Rana sylvatica (Lithobates sylvaticus) as a model to study
FV3
María J. Forzán1,2, Kathleen M. Jones2
1Canadian Cooperative Wildlife Health Centre2Atlantic Veterinary College
2nd Ranavirus SymposiumTennessee, July 2013
Laboratory vs. field research
• Laboratory vs. field research
Source: Yale University Historical Library
Species selection
• Native species • Widespread• Sympatric• Suitable for captive breeding
• Native species • Widespread• Sympatric• Suitable for captive breeding
Species selection
the wood frog
Wood frog
• Fam. Ranidae (Rana or Lithobates)• 9 species Canada
• Terrestrial• Spread across NA• Broadly sympatric
• Naturally host of ranavirus• Life span >3-5 y
Wood frogs in the lab
• Housing/feeding of pre- & post-metamorphic frogs• breeding in captivity?
• Dosing of infective virus (FV3)• Collection and evaluation of blood samples• cellular and humoral responses
• Collection of tissues• pathogenesis• PCR• virus isolation• histopathology• immunohistochemistry
Eggs for tadpoles and metamorphs
Eggs for tadpoles and metamorphs
Eggs for tadpoles and metamorphs
Froglets from tadpoles
Froglets from tadpoles
Froglets from tadpoles
Housing tadpoles-metamorphs
Adults
Adults
@AVC
Housing
• Temperature 17-23°C
• Adults fed
• CK, MW, EW
• Froglets fed
• Dm, Dh, CK, MW
• Tadpoles fed
• FF, Blet
Extremely hard!• 75% adults (44/59, 6mo)• 72% froglets (81/112, 8mo)
Date (mm-dd)
Cage Frogs Food left Fed Comments
Alive Dead
C01 4x 1CK Ø 8MW Not hungry
C02
C04
C05
C06
C07
C08
C09
C10
C03
Oral dosing 107.7 pfu / ml
Oral dosing
0.25ml / adult0.05ml / 1-y-old
LD50
1-year-old frogs (3.5 cm SV)• raised from tadpoles• oral infection – 0.05ml• euthanized when terminal (*)
Dose (pfu/frog)
105.4
104.4
103.4
102.4
101.4
100.4
Control
LD50
1-year-old frogs (3.5 cm SV)• raised from tadpoles• oral infection – 0.05ml• euthanized when terminal (*)
Dose (pfu/frog)
%(*)
105.4 100
104.4 100
103.4 80
102.4 20
101.4 0
100.4 0
Control 0
103.5 pfu/frogor
104.8 pfu/ml
Pathogenesis
• Adults (wild-caught)• 5.3 cm SV
• Killed at 0.25, 0.5, 1, 2, 4, 9 and 14 days post-infection• Blood, tissues and abdominal fluid
collected
Blood samples
Blood cells: N, Eo, B, Lym, Th, Ery
End day
Shedding – fecal swab (PCR)
Immunity – Abs, IF-α/β
Pathogenesis – PCR, H&E , IHC
Pathogenesis – PCR, H&E , IHC
Pathogenesis – PCR, H&E , IHC
Spleens: Ctrl(-), 9 dpi, 14 dpi
Done / In progress
Housing and feeding of pre- and post-metamorphic frogs
Dosing of infective virus (FV3)
Collection and evaluation of blood samples
• cellular and humoral responses
• Pathogenesis
• PCR, virus isolation, histopathology, immunohistochemistry
Acknowledgments
Unaccredited images from:Snailtail.com, Silkwormshop.com, Flukefarms.com, Animal Planet , CALA, Acadia University and Env. Canada websites
Rana sylvatica (Lithobates sylvaticus) as a model to study FV3
• Native to North America• Fam. Ranidae (Rana or Lithobates)
• Widespread (Canada, Eastern USA)• 9 species Canada • broadly sympatric
• Suitable for captive breeding (possibly)• Naturally host of ranavirus• Life span >3-5 y
Wood frogs in the laboratory• Housing/feeding of pre- and post-metamorphic frogs• recommendations• breeding in captivity (?)
• Dose of infective virus (FV3)• LD50• oral dosing
• Blood samples• cellular & humoral responses
• Tissues• pathogenesis• PCR, virus isolation• histopathology, immunohistochemistry