Dose-Titration Assay Of Walleye Dermal Sarcoma Tumor Homogenate     Rodman G. Getchell, Greg A. Wooster, Claudia A. Sutton, James W. Casey, and Paul R. Bowser   Aquatic Animal Health Program Department of Microbiology and Immunology, College of Veterinary Medicine Cornell University, Ithaca, NY     Walleye dermal sarcoma (WDS) is a seasonally limited neoplasm caused by a retrovirus.  Due to the inability to culture this virus, our pathogenesis, regression, and immunity studies have depended upon the use of a crude tumor homogenate containing an unknown number of walleye dermal sarcoma virus (WDSV) particles.  To determine the quantity of dermal sarcoma virus in these preparations, real-time reverse transcriptase-polymerase chain reaction (qRT-PCR) was performed on dilutions of the homogenate and compared to RNA standards transcribed from a plasmid containing a portion of the WDSV gag gene.  To approximate the dose of virus needed to induce walleye dermal sarcoma tumors, walleyes were challenged with a topical application of cell-free dermal sarcoma tumor filtrates.  Ten-fold dilutions of the filtrate were applied to the side of the fish, which were allowed to develop grossly visible tumors at 15°C for 20 weeks.  We have determined that there are approximately 1010 viral particles in 100 ml of walleye dermal sarcoma inoculum. There is an estimated 0.075 g of WDS tumor in the 100 ml of inoculum that we normally prepare, which converts to 13.3 X 1010 viral particles in 1 g.  The minimum dose of walleye dermal sarcoma virus that could induce tumors by the topical challenge method was the 1,000-fold dilution of this 1010 inoculum or 107 viral particles.  The ability to enumerate the WDS virus may allow renewed attention to conducting waterborne challenges of walleyes.