Pathogenesis of frog virus 3 (Ranavirus sp, Iridoviridae) in the wood frog, Rana sylvatica (Lithobates sylvaticus)

Amphibian populations suffer massive mortalities from infection with Frog Virus 3 (FV3, Ranavirus, Iridoviridae), a pathogen also responsible for mortalities of fish and reptiles. Wood frogs, Rana sylvatica, have been proposed as a research model to study diseases of native amphibians in North America. Our objectives were: 1) describe the longbterm housing, feeding and veterinary care of wildbcaught wood frogs, 2) develop a Ranavirusb wood frog model of infection with FV3, 3) review the literature on amphibian clinical pathology, 4) determine hematological reference intervals (RIs) for adult wood frogs and the hematological alterations associated with infection with FV3, and 5) establish the chronology of lesions due to a lethal infection with FV3. Wildbcaught adults and tadpoles were collected from Prince Edward Island and maintained in captivity for up to a year. Survival was good for wildbcaught individuals: 75 % for wildbcaught adults and 77 % for tadpoles raised to adulthood. A dose trial on adults raised from wildbcaught tadpoles established a lethal dose 50 (LD50) of 102.93 (2.42b3.44) pfu of FV3 for frogs averaging 35 mm in length. Onset of clinical signs occurred 6b14 days postbinfection (dpi) (median 11 dpi) and timebtobdeath 10b14 dpi (median 12 dpi). Each tenbfold increase in virus dose increased the odds of dying by 23bfold and accelerated onset of clinical signs and death by approximately 15%. Ranavirus DNA was demonstrated in skin and liver of all frogs that died or were euthanized because of severe clinical signs. Shedding of virus occurred in feces (7b10 dpi; 3b4.5 d before death) and skin sheds (10 dpi; 0b1.5 d before death) of some frogs that died from infection. Most common lesions were dermal erosion and hemorrhages, hematopoietic necrosis in bone marrow, kidney, spleen and liver, necrosis in renal glomeruli and in tongue, gastrointestinal tract, and urinary bladder mucosa. Intracytoplasmic inclusion bodies (probably viral) were present in the bone marrow and the epithelia of the oral cavity, gastrointestinal tract, renal tubules and urinary bladder. A timebcourse trial on wildbcaught adults using a lethal dose of FV3 (104.43 pfu/frog) followed by euthanasia at 0.25, 0.5, 1, 2, 4, 9 and 14 dpi established pathogenesis and hematological alterations due to infection. Infection with FV3 caused neutrophilia, increase in undifferentiated blastblike cells and relative reduction of basophils. Lymphocytes decreased at 4 and 9 dpi but increased 14 dpi. From 9 dpi onwards, nuclear deterioration and mild toxic change were present in neutrophils; cytoplasmic inclusion bodies were present in lymphocytes, monocytes, neutrophils and eosinophils. FV3 first targets hematopoietic tissue in the bone marrow and endothelial cells in the skin causing very mild microscopic lesions (1b2 dpi). Approximately 9 dpi, FV3 caused severe lesions in medullary and extamedullary hematopoietic tissue, lymphoid tissue and epithelial cells of skin and mucosae throughout the body. Direct contact (skin) and fecalboral contamination are likely effective routes of transmission.