Zebrafish may prove to be one of the best vertebrate models for innate immunology. These fish have sophisticated immune components, yet rely heavily on innate immune mechanisms. Thus, the development and characterization of mutant and/or knock out zebrafish are critical to help define immune cell and immune gene functions in the zebrafish model. The use of Severe Combined Immunodeficient (SCID) and recombination activation gene 1 and 2 mutant mice has allowed the investigation of the specific contribution of innate defenses in many infectious diseases. Similar zebrafish mutants are now being used in biomedical and fish immunology related research. This report describes the leukocyte populations in a unique model, recombination activation gene 1-/- mutant zebrafish (rag1 mutants).

The innate immune response constitutes the first line of defense against invading pathogens and consists of a variety of immune defense mechanisms including active endocytosis by macrophages and granulocytes. Endocytosis can be used as a reliable measure of selective and non-selective mechanisms of antigen uptake in the early phase of an immune response. Numerous assays have been developed to measure this response in a variety of mammalian and fish species. The small size of the zebrafish has prevented the large-scale collection of monocytes/macrophages and granulocytes for these endocytic assays. Pooled zebrafish kidney hematopoietic tissues were used as a source of phagocytic cells for flow-cytometry based endocytic assays. FITC-Dextran, Lucifer Yellow and FITC-Edwardsiella ictaluri were used to evaluate selective and non-selective mechanisms of uptake in zebrafish phagocytes. Zebrafish kidney phagocytes characterized as monocytes/macrophages, neutrophils and lymphocytes utilize macropinocytosis and Ca²⁺-dependant endocytosis mechanisms of antigen uptake. These cells do not appear to utilize a mannose receptor. Heat-killed Edwardsiella ictaluri induces cytoskeletal interactions for internalization in zebrafish kidney monocytes/macrophages and granulocytes. The proposed method is easy to implement and should prove especially useful in immunological, toxicological and epidemiological research.

Recombination activation gene 1 deficient (rag1^−/−) mutant zebrafish have a reduced lymphocyte-like cell population that lacks functional B and T lymphocytes of the acquired immune system, but includes Natural Killer (NK)-like cells and Non-specific cytotoxic cells (NCC) of the innate immune system. The innate immune system is thought to lack the adaptive characteristics of an acquired immune system that provide enhanced protection to a second exposure of the same pathogen. It has been shown that NK cells have the ability to mediate adaptive immunity to chemical haptens and cytomegalovirus in murine models. In this study we evaluated the ability of rag1^−/− mutant zebrafish to mount a protective response to the facultative intracellular fish bacterium Edwardsiella ictaluri. Following secondary challenge with a lethal dose of homologous bacteria 4 and 8 weeks after a primary vaccination, rag1^−/− mutant zebrafish demonstrated protective immunity. Heterologous bacterial exposures did not provide protection. Adoptive leukocyte transfers from previously exposed mutants conferred protective immunity to naïve mutants when exposed to homologous bacteria. Our findings show that a component of the innate immune system mounted a response that provided significantly increased survival when rag1^−/− mutant zebrafish were re-exposed to the same bacteria. Further, adoptive cell transfers demonstrated that kidney interstitial leukocytes from previously exposed rag1^−/− mutant zebrafish transferred this protective immunity. This is the first report of any rag1^−/− mutant vertebrate mounting a protective secondary immune response to a bacterial pathogen, and demonstrates that a type of zebrafish innate immune cell can mediate adaptive immunity in the absence of T and B cells.

In August and November 2010 we collected and examined peripheral blood and tissues from three species of Gulf of Mexico fish. Findings were compared to non-exposed control fish. The leukocyte counts of exposed alligator gar were not significantly different from controls, while exposed Gulf killifish and sea trout had significantly decreased lymphocyte counts. Liver ethoxyresorufin-O-deethylase (EROD) values from sea trout were significantly greater than control sea trout EROD values, suggesting poly aromatic hydrocarbon exposure. Splenic melano-macrophage centers (MMCs) from exposed sea trout and Gulf killifish showed a significant increase in number compared to non-exposed fish. Sea trout splenic MMCs were also significantly greater in size. These findings suggest that Gulf fish sampled were exposed to crude oil from the Macondo well and were in a lymphopenic or immuno-compromised state.

The use of the visible implant elastomer (VIE) tagging system in zebrafish (Danio rerio) was examined. Two tag orientations (horizontal and vertical) at the dorsal fin base were tested for tag retention, tag fragmentation and whether VIE tags affected growth and survival of juvenile zebrafish (1–4 month post hatch). Six tag locations (abdomen, anal fin base, caudal peduncle, dorsal fin base, pectoral fin base, isthmus) and 5 tag colors (yellow, red, pink, orange, blue) were evaluated for ease of VIE tag application and tag visibility in adult zebrafish. Long-term retention (1 year) and multiple tagging sites (right and left of dorsal fin and pectoral fin base) were examined in adult zebrafish. Lastly, survival of recombination activation gene 1^−/− (rag1^−/−) zebrafish was evaluated after VIE tagging.