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