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