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Rana sylvatica (Lithobates
sylvaticus) as a model to study FV3
María J. Forzán1,2, Kathleen M. Jones2
1Canadian Cooperativ...
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
• Na...
Wood frogs in the lab
• Housing/feeding of pre- & post-metamorphic frogs
• breeding in captivity?
• Dosing of infective vi...
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
Extremel...
Oral dosing
107.7 pfu / ml
Oral dosing
0.25ml / adult
0.05ml / 1-y-old
LD50
1-year-old frogs (3.5 cm SV)
• raised from tadpoles
• oral infection – 0.05ml
• euthanized when terminal (*)
Dose
(pf...
LD50
1-year-old frogs (3.5 cm SV)
• raised from tadpoles
• oral infection – 0.05ml
• euthanized when terminal (*)
Dose
(pf...
Pathogenesis
• Adults (wild-caught)
• 5.3 cm SV
• Killed at 0.25, 0.5, 1, 2, 4, 9 and 14 days post-
infection
• Blood, tis...
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 an...
Acknowledgments
Unaccredited images from:
Snailtail.com, Silkwormshop.com, Flukefarms.com, Animal Planet , CALA, Acadia Un...
Rana sylvatica (Lithobates sylvaticus) as a
model to study FV3
• Native to North America
• Fam. Ranidae (Rana or Lithobate...
Wood frogs in the laboratory
• Housing/feeding of pre- and post-metamorphic frogs
• recommendations
• breeding in captivit...
The wood frog as a model to study the pathogenesis and host-pathogen interactions of frog virus 3
The wood frog as a model to study the pathogenesis and host-pathogen interactions of frog virus 3
The wood frog as a model to study the pathogenesis and host-pathogen interactions of frog virus 3
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The wood frog as a model to study the pathogenesis and host-pathogen interactions of frog virus 3

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2013 International Symposium on Ranaviruses
by Maria Forzan

Published in: Education, Lifestyle, Technology
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The wood frog as a model to study the pathogenesis and host-pathogen interactions of frog virus 3

  1. 1. Rana sylvatica (Lithobates sylvaticus) as a model to study FV3 María J. Forzán1,2, Kathleen M. Jones2 1Canadian Cooperative Wildlife Health Centre 2Atlantic Veterinary College 2nd Ranavirus Symposium Tennessee, July 2013
  2. 2. Laboratory vs. field research • Laboratory vs. field research Source: Yale University Historical Library
  3. 3. Species selection • Native species • Widespread • Sympatric • Suitable for captive breeding
  4. 4. • Native species • Widespread • Sympatric • Suitable for captive breeding Species selection the wood frog
  5. 5. 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
  6. 6. 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
  7. 7. Eggs for tadpoles and metamorphs
  8. 8. Eggs for tadpoles and metamorphs
  9. 9. Eggs for tadpoles and metamorphs
  10. 10. Froglets from tadpoles
  11. 11. Froglets from tadpoles
  12. 12. Froglets from tadpoles
  13. 13. Housing tadpoles-metamorphs
  14. 14. Adults
  15. 15. Adults @AVC
  16. 16. 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
  17. 17. Oral dosing 107.7 pfu / ml
  18. 18. Oral dosing 0.25ml / adult 0.05ml / 1-y-old
  19. 19. 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
  20. 20. 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/frog or 104.8 pfu/ml
  21. 21. 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
  22. 22. Blood samples
  23. 23. Blood cells: N, Eo, B, Lym, Th, Ery
  24. 24. End day
  25. 25. Shedding – fecal swab (PCR)
  26. 26. Immunity – Abs, IF-α/β
  27. 27. Pathogenesis – PCR, H&E , IHC
  28. 28. Pathogenesis – PCR, H&E , IHC
  29. 29. Pathogenesis – PCR, H&E , IHC
  30. 30. Spleens: Ctrl(-), 9 dpi, 14 dpi
  31. 31. 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
  32. 32. Acknowledgments Unaccredited images from: Snailtail.com, Silkwormshop.com, Flukefarms.com, Animal Planet , CALA, Acadia University and Env. Canada websites
  33. 33. 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
  34. 34. 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

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