Materials and Methods
Skin lesion samples
Skin lesions clinically indicative of papilloma virus infection were sampled from four captive Florida manatees (Trichechus manatus latirostris) housed in Homosassa Springs Wildlife State Park (HSSWP), Homosassa, Florida. All four samples (V369, V374, V375, and P31) were obtained from adult female Florida manatees. Samples V369, V374, V375 and P31 had been collected in 1998 and preserved in 10% neutral buffered formalin at room temperature until extracted in January, 2003. Recently, samples were harvested from papillomatous skin lesions of two free-ranging Florida manatees (V378 and V396). Sample V378 was obtained from a 250 cm in length adult male manatee in Crystal River, FL. Sample V396 was harvested from a male manatee calf in Homosassa River, FL. Fresh samples of skin lesions from these two manatees were chilled in ice, transported to the laboratory and total DNA was extracted and stored frozen at – 80oC. Samples V378 and V396 were obtained and extracted in January, 2003, and February, 2003, respectively.
Extraction of total DNA from skin lesions
Total DNA was extracted from the entire skin lesion (~25 mg) using the DNeasy tissue kit (Qiagen) following the manufacturer's protocol. Tissues were grinded in 1.5 ml micro centrifuge tubes and suspended in 180 µl of digestion buffer ATL, 20 µl proteinase K and incubated at 55°C overnight. Then, 200µl of buffer AL and 200 µl of 100% ethanol were added to the samples and the mixture run through a DNeasy spin column to bind the DNA. The membrane was then washed with 500µl of buffer AW1 and 500µl of buffer AW2. Finally, 200 µl of buffer AE was added to the membrane and centrifuged to elute the DNA. The final DNA product was quantitated by spectrophotometry and stored frozen at -80°C.
Total DNA extracted from skin lesions suggestive of papillomatosis was assayed by PCR targeting the L1 capsid protein gene with primers MY09 and MY111. These primers amplify a DNA fragment of ~ 458-bp when used with DNA from most of the more than 85 types of human papilloma viruses described. The PCR reaction mixture in a final volume of 100µl contained 200 nM of each primer, 2 mM MgCl2, 100 M dNTP's, 20 mM Tris-HCl pH 8.4, 50 mM KCl, and 2 units of Taq DNA polymerase. Approximately 500ng to 1g of total DNA was incorporated in the PCR reaction. After an initial denaturation at 94°C for 1 min, reactions were subjected to 35 cycles of 94°C for 1 min, 50°C for 1 min, and 72°C for 2 min. A final cycle incorporated an extension at 72°C for 10 min. Small aliquots of the amplified DNA fragments were resolved by horizontal electrophoresis in 1.2 % agarose containing ethidium bromide (0.5 g/ml) and visualized by transillumination under UV light using a gel documentation system.
Amplified PCR fragments were resolved by electrophoresis in low melting point (LMP) agarose (1.0 %), and purified with the Wizard SV Gel kit (Promega). Approximately 200 fmol of total DNA was sequenced using the forward and reverse primers and the Beckman Coulter 2000 XL sequencing instrument. Sequences were confirmed using the Chromas software (Technelysium), and the sequences analyzed using the Gap, Seqed, Translate, and Lineup functions of the GCG Wisconsin Package (University of Wisconsin) and the EditSeq and MegAlign functions of the Lasergene software (DNASTAR, Inc.).