-mm membrane and stored at room temperature. Heavy metal stock solutions were diluted with sterilized DDW into the appropriate concentrations for testing. After mixing with an equal volume of virus solution (108 TCID50/ml), the final mixtures contained either 20 or 2 mM of ZnCl2, CdCl2 and 20, 2, 0.2 or 0.02 mM of CuCl2. These mixtures were held at 20°C until virus titration. (For reference, 20 mM ZnCl2 =2729 ppm; 20 mM CdCl2 =4387 ppm; 20 mM CuCl2 =5420 ppm.)
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groupers were then caught for a third time and returned to their original 33 ppt seawater holding tanks; in the salinity shock experiments, the original 33 ppt environment was restored by flushing with seawater for 6 h. A simplified flow diagram and details of the experimental conditions are given in the scheme below.
2.4. Effect of three hea6y metal cations on susceptibility of grouper to T42G 6irus The metal concentrations used reflect government specified concentrations for heavy metal contaminants in water discharges from factories or mines in Taiwan. Based on the preliminary results of short-term exposure (48 h followed by observation for 7 days) of grouper to zinc, cadmium and copper solutions which had no influence on grouper survival, 5 ppm ZnCl2, 3 ppm CdCl2 and 1 ppm CuCl2 concentrations were selected and used in the subsequent experiments. In these infection trials, which were carried out by means of waterborne inoculation, the effect of exposure of groupers to seawater containing zinc, cadmium and copper before or after T42G virus infection was investigated. Two replicates of 20 groupers were used in every treatment. Water temperature and salinity were respectively 259 1°C and 33 ppt throughout the experiments unless otherwise stated. The basic protocols for all four of the studies described here were similar. Grouper fry (1 g average weight, 3.3 cm average length) were netted and transferred from seawater holding tanks to the first experimental environment (i.e. virus supernatant, environmental stressor or control). After 24 or 48 h, the groupers were recaptured and transferred to the second experimental environment. In the heavy metal experiment,
In experiment I, groupers were immersed in 105.0 TCID50/ml T42G virus suspension for 24 h. Control groups were exposed to supernatant from a CHSE-214 cell culture which did not contain the virus. Afterwards, the experimental groups were transferred to tanks of seawater which contained ZnCl2, CdCl2 and CuCl2, respectively. Mortality was observed daily for 42 days. In experiment II, the experimental groups (40 groupers each) were exposed to seawater which contained ZnCl2, CdCl2 and CuCl2, respectively, for 48 h. Half of each group was then infected with virus by immersion (105.0 TCID50/ml for 24 h), while half served as controls for the heavy metal treatment. Mortality was recorded daily for 42 days. Dead and moribund fish were collected and attempts were made to re-isolate the virus. Some of the fish collected had already been partially eaten (groupers are cannibalistic), but only complete fish were used for virus re-isolation. The viscera of each fish were removed, weighed and homogenized. HBSS (Hank’s buffered saline solu-
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tion) was added to homogenates in the ratio of 4:1. After being centrifuged at 1300×g (Sigma 2K15 rotor 12145) for 20 min, the supernatant fluid was removed to a new vial and mixed with antibiotics (1000 IU/ml penicillin, 1000 mg/ml streptomycin and 50 mg/ml fungizone). After 4 h of continuous shaking at 4°C, the mixture was inoculated into 24-well plates (50 ml/well) containing confluent monolayers of CHSE-214 cells prepared 24 h previously. The plates were then incubated at 20°C until a cytopathic effect (CPE) was observed.
2.5. Copper analysis At 2 days and 7 days after a 33 ppt seawater environment had been restored, the gill, viscera and muscle were removed from three groupers (5.3 g average weight, 7.5 cm average length) from each experimental group and from each corresponding control group. After being allowed to dry out at 60°C overnight, the tissues were then digested in concentrated nitric acid (15 ml per 0.5 – 1 g dry weight) and superoxide (5 ml per 0.5 – 1 g dry weight) for 2 h at 80°C and then for a further 48 h at room temperature. DDW was added to the mixtures to make a total volume of 10 ml, and the fluids were filtered through a 0.45-mm membrane. Flame atomic absorption spectrophotometry (HITACHI, Z-8200, Polarized Zeeman Atomic Absorption Spectrophotometer) was then used to analyze the copper content.
2.6. Effect of salinity shock on susceptibility of grouper to T42G 6irus The effect of salinity shock (33 ppt to 20 ppt, or 33 ppt to 40 ppt) before or after virus infection on the susceptibility of groupers to the T42G virus was investigated. Two replicates of 20 groupers (mean weight 1 g) were used in every treatment. In experiment III, groupers were immersed in 105.0 TCID50/ml T42G virus solution for 24 h. Control groups were exposed to supernatant from a CHSE-214 cell culture which did not contain the virus. Afterwards, the groupers were transferred to tanks of seawater in which salinity was 20 ppt, 33 ppt or 40 ppt for 48 h. A 33 ppt seawater
environment was restored by flushing for 6 h. Mortality was recorded daily for 28 days. In experiment IV, groups of 40 groupers were kept in seawater with salinity of 20 ppt, 33 ppt and 40 ppt, respectively, for 48 h. Half of each group were then transferred to tanks of virus solution (105.0 TCID50/ml), prepared in 20 ppt, 33 ppt and 40 ppt, respectively. The other half were transferred to tanks of seawater with the same three salinities and served as controls for the salinity shock treatment. After 24 h, the original 33 ppt seawater environment was restored by flushing for 6 h. Mortality was recorded daily for 28 days.
3. Results
3.1. Virus sur6i6al studies Fig. 1 shows the results of T42G virus survival in several concentrations of ZnCl2, CdCl2 and CuCl2 solutions. In ZnCl2/DDW and CdCl2/ DDW solutions, T42G virus was stable, with infectivity being sustained at 85–90% after 28 days. T42G was not stable in CuCl2 solution: virus infectivity declined rapidly in the 20 mM, 2 mM and 0.2 mM CuCl2 solutions, and more slowly in the 0.02 mM CuCl2 solution, although infectivity survived for 28 days in the 0.02 mM CuCl2 solution.
3.2. Effect of three hea6y metal cations on susceptibility of groupers to T42G 6irus In experiment I, mortalities increased sharply in the fifth or sixth week, and after 42 days cumulative mortalities had reached 96–100% in the experimental groups (Fig. 2). In experiment II, in which the groupers which had been stressed with the heavy metals first, onset of mortality was earlier than experiment I, and cumulative mortalities reached 100% in 42 days (Fig. 3). In both experiments I and II, only 10–15% mortalities were observed in the groups that were exposed to either heavy metals or virus infection, but not both. No groupers died in the control groups during the experimental period. Mortality
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among the experimentally infected groupers under stress was higher than that of groupers which were infected with virus only or exposed to heavy metals only. As Table 1 shows, virus was successfully re-isolated from almost all of the tested samples.
Fig. 2. Cumulative mortality (%) of grouper (1 g average weight, 3.3 cm average length) experimentally infected by immersion in T42G virus suspension (105.0 TCID50/ml) for 24 h, subsequently transferred to tanks of seawater containing either 5 ppm ZnCl2, 3 ppm CdCl2 or 1 ppm CuCl2, and reared in seawater at 25°C. Data shown are the mean of two replicates.
3.3. Copper analysis Fig. 1. Survival of T42G virus (107.5 TCID50/ml) in different concentrations of ZnCl2, CdCl2 and CuCl2 solutions.
Low copper contents (B5 mg/g) were measured in gill and viscera, and almost no copper was
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detected in muscle. The results of the 2-day and 7-day copper content analysis were very similar in the groupers from experimental and control groups (Fig. 4).
3.4. Effect of salinity shock on susceptibility of grouper to T42G 6irus After 28 days, cumulative mortalities of groupers exposed to the virus first (experiment III) were about 80% (Fig. 5). In experiment IV, survival time shortened and the mortalities reached 90–100% within 2 weeks (Fig. 6). Although mortality was also quite high (45%) in fish subjected to increased salinity shock, only 10% mortalities were observed in the other groups that were treated either with salinity shock or virus infection, but not both. No fish died in the control groups.
4. Discussion
Fig. 3. Cumulative mortality (%) of grouper (1 g average weight, 3.3 cm average length) experimentally exposed to either 5 ppm ZnCl2, 3 ppmCdCl2 or 1 ppm CuCl2 for 48 h, subsequently infected by immersion in T42G virus suspension (105.0 TCID50/ml) for 24 h, and then reared in seawater at 25°C . Data shown are the mean of two replicates.
Heavy metal poisoning alone cannot account for the results seen in experiments I and II (Fig. 2). Firstly, since milkfish fry (Chanos chanos) can survive copper levels of 250 ppm (Lai, 1987) and carp (Cyprinus carpio) can survive similar conditions (Lin and Tang, 1990), the much lower levels found in the present study ( B 5 mg/g; Fig. 3) appear to be well below lethal concentrations. The high mortalities found in the present study (see Figs. 2, 3, 5 and 6), however, can be accounted for if the virus and the environmental stressor— whether heavy metal or salinity shock—are having a combined effect. In fact, fishery biologists have long recognized that stresses such as rapid temperature changes, handling, crowding and deterioration of water quality adversely affect fish health and increase susceptibility to disease (Wedemeyer, 1970; Snieszko, 1974). A number of mechanisms by which pollutants may act to increase disease incidence in fish have been speculated, many suggesting immunosuppression as a factor in the impact of infectious disease among fishes in highly contaminated areas (Anderson et al., 1984; Moller, 1985; Sindermann, 1993; Zelikoff, 1993). Ross et al. (1996) have defined immunotoxicity as a toxicant-induced injury to part of the immune system that may lead to a diminished capacity of an organism to defend itself against infection by pathogens. They also demonstrated
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Table 1 Positive detection of re-isolated virus from dead or moribund groupers exposed to heavy metals before or after virus infection
Samplesa a
V/Zn
Zn/V
V/Cd
Cd/V
V/Cu
Cu/V
V
7/7 (100)
14/14 (100)
12/12 (100)
22/24 (92)
14/14 (100)
7/7 (100)
2/2 (100)
Number positive/total number tested; % in parentheses.
that environmental contaminants such as polychlorinated biphenyls (PCBs), polychlorinated dibenzo-p-dioxins (PCDDs), and polychlorinated dibenzofurans (PCDFs) can be immunotoxic at low doses in marine mammals (Ross et al., 1996). Although the immune system of grouper bears little similarity to that of a marine mammal, an immunotoxic effect (albeit via different pathways) may well account for the results of the present study. Some corroboration is provided by the virus titres that were taken at the end of experiments I and II (data not shown); although virus was not found in surviving fish that were exposed to virus only, a low virus titre was found in the dead fish of these same virus-only groups. Furthermore, virus titres of almost exactly the same low levels were found in all tested fish in the heavy metal/virus and virus/heavy metal groups. This suggests: (1) that the virus did not replicate in any of the fish; (2) that the fish’s immune system effectively eliminated the virus in most of the unstressed fish; (3) that, when the virus was not eliminated, even these low virus levels were sufficiently high to cause death in most cases; and (4) that the increased susceptibility of the fish exposed to heavy metals was due to their inability to eliminate the virus. These considerations support the conclusion that the high mortalities were the result of an immunotoxic effect. Other explanations are nonetheless possible, and indeed in the salinity stress studies (experiments III and IV) immunotoxicity seems an unlikely explanation. The earlier onset of mortality in experiments III and IV (cf. Figs. 2, 3, 5 and 6) also needs to be accounted for. In the absence of any specific mechanism for this immune suppression in this study, other explanations cannot be ruled out. More work to establish a link between environmental stressors, IPNV infection and immune responses of grouper is required. Since so
Fig. 4. Copper contents found in gill, viscera and muscle of groupers from experimental groups and their corresponding control groups at 2 days and 7 days. Data shown are the mean of three pooled samples from each group.
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Fig. 5. Cumulative mortality (%) of grouper (1 g average weight, 3.3 cm average length) experimentally infected by immersion in T42G virus suspension (105.0 TCID50/ml) for 24 h, subsequently transferred to lower (from 33 ppt to 20 ppt) or higher (33 ppt to 40 ppt) salinity for 48 h, and flushed with seawater until salinity returned to 33 ppt in 6 h. Water temperature was 25 91°C during the experiment. Data shown are the mean of two replicates.
many different environmental stressors in combination with a virus produce such similar effects, it might also prove worthwhile to continue the search for stress proteins analogous to those that are already well documented in humans.
Acknowledgements This work was supported by the National Science Council under Grant No. NSC86-2311-B019-003-B23. We also thank N.-H. Yu for providing the fish, and to P. Barlow for his helpful comments on the manuscript.
References
Fig. 6. Cumulative mortality (%) of grouper (1 g average weight, 3.3 cm average length) experimentally subjected to lower (33 ppt to 20 ppt) or higher (33 ppt to 40 ppt) salinity for 48 h and then infected by immersion in T42G virus suspension (105.0 TCID50/ml) for 24 h before being returned to 33 ppt seawater at 2591°C. Data shown are the mean of two replicates.
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