Emerging infectious diseases and
amphibian population declines:
How are we going?
Rick Speare
Emeritus Professor
James Coo...
• Amphibians suffer from two formidable
infectious diseases, chytridiomycosis and
ranaviral disease
• Both can cause high ...
My contribution to research on
amphibian diseases
• 1989 discovered Bohle iridovirus (Speare &
Smith 1992)
• 1989 discover...
Source of funding affects the
philosophical approach of research
• Australian ranavirus research funded to
improve scienti...
Ranavirus genus
• Many types
• Three species (possibly)
– Frog virus 3 (FV3)
– Ambystoma tigrinum virus (ATV)
– Bohle irid...
Batrachyochytrium
dendrobatidis
• Kingdom: Fungi
• Class: Chytridiomycetes
• Order: Chytridiales
• Phylum: Chytridiomycota...
Batrochytrium dendrobatidis (Bd)
• One species
• Many strains
• Global panzootic lineage
(Bd-GPL-1 & Bd-GPL-2)
• Virulence...
Understanding of the taxonomy and
genetics of the two pathogens
• No Global Panzootic Lineage for ranaviruses
• Comparable...
Key Ranavirus question
• What gene(s) controls the temperature
sensitivity?
• Ranaviruses can kill mammalian cells in
vitr...
Ranaviruses in Australia
• Epizootic haemopoetic necrosis virus
(EHNV) – bony fish only – 1986
• Bohle Iridovirus (BIV) – ...
Distribution of anti-ranaviral antibodies
in introduced Bufo marinus
Overall prevalence
2.7%
Regional range
0-13%
Zupanovi...
The search for Tadpole
Edema Virus (TEV)
• 1988-1989 Biological control of cane toads
(Rhinella marina (Bufo marinus))
• S...
Tadpole edema virus (TEV)
• Ken Wolf et al’s work (J Inf Dis 1968;118:253)
• Initial isolate from West Virginia
• Found in...
End of wet season April 1989
Townsville: Bohle
Picture of Tvlle in wet season
• Metamorphs of Limnodynastes
ornatus dying from ranaviral
disease in Townsville (1989)
Bohle iridovirus (BIV)
“Bohle iridovirus (BIV)”
First use of name!
Comparison with EHNV
• Epizootic haematopoetic
necrosis virus (EHNV)
isolated from fish
• First ranavirus found in
Austral...
Histopathology due to BIV
• Widespread necrosis of haematopoeitic
and interstitial cells
• Severe necrosis in bone marrow,...
Kidney with focal necrosis
Kidney with necrosis of haemopoetic tissue
• Antigen of BIV could be detected by
immunoperoxidase
• Pathology was extensive in fatal
cases
• Many organs damaged
Liver with
necrosis
Ranavirus
pathology
• Widespread focal necrosis in many organs
± haemorrhage
• Some species have skin ulcers; some
have sk...
Pathology of Bd
• Fungus invades superficial layers of the
epidermis
• Grows inside cells of the top two layers
(stratum c...
Skin slough is occasionally visible
Myxophes fasciolatus with chytridiomycosis
showing sloughing of epidermis
Effects on frogs and toads
Clinical signs range from none to
death
Superficial layer of skin is
microscopically thickene...
All skin sloughing is NOT due to
chytridiomycosis
Normal skin of a Litoria caerulea
• Note epidermis
Epidermis
Dermis
Note the epidermis has a smooth surface
and is about 6...
Chytridiomycosis: Cells of stratum
corneum proliferate
• Look for thickened epidermis with cluster of
sporangia
The cells under
the surface
“dissolve”
Top layer of epithelium
sloughs off
No local inflammatory response
Is Bd immunosuppressive?
Surface of skin with
chytridiomycosis
Some discharge papillae of sporangia
project above the epithelial surface
Normal
Sporangia in epidermal cell
showing cross-section through
discharge papilla with plug (TEM)
Systemic Effects of B.
dendrobatidis
• Behavioural
changes
• Mild neurological
effects
• Severe
neurological
effects
• Noc...
Two pathogenic mechanisms
proposed
• Osmotic effect
OR
• Toxic effect
OR
• Both
Berger et al 1998
3. Organ functioning
Bd INFECTION
1. Epidermal functioning
2. Cutaneous osmoregulation
DEATH
Jamie Voyles
1. Epidermal Functioning
Outside <- Skin Cross Section -> Inside
Na+
H+
K+
ATP
Cl-
HCO3
-
Na+
ATP
HCO3
-
Cl-
Na+
Na+
K+
AT...
Frogs die from blood electrolyte
abnormalities
• Low potassium (reduced
to 50%)
• Low sodium (reduced to
80%)
1
2
3
4
5
6
...
Hypokalaemia
results in
cardiac
asystole
Voyles et al 2009
18 hrs before death
3 hrs before death
2 hrs before death
0.5 h...
Electrolyte replacement
• Increasing the serum K and Na partly
corrected cardiac and neurological signs
• Treatment of Bd ...
Understanding of pathogenesis
• Much more advanced for chytridiomycosis
• Proteases, lipases and other proteins activated
...
Eradicating the pathogen
• Very important when establishing captive
populations from wild caught amphibians
• For Bd treat...
Routes of Transmission
• Both pathogens transmit horizontally
• Water is the major medium; more critical
for Bd since zoos...
Bath exposure
1st
transmission exp for BIV (1989)
LO = Limnodynastes ornatus; BM = Bufo marinus
Tadpoles are carriers
of Bd
• Bd is found in
keratinised tissue
• Mainly in teeth and
jaws, but varies with
Gosner stage
•...
Infection oral & by injection
2nd
transmission exp for BIV (1989)
BM = Bufo marinus; sc = subcutaneous injection
Oral infection
2nd
transmission exp for BIV (1989)
LO = Limnodynastes ornatus
Could BIV kill adult cane toads?
• Exp 3: sc inoculation of 4 adult toads
– 100% mortality 9-10 d post-inoculation
• Subse...
Host range for BIV
• Amphibians: Bufo marinus and a range of
native species (Limnodynastes, Litoria,
Pseudophrenye, Taudac...
Host susceptibility to disease
• Distinguish between infection versus disease
• For both pathogens susceptibility to disea...
Bd patterns of host susceptibility
to disease
1. Able to be infected
but eliminates
infection; eg,
Leiopelma spp.
2. Infec...
Epidemiology is dynamic
• RV epidemiology / ecology is complex
• Poorly understood in Australia; better
understanding in n...
Co-infections
• Recent hypotheses that ranaviruses may
be devastating to amphibian populations
already compromised by ende...
Where to now?
• Need to offer solutions to wildlife managers
• Action to them is:
– Policy – macro or micro
– Intervention...
Control options: research needed
• Bd is possibly easier
– Strategic use of agricultural fungicides
– Biological control t...
Lessons from both diseases
• Both are complex, highly dynamic and
evolving
• Host species is important; so be wary
extrapo...
Thank you!
Sources of images not original
• Paris is September -
http://www.123rf.com/photo_16463061_paris--september-30-eiffel-tower...
Speare ranavirus symEmerging infectious diseases and amphibian population declines: How are we going?posium-web
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Speare ranavirus symEmerging infectious diseases and amphibian population declines: How are we going?posium-web

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

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  • I have had the privilege of working on both diseases Also involved in the global notification
  • Speare ranavirus symEmerging infectious diseases and amphibian population declines: How are we going?posium-web

    1. 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. 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
    3. 3. 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
    4. 4. 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
    5. 5. 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
    6. 6. 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
    7. 7. 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?
    8. 8. 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
    9. 9. 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
    10. 10. 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!
    11. 11. 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
    12. 12. 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)
    13. 13. 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?
    14. 14. End of wet season April 1989 Townsville: Bohle
    15. 15. Picture of Tvlle in wet season
    16. 16. • Metamorphs of Limnodynastes ornatus dying from ranaviral disease in Townsville (1989)
    17. 17. Bohle iridovirus (BIV)
    18. 18. “Bohle iridovirus (BIV)” First use of name!
    19. 19. 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
    20. 20. 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
    21. 21. Kidney with focal necrosis
    22. 22. Kidney with necrosis of haemopoetic tissue
    23. 23. • Antigen of BIV could be detected by immunoperoxidase
    24. 24. • Pathology was extensive in fatal cases • Many organs damaged Liver with necrosis
    25. 25. 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!
    26. 26. 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
    27. 27. Skin slough is occasionally visible Myxophes fasciolatus with chytridiomycosis showing sloughing of epidermis
    28. 28. 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
    29. 29. All skin sloughing is NOT due to chytridiomycosis
    30. 30. 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
    31. 31. Chytridiomycosis: Cells of stratum corneum proliferate • Look for thickened epidermis with cluster of sporangia
    32. 32. The cells under the surface “dissolve”
    33. 33. Top layer of epithelium sloughs off No local inflammatory response Is Bd immunosuppressive?
    34. 34. Surface of skin with chytridiomycosis Some discharge papillae of sporangia project above the epithelial surface Normal
    35. 35. Sporangia in epidermal cell showing cross-section through discharge papilla with plug (TEM)
    36. 36. 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?
    37. 37. Two pathogenic mechanisms proposed • Osmotic effect OR • Toxic effect OR • Both Berger et al 1998
    38. 38. 3. Organ functioning Bd INFECTION 1. Epidermal functioning 2. Cutaneous osmoregulation DEATH Jamie Voyles
    39. 39. 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
    40. 40. 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
    41. 41. 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
    42. 42. 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
    43. 43. 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
    44. 44. 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?
    45. 45. 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
    46. 46. Bath exposure 1st transmission exp for BIV (1989) LO = Limnodynastes ornatus; BM = Bufo marinus
    47. 47. 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
    48. 48. Infection oral & by injection 2nd transmission exp for BIV (1989) BM = Bufo marinus; sc = subcutaneous injection
    49. 49. Oral infection 2nd transmission exp for BIV (1989) LO = Limnodynastes ornatus
    50. 50. 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
    51. 51. 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
    52. 52. 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
    53. 53. 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?
    54. 54. 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?
    55. 55. 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.
    56. 56. 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
    57. 57. 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
    58. 58. 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
    59. 59. Thank you!
    60. 60. 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

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