c e l l s a n d t i s s u e d o m a i n ■

ihbi

Chlamydia and koalas : a

battle to be won .... or lost

Range of infections & disease caused by

Chlamydia in koalas

Inapparent / sub-clinical

Ocular

– Mild conjunctivitis

– Complete blindness

Urogenital

– Bladder infections, cystitis, cysts, prostatitis

Respiratory

– Rhinitis, sneezing, cough, pneumonia

Chlamydial infections in koalas

Cystitis

“Wet bottom”

Reproductive tract

Cysts “Mars Bar”

Conjunctivitis

“Blindness”

Chlamydial developmental cycle

Stress:

- Penicillin

- Heat

- IFN – γ etc

Persistence

Aberrant body

(Belland et al., 2003)(Polkinghorne et al., 2006)

Normal inclusion

Old Taxonomy

Family

Chlamydiaceae

Genus and species

Chlamydia

C. trachomatis

C. psittaci

C. pecorum

C. pneumoniae

Family

Chlamydiaceae

Genus and species

Chlamydia

C. trachomatis

C. suis

C. muridarum

Chlamydophila

Cp. psittaci

Cp. abortus

Cp. caviae

Cp. pecorum

Cp. pneumoniae

Cp. felis

New Taxonomy(Everett et al., 1999)

Current Taxonomy(Stephens et al., 2009)

Family

Chlamydiaceae

Genus and species

Chlamydia

• C. trachomatis

• C. suis

• C. muridarum

• C. psittaci

• C. abortus

• C. caviae

• C. pecorum

• C. pneumoniae

• C. felis

Chlamydia testing methods

Clinical signs : eg. conjunctivitis, wet bottom

Cell culture : very specialised

Antibody detection : serology; ELISA

Antigen detection : Clearview test kit

DNA probes

PCR / qPCR

Epizootiology of chlamydial infection in Australia’s

wild koala population

SummaryPopulation % +ve

Magnetic Island 0%

Narangba 50%

Mutdapilly 85%

Koala Coast 87%

Coombabah 10%

East Coomera 35%

Koala Beach 67%

Port Macquarie 45%

Pilliga State Forest 12%

Pine Creek State Forest 72%

Ballarat 100%

French Island 0%

Mt Lofty Ranges 90%

Kangaroo Island 0%

SummaryPopulation % +ve C.pecorum C.pneumoniae

Magnetic Island 0% 0% 0%

Narangba 50% 50%

Mutdapilly 85% 73% 21%

Koala Coast 87% 87% 4%

Coombabah 10% 10% 10%

East Coomera 35% 35%

Koala Beach 67% 67% 10%

Port Macquarie 45% 38% 23%

Pilliga State Forest 12% 8% 8%

Pine Creek State Forest 72% 52% 12%

Ballarat 100% 90% 20%

French Island 0% 0% 0%

Mt Lofty Ranges 90% 90% 53%

Kangaroo Island 0% 0% 0%

C.pecorum in koalas

1. What is the diversity of C.pecorum

infections in koalas ?

2. Where did koalas get their C.pecorum

from ?

What is the origin of C. pecorum in koalas?

Ancient USA / Europe / Asia

strains

Australia (1788)

Phylogenetically diversesheep / cattle strains

birds / animals

C. pecorum line

Range of hosts

Major outer membrane protein (ompA)

ompA

– MOMP

Important functions:

– Porin

– Attachment

– Structural stability

(Kaltenboeck et al., 1993)

CDs and VDs

– Target of molecular studies

Kaltenboeck et al.,

1993

Kaltenboeck et al.,

1993

C.pecorum phylogeny using the ompA gene only

ompA, incA, ORF663ompA

C.pecorum phylogeny using several genes

C.pneumoniae infections in koalas

1. Is C.pneumoniae significant in koalas ?

2. How does the koala strain of

C.pneumoniae relate to the human

strains of C.pneumoniae ?

1999

Chlamydia much more widespread than first

thought

Berger ……….. & Timms (1999)

C.pneumoniae identified in a free-ranging Giant Barred frog

Gene sequences extremely similar to koala C.pneumoniae

strains

Koala C.pneumoniae – strain LPCoLN

Respiratory disease outbreak in a large captive koala colony– 70% of animal affected over 6-9 month period

– Rhinitis …. Coughing/sneezing ….. Serous/purulent discharge

– spread through the colony via respiratory route

No other bacteria or viruses isolated

Chlamydia / C.pneumoniae diagnosed via antigen detection and PCR

Nasal swabs from Connor cultured in Hep2 cells at 37C →LPCoLN

Host range of C.pneumoniae

Humans

Horses (Storey et al., 1993)

Marsupials

– koalas (Jackson et al., 1997; Wardrop et al., 1998)

– bandicoots (Bodetti et al., 2002)

– gliders (Bodetti et al., 2002)

Amphibians

– frogs (Berger et al., 2000; Reed et al., 2000; Hotzel et al., 2001; Blumer et al., 2007)

Reptiles

– snakes (Bodetti et al., 2002; Jacobsen et al., 2004)

– crocodiles

– iguanas (Bodetti et al., 2002)

Current status of C.pneumoniae evolution

Some recent epidemiology

lessons from SE Queensland

populations

Populations analysed

Narangba (A)

Brendale (B)

East Coomera (C)

Epizootiology

Populationq PCR +ve

Animals*

Male/Female

qPCR +ve

5yrs

Population A

63 swabs from 16

animals

8 (50%) 3:5 1/8 1/8 6/8

Population B

83 swabs from 22

animals

11 (50%) 5:6 0/11 3/11 8/11

Population C

51 swabs from 17

animals

6 (35%) 4:1 0/5 2/5 3/5

•1 = < 10 copies/ul; 2 = 10-100 copies/ul; 3 = 100-1000 copies/ul; 4 = 1000-10000 copies/ul; 5 = >10000 copies/ul

Genetic diversity of C.pecorum infections

VD1 VD2 VD3 VD4

CpeNTVD3 CpeNTVD4

331bp

MOMP

Genotype Sample Geographical location Year of Study

A

LPDowellV495 Lone Pine Koala Sanctuary, QLD

MT113V189 Mutdapilly, QLD

MT213V189 Mutdapilly, QLD

RBNarO1095 Redland Bay, QLD Jackson et al., 1997

CS9745V496 Currumbin Sanctuary, QLD

CS9762V496 Currumbin Sanctuary, QLD

PMKalaO594 Port Macquarie, NSW

B RI28Vna Raymond Island, VIC Jackson et al., 1997

C FWPBilbPna Featherdale Wildlife Park, NSW Jackson et al., 1997

D K4 Australia Jackson et al., 1997

E

K13 Australia

EMDuarV496 Emerald, QLD

PMRiorV295 Port Macquarie, NSW Jackson et al., 1997

PMScarVna Port Macquarie, NSW

PMBossPna Port Macquarie, NSW

E'

Nar/Edna/UGT

Population A Current Study

Nar/Dion/L/E

Nar/Dion/UGT

Nar/Frankie/UGT

Nar/Felix/UGT

Nar/Greg/Nasal

Nar/Kaia/UGT

F

Bre/Pnau/UGT

Population B

Current Study

Bre/Kurt/UGT

Bre/Poppy/UGT

Bre/Michael/UGT

Bre/Xena/UGT

Bre/Nigel/UGT

EC/Ned/L/E

Population CEC/Ned/R/E

EC/Graeme/UGT

G

Bre/Miss Radio/UGT Population B

Current StudyEC/Steve/UGT Population C

EC/James/UGT Population C

Mars Bar/UGT Mt.Cotton, Brisbane

HEC/Ned/UGT

Population C Current StudyEC/Steve/R/E

F

G

H

F

G

F

GE’

Population APopulation B Population C

Diversity of MOMP genotypes

SEQ Map

OmpA genotypes of koala C. pecorum isolates

Genotypes A,B,C and D identified in

1999 by Jackson et al. Vet Micro

Genotypes E,E’,F,G and H identified

in 3 study populations

Collaborators

QUT

Peter Timms

Ken Beagley

Alison Carey

Adam Polkinghorne

Avinash Kollipara

Lone Pine Koala Sanctuary

Galit, Jacqui & Karen

Australian Wildlife Hospital

Jon & Jo

Gold Coast City Council

John & team

Pfizer Animal Health

John Walker

Friends of the Koala (Lismore)

Lorraine Vaas

Supported by ARC, Qld Gov