O.W. Dixon

In vitro immunostimulation of rainbow trout (Oncorhynchus mykiss) spleen cells with levamisole.

An in vitro immunization and cultivation method for fish spleen organ sections was used to investigate the effects of levamisole on the immune response. After 10 days of culture with either 50 micrograms/ml, 25 micrograms/ml, 5 micrograms/ml or no levamisole in the media, the nonspecific defense reactions were measured by determining the metabolic activity of neutrophils by using the nitroblue tetrazolium test, and phagocytic and adherence indexes by incubating the fish cells with suspensions of formalin-killed Staphylococcus aureus. The specific immune system activity was shown by the passive hemolytic plaque assay demonstrating the numbers of antibody-producing cells. Elevations were found in nonspecific defense and specific immune response in the 5 micrograms/ml levels of levamisole. The 25 micrograms/ml levels instigated a slight elevation in some nonspecific levels but showed suppression in the specific immune response. The 50 micrograms/ml levels suppressed all indicators whether levamisole was given alone or combined with Yersinia ruckeri O-antigen or DNP-Ficoll. These antigens are themselves nonspecific defense stimulators, and a synergistic effect was evident when combined with 25 micrograms/ml and 5 micrograms/ml levels of levamisole.

Comparisons of nonspecific and specific immunomodulation by oxolinic acid, oxytetracycline and levamisole in salmonids

Oxolinic acid, a promising drug for the treatment of bacterial fish disease agents, was tested for possible immunomodulatory effects on fish. Another antibiotic oxytetracycline, known to be immunosuppressive at higher treatment doses, and levamisole, a known immunostimulator for higher vertebrates, were also compared for causing changes in the nonspecific defense compartment and the specific immune system in rainbow trout. Groups of fish were immunized with Yersinia ruckeri O-antigen bacterin in combination with selected doses of the drugs. The nonspecific defense activity was measured by demonstrating neutrophil metabolic activity by the nitroblue tetrazolium assay, by counting engulfed bacterial cells for a phagocytic index and by counting leukocytes with adherent bacterial cells for the adherence index. The specific immune response was monitored by the passive hemolytic plaque assay demonstrating the numbers of antibody-producing cells. The results showed that oxolinic acid, used at recommended doses for the treatment of bacterial diseases, did not cause immunosuppression in either the nonspecific defense or specific immune system compartments, whereas tetracycline at 10 mg/kg caused reduced activity in both. Fish given levamisole injections before the antigen injection showed a stimulated nonspecific defense but a much reduced specific immune response.

Immunization and culture of rainbow trout organ sections in vitro

Splenic and anterior kidney sections or whole organs were excised from large (1 kg) or small (200 g) rainbow trout (Salmo gairdneri) and placed in sterile 60 mm plastic plates containing 10 ml of Eagles minimal essential medium (EMEM) supplemented with normal or fetal calf serum for in vitro culture. The organ samples were immunized in vitro by direct injection or by mixing in the medium Yersinia ruckeri O-antigen or dinitrophenyl-Ficoll. The medium was changed once during the 10-day incubation at 15 C. The passive hemolytic plaque assay demonstrated antibody production from the plaque-forming cells (PFC); passive hemagglutination was used to measure antibody titers in the media. High numbers of PFC occurred in cultures of either kidney or spleen, demonstrating that these organs can function independently for antibody production. Splenic sections from large fish produced more PFC than comparable whole organs from small fish. EMEM supplemented with 2% normal calf serum was a satisfactory culture medium. 2-hydroxyethyl-mercaptan an ingredient used in mammalian cell culture, inhibited antibody production in trout cells. These techniques are being used in the culture of organs and cells to elucidate pathways and sequences of antigen uptake and delivery of the immunopoietic tissues in trout.

In vivo to in vitro transfer of trout spleen sections for early analysis of the immune response

To determine the earliest time after in vivo immunisation that the spleen could be excised and held in vitro to detect an immune response, lake trout ( Salvelinus namaycush ) were exposed to DNP-Ficoll or Yersinia ruckeri O antigen administered by intraperitoneal injection or by bath. The spleens were excised from the fish at selected times after immunisation, placed in vitro and held in tissue culture media at 14°C until 10 days after the in vivo immunisation. They then were analysed for an immune response by counting the numbers of plaqueforming cells (PFC). PFC were first detected in samples taken 3 days after injection with either antigen. With bath immunisation, however, PFC were first seen 6 days post-immunisation when using the Y. ruckeri O antigen and no PFC above background levels appeared in fish bathed in the DNP-Ficoll solution. On day 10 after immunisation, the spleens taken directly from immunised fish always produced more PFC than when the spleens were taken through in vitro culture. A unique feature of this method, is that an immune response to an antigen may be detected by excising the spleen and holding it in in vitro culture without necessarily holding the fish for long periods of time after antigen injection or bath. This technique also adds more information on how rapidly the bacterins or antigens are processed by fish to initiate an immune response.