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Snake fungal disease (SFD) is a clinical syndrome associated with dermatitis, myositis, osteomyelitis, and pneumonia in several species of free-ranging snakes in the US. The causative agent has been suggested as Ophidiomyces ophiodiicola, but other agents may contribute to the syndrome and the pathogenesis is not understood. To understand the role of O. ophiodiicola in SFD, a cottonmouth snake model of SFD was designed. Five cottonmouths (Agkistrodon piscivorous) were experimentally challenged by nasolabial pit inoculation with a pure culture of O. ophiodiicola. Development of skin lesions or facial swelling at the site of inoculation was observed in all snakes. Twice weekly swabs of the inoculation site revealed variable presence of O. ophiodiicola DNA by qPCR in all five inoculated snakes for 3 to 58 days post-inoculation; nasolabial flushes were not a useful sampling method for detection. Inoculated snakes had a 40% mortality rate. All inoculated snakes had microscopic lesions unilaterally on the side of the swabbed nasolabial pit, including erosions to ulcerations and heterophilic dermatitis. All signs were consistent with SFD; however, the severity of lesions varied in individual snakes, and fungal hyphae were only observed in 3 of 5 inoculated snakes. These three snakes correlated with post-mortem tissue qPCR evidence of O. ophiodiicola. The findings of this study conclude that O. ophiodiicola inoculation in a cottonmouth snake model leads to disease similar to SFD, although lesion severity and the fungal load are quite variable within the model. Future studies may utilize this model to further understand the pathogenesis of this disease and develop management strategies that mitigate disease effects, but investigation of other models with less variability may be warranted.

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The ability to produce cloned livestock using postmortem tissue could incorporate an additional application into the field of nuclear transfer. This study examined the feasibility of producing cloned cattle using a primary cell line established from a postmortem beef carcass. A market beef heifer processed at a USDA-certified slaughterhouse was used to develop a primary somatic cell line. Tissue samples were taken from the kidney and forelimb regions either 1) immediately following slaughter (fresh) or 2) 48 h postslaughter (cooled) where the carcass was housed at 2 to 4 • C. Tissue was removed and placed on ice in PBS + 5.0% (v:v) penicillin/streptomycin. A primary culture was established using standard techniques and cultured in supplemented DMEM F-12 medium. Once established, cells were trypsinized and either frozen or continually passaged. Cells used for nuclear transfer (NT) were passaged (48 h before use) and cultured with 15 µM roscovitine roughly 24 h prior to nuclear transfer. Cells were approximately 80% confluent and between passage numbers 1 and 11 at the time of NT. Selected slaughterhouse-derived oocytes were matured in supplemented TCM 199 medium for 18-20 h at 39 • C in 5.0% CO 2 and air. Mature Metaphase II oocytes were vortexed and stained with Hoechst 33342 to help with chromatin removal. Following enucleation, roscovitine-treated carcass cells were placed in the perivitelline space of the oocyte. Reconstructed NT embryos were fused in Zimmermann's medium and pulsed using needle-like electrodes. This was followed by activation using a combination of calcium ionophore (5 µM), cytochalasin D (5 µg mL −1 ), and cycloheximide (10 µg mL −1 ) in TCM + 10% FBS. Fused NT embryos were cultured in 50-µL drops of BARC medium (USDA, Beltsville, MD) for 7 days at 39 • C in a 5% CO 2 , 5% O 2 and 90% N 2 environment. Embryo development for all four groups (Table 1) was assessed with blastocysts (grade 1 or 2) being transferred into recipient cows 7 days post-estrus. Cleavage rates were not significantly different between groups, and the use of either fresh or cooled cells did not impact blastocyst formation. However, there was a significant difference (P = 0.05) in % blastocyst based on the source of the donor cell. Overall, one live calf resulted from 34 transferred NTs produced using kidney cells taken from a 48 h cooled carcass. These results display the feasibility of producing cloned calves from cells collected post mortem, which ultimately could be used as a tool to select breeding bulls based on their own steer carcass characteristics.