Binding Of Bacterial Genomic DNA And Synthetic Oligodeoxynucleotides Leads To Activation Of Tilapia Nonspecific Cytotoxic Cells (NCC)     Mustapha Oumouna, Liliana Jaso-Friedmann and Donald L. Evans   College of Veterinary Medicine, University of Georgia, Athens, GA     Previous studies in mammals have shown that nonmethylated bacterial DNA (bDNA) in the form of oligodeoxynucleotides (ODNs) activates many different types of cells including lymphocytes. In the present study genomic bDNA from Streptococcus iniae and synthetic ODNs containing CpG dinucleotide motifs were tested for activation of tilapia nonspecific cytotoxic cells (NCC). Bacterial DNA treatment of NCC (in vitro) activated the killing of tumor cells. Calf thymus and tilapia genomic DNA treatments had no effects on NCC activity. Oligodeoxynucleotides containing CpG motifs and oligodeoxyguanosine 20-mers increased the cytotoxicity more than the ODN composed of GpC motifs. In vitro binding experiments demonstrated that bacterial DNA as well as synthetic ODNs bound to splenic NCC. Specific binding of bDNA to NCC was demonstrated by reciprocal cold target inhibition experiments. To determine if binding occurred in vivo, tilapia were injected (IV) with Rhodamine labelled ODNs and purified NCC from different tissues were examined by flow cytometry. Percentage positive NCC for ODN binding was: peripheral blood (38%), anterior kidney (12%), spleen (17%) and liver (50%). One mechanism of regulation of NCC activity by bDNA was protection from apoptosis detected by finding a reduction in the production of DNA hypoploidy in bDNA treated NCC. NCC treated with calf thymus DNA had a 73% reduction in 2N DNA compared to 31% in cells treated with bDNA. These data demonstrated that both in vitro and in vivo, bDNA may participate in the activation of NCC and as such stimulate anti-bacterial innate immunity.