Abstract

At low levels, selenium in combination with vitamin E has been shown to be an immunopotentiator. However, at slightly higher levels (15-20 mg/L) selenium is toxic. Studies have shown that a major organ of bioaccumulation in fish is the spleen. Concentrations of selenium in the spleen represent potential harmful consequences on normal immune functions and erythropoiesis. Resistance to bacterial infections, particularly in intensive culture conditions, may be seriously impaired.

This study was designed to correlate the effect of selenium on the immune response with the accumulation in tissues. Channel catfish were exposed to various levels of selenium, and the effect on the phagocytic response was measured. The levels of selenium in the water and various fish tissue were determined by atomic absorption. Water quality parameters were also monitored to determine if pH influenced the uptake of selenium by the fish.

Introduction

In recent years much interest has been generated in piscine immunology, both from a comparative (phylogenetic) and a practical (vaccination) viewpoint. There has been remarkable development in fish immunology mainly due to the worldwide increase in aquaculture. This intensive growing and harvesting of certain fish is seen as a way to provide animal protein for a growing human population (Van Muiswinkel, 1982; Dixon, 1986).

Although advances have been made in the understanding of the fish immune system, there is still much basic research required. Such factors as diet and metal content of water have only recently been observed to greatly influence the immune response of fish. Researchers have demonstrated that feed with trace elements (Fe, Cu, Mn) and vitamins such as A and C have significantly lowered the incidence of bacterial disease in several species of fish such as salmon and trout (Blazer and Wolke, 1984). Although reports show that disease incidence and diet are related, few attempts have been made to demonstrate the basis of this relationship.

Conversely, elements such as zinc and cadmium have evidenced an adverse effect on the immune system. For example, experiments have shown a toxic effect of zinc on the immune response, with zinc treated fish not producing detectable evidence of acquired humoral immunity (Sarot and Perlmutter, 1976). Cadmium and zinc have also been implicated in decreasing the nonspecific immune response, in particular the phagocytic response of macrophages (McCarty, et al, 1978).

A complex physiological link between vitamin E and selenium has long been established in veterinary medicine (Smith, et al, 1972). For example, it is well known that hypovitaminosis E and selenium deficiencies produce similar lesions leading to necrosis in skeletal and cardiac muscles, hepatic and renal tissue. The recent evidence implicating the role of vitamin E in the immune response may also suggest the involvement of selenium in the immune system.

This research proposes to examine the effect of selenium on the immune response of fish. Deficiencies of selenium, like that of vitamin E, may decrease the effectiveness of the immune response. The possibility also exists that a synergistic effect between selenium and vitamin E could potentiate nonspecific immunity.

Materials and Methods

The fish used in this experiment were Channel Catfish (Ictalurus punctatus) averaging in weight from 12-63 grams. Six fish per tank were maintained in 10 gallon (38 liter) glass aquaria equipped with undergravel filters to provide biological filtration. Fish were fed formulated pellets (Star Milling, Perris, Ca) ad libitum. The water quality parameters of ammonia, nitrite, pH and temperature were monitored throughout the exposure period. The temperature ranged between 19-22°C.

Fish were exposed for fourteen days to selenium in the form of sodium selenite (Aldrich Chemical Co), added to provide the following concentrations: 5.0 mg/L, 2.5 mg/L, 1.0 mg/L, 0.5 mg/L and 0.25 mg/L. Total selenium levels were 2.35 mg/L, 1.17 mg/l, 0.4 mg/L, 0.23 mg/L and 0.012 mg/L respectively. Each concentration level was run in duplicate tanks including untreated controls. On the seventh day of exposure, a second dose was added.

Samples of tap water, tank water before and after treatment, and on the final day of exposure were collected. The samples were acidified with nitric acid for selenium analysis by atomic absorption.

On the fourteenth day of exposure, fish were killed by severance of the cervical vertebrae. Total and spleen weight and hematocrit, were measured. Blood smears and spleen imprints were also made on every fish. The Phagocytic Index was determined by the Standard Nitro Blue Tetrazolium Test (Peacock and Tomar, 1980). Fish tissues preserved in 95% ethanol were assayed for selenium by atomic absorption.

Results

Five days following initial exposure, fish in the highest dosage tanks (5 mg/L) began to die. Within two (2) days all fish in this group were dead. Post mortum examination revealed mottled liver and spleen and pale gills. The kidneys were distended, indicating congestion with resulting abdominal ascites.

Fish in the 2.5 mg/L dosage began to die following the addition of the second dose. Nitrite levels began to increase in these tanks from 0 to 0.15 mg/L, following initial exposure. It is speculated that the selenium interfered with the nitrification cycle, in particular with the conversion of nitrite to nitrate by Nitrobacter sp. The increase in all nitrite may have been a contributing factor to death in this dosage level. To further evidence this, no detectable ammonio was measured.

Discussion

It has been previously shown, that the uptake of selenium in fish occurs in the gills and other thin membranous tissue. The major pathway appears to be erythrocyte transport mediated by active oxo-groups on the selenium anion (Lemley, 1982). Selenium eventually bonds available sulfur groups in amino acids with resulting incorporation into proteins. Several sublethal effects on fish have been described, including decreased growth rates, edema, decreased blood iron concentration and decreased erythrocyte volumes (Davis et al, 1988). In this study, abdominal ascites was noted at the two highest doses of the 5.0 and 2.5 mg/L. Fish at these dosage levels also had swollen kidneys and pale gills. Other studies have noted proliferative glomerulonephritis and vacuolation of parenchymal hepatocytes (Hamiton, et al, 1985). Damage in these organs may account for edema and passive congestion observed in this study. Unfortunately, fish at the highest dose died prior to the completion of the study. The two surviving fish in the 2.5 mg/L dosage had a hemocrit of 20 and 11, respectively, indicating a decrease in erythrocyte volume. Average hematocrit in channel catfish ranges from 30 to 46 (Grizzle and Rogers, 1976).

Unlike other metals, selenium is a required micronutrient for fishes (Lemley, 1982). It has been well documented that in other animals, that selenium promoted increased numbers of IgM-producing cells resulting in increased synthesis of IgM. Selenium is also required in maintenance of erythrocyte membrane integrity. Mechanisms associated with membrane fluidity of lymphoid cells have also been postulated. However, at elevated or prolonged exposure, selenium produces toxic effects similar to other heavy metals. Compounds such as boron enhance selenium toxicity while arsenic actually decreases toxicity. Conversely, selenium reduces the toxic effects of several metals including mercury lead and cadmium which can interfere in the immune process. Another factor that alters toxicity is the rate at which selenium is taken into living systems. Parameters such as pH, salinity, water hardness, temperature and dissolved oxygen can affect the biological uptake of selenium.

The results from this preliminary study regarding the effects of selenium on the immune response are inconclusive. In addition, the literature contains a paucity of studies dealing with chronic and sublethal effects. Wide discrepancies exist as to the toxic dosages in acute and chronic exposure. Over the years, severe losses in fish populations have been directly associated with selenium toxicity. Further research is needed to establish the relation between environmental selenium levels, selenium residue concentrations, and toxic effects on fish. Research in our lab is continuing to focus on the immune response, including eventual direct challenge with bacterial pathogens.

References

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