Keynote address at the Second International Ranavirus Symposium, Knoxville, Tennessee, 27 July 2013

1. Emerging infectious diseases and
amphibian population declines:
How are we going?
Rick Speare
Emeritus Professor
James Cook University, Townsville, Australia
&
Director, Tropical Health Solutions
27 July 2013
rickspeare@gmail.com

2. • Amphibians suffer from two formidable
infectious diseases, chytridiomycosis and
ranaviral disease
• Both can cause high mortality in wild and
captive populations
• Both can cause morbidity in wild
amphibians
• Both are emerging infectious diseases
• Both are globally notifiable diseases with
the World Organisation for Animal Health

4. My contribution to research on
amphibian diseases
• 1989 discovered Bohle iridovirus (Speare &
Smith 1992)
• 1989 discovered a large “iridovirus” in Bufo
marinus in Costa Rica (Speare et al 1991)
• Research on Ranaviruses continued until
1998
• Investigating amphibian declines since 1993
• 1997 discovered chytridiomycosis (Berger et al
1998)
• Research on chytridiomycosis to present

5. Source of funding affects the
philosophical approach of research
• Australian ranavirus research funded to
improve scientific knowledge
– Basic and applied research
• Aust chytridiomycosis research funded to give
evidence-based advice to wildlife managers to
enable them to formulate best possible policy
– Operational research, applied research, basic
research
– Action based on hypotheses and imperfect
evidence

6. Ranavirus genus
• Many types
• Three species (possibly)
– Frog virus 3 (FV3)
– Ambystoma tigrinum virus (ATV)
– Bohle iridovirus (BIV)
• Distribution
– FV3 – “global” (not Australia)
– ATV – USA & Canada
– BIV – Australia

7. Batrachyochytrium
dendrobatidis
• Kingdom: Fungi
• Class: Chytridiomycetes
• Order: Chytridiales
• Phylum: Chytridiomycota
• Genus: Batrachyochytrium
• Species: dendrobatidis
Berger et al 1998. PNAS 95:903
Nichols et al 1998 Proc Am Assoc Zoo Vet 1998:269
Longcore et al 1992. Mycologia 91:219
Joyce
Longcore
zoospore
zoosporangium

8. Batrochytrium dendrobatidis (Bd)
• One species
• Many strains
• Global panzootic lineage
(Bd-GPL-1 & Bd-GPL-2)
• Virulence differs with
strain
• At least two whole
genomes sequenced
• Distribution: all continents
with amphibians Schoegel et al 2012. Mol Ecol 21(21):5162
How does Bd
hybridise?

9. Understanding of the taxonomy and
genetics of the two pathogens
• No Global Panzootic Lineage for ranaviruses
• Comparable status in understanding taxonomies
• Search in both for virulence genes / proteins
• Bd: comparison with non-pathogenic chytrid &
other fungi (Bd has 1,974 unique protein
encoding genes!) (Joneson et al 2011 PloS Path 7:e1002338)
• Bd: increasing understanding of what genes are
important for virulence (but complex)
• Much better understanding for ranaviruses of
what antigens could be used to develop vaccine

10. Key Ranavirus question
• What gene(s) controls the temperature
sensitivity?
• Ranaviruses can kill mammalian cells in
vitro at <34ºC
• FV3 given iv to mice kill within 19-30 hrs
(without even multiplying!)
• Genetic modification could have
disasterous consequences by making a
ranavirus capable of infecting
homeothermic vertebrates

11. Ranaviruses in Australia
• Epizootic haemopoetic necrosis virus
(EHNV) – bony fish only – 1986
• Bohle Iridovirus (BIV) – amphibians –
1992
• Mahaffey Road virus (MHRV) –
amphibians – 2012 – BIV-like
• No major mortality in wild populations
Very elusive viruses!

12. Distribution of anti-ranaviral antibodies
in introduced Bufo marinus
Overall prevalence
2.7%
Regional range
0-13%
Zupanovic et al 1998
BIV
Ellen Ariel: RV
antibodies common in
freshwater turtles &
freshwater crocodiles
MHRV

13. The search for Tadpole
Edema Virus (TEV)
• 1988-1989 Biological control of cane toads
(Rhinella marina (Bufo marinus))
• Search for diseases in Australian toads
• Identify and isolate pathogens
• Evaluate for suitability as biological control
agent for R. marina
• Looking for an Australian Tadpole
Oedema Virus (TOV)

14. Tadpole edema virus (TEV)
• Ken Wolf et al’s work (J Inf Dis 1968;118:253)
• Initial isolate from West Virginia
• Found in Rana catesbiana tadpoles
• Could infect by injection, bath exposure,
and by feeding contaminated insects
• Toads more susceptible than bullfrogs
• Carriage of virus by adults
Why is Wolf’s work forgotten?

15. End of wet season April 1989
Townsville: Bohle

16. Picture of Tvlle in wet season

17. • Metamorphs of Limnodynastes
ornatus dying from ranaviral
disease in Townsville (1989)

18. Bohle iridovirus (BIV)

19. “Bohle iridovirus (BIV)”
First use of name!

20. Comparison with EHNV
• Epizootic haematopoetic
necrosis virus (EHNV)
isolated from fish
• First ranavirus found in
Australia
• BIV and EHNV were
distinct species
Henstberger et al (1993)
EHNV
BIV
Alex Hyatt

21. Histopathology due to BIV
• Widespread necrosis of haematopoeitic
and interstitial cells
• Severe necrosis in bone marrow, spleen,
haematopoeitic cells of kidney and liver,
glomeruli, limb buds in tadpoles.
• Karyorrhexis, karyolysis common
• Basophilic intracytoplasmic inclusions
• Index case metamorphs also had bacteria
in many tissues

22. Kidney with focal necrosis

23. Kidney with necrosis of haemopoetic tissue

24. • Antigen of BIV could be detected by
immunoperoxidase

25. • Pathology was extensive in fatal
cases
• Many organs damaged
Liver with
necrosis

26. Ranavirus
pathology
• Widespread focal necrosis in many organs
± haemorrhage
• Some species have skin ulcers; some
have skin growths
• Frogs die from “organ failure”, but
pathophysiology is not understood
Makes treatment for RV
disease difficult!

27. Pathology of Bd
• Fungus invades superficial layers of the
epidermis
• Grows inside cells of the top two layers
(stratum corneum, stratum granulosum)
• Does not invade the body
• Causes minor morphological epidermal
pathology
• Causes a minimal local inflammatory
reaction
• No internal histopathological changes

28. Skin slough is occasionally visible
Myxophes fasciolatus with chytridiomycosis
showing sloughing of epidermis

29. Effects on frogs and toads
Clinical signs range from none to
death
Superficial layer of skin is
microscopically thickened
This layer often sloughs off in
fragments
Frogs die with neurological signs

30. All skin sloughing is NOT due to
chytridiomycosis

31. Normal skin of a Litoria caerulea
• Note epidermis
Epidermis
Dermis
Note the epidermis has a smooth surface
and is about 6-8 cell layers thick
Subdermal lymph
sinus
Lee Berger

32. Chytridiomycosis: Cells of stratum
corneum proliferate
• Look for thickened epidermis with cluster of
sporangia

33. The cells under
the surface
“dissolve”

34. Top layer of epithelium
sloughs off
No local inflammatory response
Is Bd immunosuppressive?

35. Surface of skin with
chytridiomycosis
Some discharge papillae of sporangia
project above the epithelial surface
Normal

36. Sporangia in epidermal cell
showing cross-section through
discharge papilla with plug (TEM)

37. Systemic Effects of B.
dendrobatidis
• Behavioural
changes
• Mild neurological
effects
• Severe
neurological
effects
• Nocturnal frog in
daylight / fossorial
frog on surface
• Abnormal posture
• Reluctance to move
• Loss of righting reflex
• Fitting
• Death
How does such a superfical pathogen kill?

38. Two pathogenic mechanisms
proposed
• Osmotic effect
OR
• Toxic effect
OR
• Both
Berger et al 1998

39. 3. Organ functioning
Bd INFECTION
1. Epidermal functioning
2. Cutaneous osmoregulation
DEATH
Jamie Voyles

40. 1. Epidermal Functioning
Outside <- Skin Cross Section -> Inside
Na+
H+
K+
ATP
Cl-
HCO3
-
Na+
ATP
HCO3
-
Cl-
Na+
Na+
K+
ATP
Na+
uptake & Cl-
secretion are
inhibited in the
skin of infected
frogs

41. Frogs die from blood electrolyte
abnormalities
• Low potassium (reduced
to 50%)
• Low sodium (reduced to
80%)
1
2
3
4
5
6
Final Potassium
Control N = 7
Aclinical N = 7
Diseased N = 10
ANOVA
p = 0.001*
Voyles et al 2007, 2009 Images from Jamie Voyles

42. Hypokalaemia
results in
cardiac
asystole
Voyles et al 2009
18 hrs before death
3 hrs before death
2 hrs before death
0.5 hrs before death

43. Electrolyte replacement
• Increasing the serum K and Na partly
corrected cardiac and neurological signs
• Treatment of Bd with chloramphenicol and
electrolyte therapy cured frogs (Young et al
2012 JZWM 43:330)
• This is not the complete story about
pathophysiology
• Bd killed by itraconazole, but corroboree
frogs died from bacterial overgrowth
• Bd is immunosuppressive

44. Understanding of pathogenesis
• Much more advanced for chytridiomycosis
• Proteases, lipases and other proteins activated
on exposure to host tissue are being implicated
• Better understanding of RV pathogenesis
needed
• For chytridiomycosis ill frogs can be treated
– Correct pathophysiology
– Kill Bd
– Control secondary bacterial invasion

45. Eradicating the pathogen
• Very important when establishing captive
populations from wild caught amphibians
• For Bd treatment with fungicides
(itraconazole), but ideal regime is elusive
• Heat works! 32ºC for 5 days (Retallick & Miera
2007 DAO 75:201)
Could heat be used to clear RVs carriers?
Heat unlikely to kill RV, but will it stop
replication and allow elimination?

46. Routes of Transmission
• Both pathogens transmit horizontally
• Water is the major medium; more critical
for Bd since zoospores are aquatic
• Bd penetrates into epidermal cells
• RVs transmit orally Can RVs infect
skin directly?
Rooij et al 2012 PLoS One
7:e41481

47. Bath exposure
1st
transmission exp for BIV (1989)
LO = Limnodynastes ornatus; BM = Bufo marinus

48. Tadpoles are carriers
of Bd
• Bd is found in
keratinised tissue
• Mainly in teeth and
jaws, but varies with
Gosner stage
• May damage these
• No direct mortality
• May reduce fitness
Marantelli et al 2005 Pac Cons Biol 10:173

49. Infection oral & by injection
2nd
transmission exp for BIV (1989)
BM = Bufo marinus; sc = subcutaneous injection

50. Oral infection
2nd
transmission exp for BIV (1989)
LO = Limnodynastes ornatus

51. Could BIV kill adult cane toads?
• Exp 3: sc inoculation of 4 adult toads
– 100% mortality 9-10 d post-inoculation
• Subsequent experimental infections
showed:
– Mortality variable
– Toads in contact with inoculated toads could
become infected and die
• BIV in adult toads could be isolated from:
– Blood, many internal organs, muscle, fat

52. Host range for BIV
• Amphibians: Bufo marinus and a range of
native species (Limnodynastes, Litoria,
Pseudophrenye, Taudactylus) could be infected
with BIV, but mortality variable (Cullen et al 1995;
Cullen & Owens 2002)
– Juveniles more susceptible (x66)
• Fish: mortality in barramundi (Lates calcarifer);
infect tipalia (Oreochromis mossambicus)
(Moody & Owens 1994; Ariel & Owens 1997)
• Reptiles: kill tortoise hatchlings (Ariel 1997)
– Elseya latisternum & Emydura krefftii
Ellen Ariel
Leigh Owens

53. Host susceptibility to disease
• Distinguish between infection versus disease
• For both pathogens susceptibility to disease
depends on life stage
– RVs: > for larvae and metamorphs, low for adults
– Bd: low for larvae; >metamorphs, adults
• For both pathogens the susceptibility
depends on host species
• Susceptibility is not predictable from host
taxonomy

54. Bd patterns of host susceptibility
to disease
1. Able to be infected
but eliminates
infection; eg,
Leiopelma spp.
2. Infected and
generally dies
3. Infected and
usually aclinical
(ie, carrier)
Shaw et al 2010 DAO 92:150
Similar patterns for both pathogens.
What determines host susceptibility?

55. Epidemiology is dynamic
• RV epidemiology / ecology is complex
• Poorly understood in Australia; better
understanding in north America & Europe
• Both diseases have multiple hosts of different
susceptibilities interacting with local environment
• RVs much more complex: non-amphibian hosts,
greater environmental persistence of pathogen
and bigger role for acquired immunity
How can local outbreaks be predicted?

56. Co-infections
• Recent hypotheses that ranaviruses may
be devastating to amphibian populations
already compromised by endemic
chytridiomycosis.
• The impact of these two pathogens (and
others) certainly needs more clarification
in a range of environments and
populations.

57. Where to now?
• Need to offer solutions to wildlife managers
• Action to them is:
– Policy – macro or micro
– Interventions to prevent negatives
– Justification for what they do
• Models indicating best “bang for the buck”
• Surveillance is valued if something can be
done
• Prevent outbreaks through long term strategies
• Stop outbreaks through timely interventions

58. Control options: research needed
• Bd is possibly easier
– Strategic use of agricultural fungicides
– Biological control through Bd-specific viruses
– Exclusion of carrier species from contact with
susceptible species during critical life stages
• For RVs
– Vaccines
• Use of attenuated vaccine virus in hot spots
– Vaccination of replacement adults
• For both
– Selection for resistant hosts

59. Lessons from both diseases
• Both are complex, highly dynamic and
evolving
• Host species is important; so be wary
extrapolating results across species
• Differentiate infection from disease
• Epidemiology varies with locality
• Do research to make a difference
• Work in multidisciplinary teams with team
members respecting and valuing the
special skills of each discipline

60. Thank you!

61. Sources of images not original
• Paris is September -
http://www.123rf.com/photo_16463061_paris--september-30-eiffel-tower-a
• Green frog cartoon - http://blog.tombowusa.com/2012/02/
• Archey’s frog -
http://www.arkive.org/archeys-frog/leiopelma-archeyi/image-G22123.html
• Litoria ewingii -
http://calphotos.berkeley.edu/cgi/img_query?enlarge=0000+0000+0203+0
• Alpine tree frog drawing -
http://www.redbubble.com/people/lauragrogan/works/6520637-alpine-tree