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

• 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

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

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

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

Batrachyochytrium dendrobatidis

• Kingdom: Fungi

• Class: Chytridiomycetes

• Order: Chytridiales

• Phylum: Chytridiomycota

• Genus: Batrachyochytrium

• Species: dendrobatidis

Berger et al 1998. PNAS 95:903Nichols et al 1998 Proc Am Assoc Zoo Vet 1998:269Longcore et al 1992. Mycologia 91:219

Joyce Longcore

zoospore

zoosporangium

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?

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

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

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!

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

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)

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?

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 Australia

• BIV and EHNV were distinct species

Henstberger et al (1993)

EHNV

BIV

Alex Hyatt

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

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 skin growths

• Frogs die from “organ failure”, but pathophysiology is not understood

Makes treatment for RV disease difficult!

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

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 thickened

This layer often sloughs off in fragments

Frogs die with neurological signs

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 6-8 cell layers thick

Subdermal lymph sinus

Lee Berger

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 responseIs 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

• 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?

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

DEATHJamie Voyles

1. Epidermal FunctioningOutside <- 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

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 = 7Diseased N = 10

ANOVA p = 0.001*

Voyles et al 2007, 2009 Images from Jamie Voyles

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

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

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

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?

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

Bath exposure1st 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

• May damage these

• No direct mortality

• May reduce fitness

Marantelli et al 2005 Pac Cons Biol 10:173

Infection oral & by injection2nd 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

• 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

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

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

Bd patterns of host susceptibility to disease1. 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 92:150Similar patterns for both pathogens. What determines host susceptibility?

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?

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.

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

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

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

Thank you!

Sources of images not original

• Paris is September - http://www.123rf.com/photo_16463061_paris--september-30-eiffel-tower-at-night-on-september-30-2012-in-paris-night-in-paris-with-eiffel-t.html

• 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+0045• Alpine tree frog drawing -

http://www.redbubble.com/people/lauragrogan/works/6520637-alpine-tree-frog-litoria-verreauxii-alpina