2013 International Symposium on Ranaviruses by Maria Forzan

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. Laboratory vs. field research
• Laboratory vs. field research
Source: Yale University Historical Library

3. Species selection
• Native species
• Widespread
• Sympatric
• Suitable for captive breeding

4. • Native species
• Widespread
• Sympatric
• Suitable for captive breeding
Species selection
the wood frog

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. 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. Eggs for tadpoles and metamorphs

8. Eggs for tadpoles and metamorphs

9. Eggs for tadpoles and metamorphs

10. Froglets from tadpoles

11. Froglets from tadpoles

12. Froglets from tadpoles

13. Housing tadpoles-metamorphs

14. Adults

15. Adults
@AVC

18. 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

19. Oral dosing
107.7 pfu / ml

20. Oral dosing
0.25ml / adult
0.05ml / 1-y-old

21. 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

22. 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

23. 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

24. Blood samples

25. Blood cells: N, Eo, B, Lym, Th, Ery

26. End day

27. Shedding – fecal swab (PCR)

28. Immunity – Abs, IF-α/β

29. Pathogenesis – PCR, H&E , IHC

30. Pathogenesis – PCR, H&E , IHC

31. Pathogenesis – PCR, H&E , IHC

32. Spleens: Ctrl(-), 9 dpi, 14 dpi

33. 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

34. Acknowledgments
Unaccredited images from:
Snailtail.com, Silkwormshop.com, Flukefarms.com, Animal Planet , CALA, Acadia University and Env. Canada websites

36. 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

37. 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