Abstract

The most common bacterial fish pathogens including Aeromonas, Edwardsiella and Flexibacter are gram negative lactose nonfermenters. These organisms, isolated from fish, were grown in various concentrations of sodium selenite. Growth was measured by standard plate counts of colony forming units and spectrophotometric analysis of optical density. These parameters were then used to generate growth curves for the bacteria, in various concentrations of sodium selenite. Some organisms grown in levels as low as 0.4 mg/L produced a brick red pigment. This pigment was extracted and characterized by standard chemical procedures.

Introduction

In 1936, Leifson developed sodium selenite broth for the growth and isolation of pathogenic salmonellae. Ever since, sodium selenite broth has been used as an enrichment medium for various gram negative enteric pathogens. This enrichment medium works on the premise that Escherichia coli and other lactose fermenting organisms making up the normal intestinal flora, are maintained in a prolonged lag phase by the selenium. Organisms such as Salmonella and Shigella are less inhibited and enter into the lag phase of growth faster than the lactose fermenters. Hence, the prolonged lag phase temporary prevents the overgrowth of these organisms and allows for an increased recovery of the pathogens from fecal samples. However, the inhibitory effect is temporary and only maintained for 4-12 hours (Koneman et al, 1983). Eventually the bacteria methylate the selenium to rid themselves of the excess (Davis et al, 1988).

The continuous uptake of small dosages of selenium has been shown to result in bioaccumulation in animals. Accumulation may occur either through natural means in areas where soil levels are high or through additives in feed. It has been speculated that the bioaccumulation of selenium may result in concentrations sufficient to act as a culturing agent for Salmonella. These increased levels of selenium in animal tissue may enhance the growth of this pathogen (Kilness, 1987).

Little is known about the effect of this heavy metal on the growth of bacterial fish pathogens, or the nitrifying bacteria important for biological filtration. This research was undertaken to delineate the effect of selenium on the growth of bacteria important to aquaculture.

Materials and Methods

Organisms: The bacteria used for this experiment were Aeromonas hydrophila isolated from an epiichthyotic of channel catfish (Ictalurus punctatus). Edwardsiella sp. isolated from the kidney of infected common goldfish (Carassius auratus); Escherichia coli isolated from tank water; Salmonella sp. from a clinical isolate, Nitrosomonas europaea (#19718) and Nitrobacter agilis (#14123) obtained from American type tissue collection (Rockville, MD).

Heterotrophic organisms were maintained on brain heart infusion (Difco Co.), autotrophic nitrifiers were maintained on specialized media according to ATCC techniques. All organisms were incubated at 25°C.

Test Procedures

Twenty four hour cultures were inoculated into BHI broth containing concentrations of sodium selenite (Aldrich Chemical Co.) at 5.0 mg/L, 2.5 mg/L, 1.0 mg/L, 0.5 mg/L and 0.25 mg/L. Final concentrations of selenium were 2.35 mg/L, 1.17 mg/L, 0.47 mg/L, 0.24 mg/L and 0.12 mg/L respectively. Two hundred fifty milliliter Erlenmeyer flasks were maintained at 25°C on a shaker. Optical density readings at 600 nm were taken every 30 minutes for four to ten hours. Simultaneous plate counts were made on BHI agar.

Characterization of Pigment

Pigment produced by various organisms was extracted from culture medium with carbon tetrachloride. Standard methods of analysis such as gas and thin layer, chromotography, and infrared and ultraviolet spectrum analysis were performed.

Results

Preliminary results indicate that Salmonella sp. was not inhibited at any of the test concentrations of selenium. Edwardsiella sp. appeared to be initially inhibited for four hours. Escherichia coli and Aeromonas hydrophilia did not appear to be inhibited.

The cellular morphology of bacteria grown in selenium was atypical compared to controls. Morphological changes such as elongation, filament formation and cell shrinkage were observed on oil immersion light microscopy.

Pigment characterization is as of yet undetermined.

Discussion

As a result of acid precipitation and industrial contamination, fish are increasingly exposed to metal contaminants. The importance of heavy metal toxicity as a stressor that exacerbates pathogenesis is beginning to emerge (MacFarlane et at, 1986). The inadvertant exposure of fish to sublethal concentrations of selenium in the presence of bacterial pathogens may be a predisposing factor to disease. It is well documented in the literature that selenium is required as a micronutrient for fish. Selenium is thought to act as an immunopotentiator in a manner similar to Vitamin E. However, at higher levels, selenium produces toxicity similar to other heavy metals.

Few studies have been carried out on the effect of heavy metals on the growth of bacteria. MacFarlane et al (1986) reported that striped bass (Morone saxatilis) were protected from experimental infection with Flexibacter columnaris, when exposed to a mixture of metals including arsenic, cadmium, copper, lead, and selenium at dosages higher than environmental concentrations. The complex interaction of selenium on the immune response, effect on pathogen growth, and environmental exposure have yet to be delineated. These relationships may prove to be important for several reasons. For example, in some natural alkaline waters, selenium occurs at high levels. Therefore, a potential situation may arise where fish immunity is depressed with a simultaneous enhancement of pathogen growth.

References

1.  Davis, E. A., Maier, K. J., and Knight, A. W. The Biological Consequences of Selenium in Aquatic Ecosystems. Calif. Agricult. 42, pp. 18-20, 1988.

2.  Kilness, A. W. Selenium and Salmonella. Medical Tribune, 28, #1, 1987.

3.  Koneman, E. W., Allen, S. D., Dowell Jr., V. R. and Somers, H. M. Color Atlas and Textbook of Diagnostic Microbiology. Second Ed. J. B. Lippincott Co., N.Y., pp. 72-73, 1983.

4.  MacFarlane, R. D., Bullock, G. L., and McLaughlin, J. J. A. Effects of Five Metals on Susceptibility of Striped Bass to Flexibacter columnaris. Trans. Am. Fish. Soc. 115, pp. 227-231, 1986.