An in vitro technique for surveying immunostimulants in fish

In vitro assays were developed to investigate the effects of immunostimulants on leukocytes in spleen sections of rainbow trout (Oncorhynchus mykiss). Fish spleens were divided, and individual sections were placed in 10 ml of tissue culture media and 0.1, 1, 10, or 100 μg/ml dilutions of Levamisole, QAC (quaternary ammonium compound), or ISK (a polypeptide). After 4 days of incubation, cell suspensions were prepared to examine the effects of the immunostimulants on neutrophil oxidative activity using the nitroblue tetrazolium (NBT) assay and on phagocytic activity by engulfment of glutaraldehyde-fixed sheep red blood cells. QAC induced a heightened activity in plates receiving 0.1 and 1.0 μg/ml, but suppressed activity at 10 and 100 μg/ml levels. Sections incubated with ISK showed heightened responses at all doses except 100 μg/ml. Levamisole showed heightened responses at all doses in the NBT assay, and at the 1.0 and 10 μg/ml doses in the phagocytic assay.