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DISEASE IN WILDLIFE OR EXOTIC SPECIES

Pneumonia due to Chlamydia pecorum in a Koala

(Phascolarctos cinereus)
J. T. Mackie*, A. K. Gillett†, C. Palmieri‡, T. Fengx and D. P. Higginsk
* Vepalabs, Woolloongabba, Queensland, † Australia Zoo Wildlife Hospital, Beerwah, Queensland, ‡ School of Veterinary
Science, x School of Agriculture and Food Sciences, The University of Queensland, Gatton, Queensland and k School of Life and
Environmental Sciences, Faculty of Veterinary Science, The University of Sydney, Sydney, New South Wales, Australia

Summary
Chlamydiosis is a common infectious disease of koalas (Phascolarctos cinereus), but Chlamydia spp. have not yet
been demonstrated to cause pneumonia in these animals. A juvenile male koala died following an episode of
respiratory disease. At necropsy examination, the lung tissue was consolidated. Microscopical lesions in the
lung included pyogranulomatous bronchopneumonia, proliferation of bronchiolar and alveolar epithelium
and interstitial fibrosis. Hyperplastic bronchiolar epithelial cells contained aggregates of small basophilic punc-
tate organisms, which were confirmed as chlamydiae by transmission electron microscopy and immunohisto-
chemistry. Real-time polymerase chain reaction identified these as Chlamydia pecorum. This report provides the
best evidence to date of chlamydial infection causing pneumonia in a koala, and the first evidence that C. pe-
corum is capable of infecting the bronchiolar epithelium of the koala.

Ó 2016 Elsevier Ltd. All rights reserved.

Keywords: Chlamydia; chlamydiosis; koala; pneumonia

Chlamydiosis is the most common infectious disease of
koalas (Phascolarctos cinereus). Chlamydial organisms
or DNA have been recovered from a number of con-
ditions, including keratoconjunctivitis, urinary tract
inflammation and incontinence, reproductive tract
inflammation in males and females resulting in infer-
tility in females, colitis and rhinitis/pneumonia com-
plex (Cockram and Jackson, 1974; Brown and
Grice, 1984; McColl et al., 1984; Brown et al., 1987;
Obendorf and Handasyde, 1990; Hemsley and
Canfield, 1996; Johnston et al., 2015). For all but
respiratory disease, a causative relationship has been
confirmed by experimental infection and
immunohistochemistry (Brown and Grice, 1986;
Kempster et al., 1996; Hemsley and Canfield, 1997;
Higgins et al., 2005). Chlamydial inclusions have
not yet been demonstrated in situ in diseased
respiratory epithelium of the koala.
Correspondence to: J. T. Mackie (e-mail: john.mackie@qml.com.au).
Most wild (and some captive) koala populations in
Australia are infected with chlamydiae, although the
level of clinical disease reported varies considerably
(Polkinghorne et al., 2013). Chlamydial organisms
isolated from koalas were initially classified as Chla-
mydia psittaci, but chlamydial taxonomy has subse-
quently undergone significant revision (Brown and
Grice, 1984). A proposal to split the family Chlamy-
diaceae into two genera (Chlamydia and Chlamydophila)
was not widely adopted, and currently accepted
nomenclature recognizes nine species in the single
genus Chlamydia (Everett et al., 1999; Stephens et al.,
2009; Kuo et al., 2011; Polkinghorne et al., 2013).
Koala isolates include Chlamydia pecorum, Chlamydia
pneumoniae (koala biovar) and a number of
Chlamydia-like organisms of unknown significance
(Glassick et al., 1996; Everett et al., 1999; Devereaux
et al., 2003). C. pecorum is the most common
chlamydial species infecting koalas and is thought to
be responsible for most of the clinical disease,

0021-9975/$ - see front matter Ó 2016 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jcpa.2016.07.011

Please cite this article in press as: Mackie JT, et al., Pneumonia due to Chlamydia pecorum in a Koala (Phascolarctos cinereus), Journal of
Comparative Pathology (2016), http://dx.doi.org/10.1016/j.jcpa.2016.07.011
2 J.T. Mackie et al.
although C. pneumoniae is also widespread in many
populations and has been suggested to be more
closely associated with ocular and respiratory
infection (Jackson et al., 1999; Polkinghorne et al.,
2013). This report describes direct involvement of C.
pecorum in a case of pneumonia in a koala.
A juvenile male koala (approximately 14 months
old) was presented to Australia Zoo Wildlife Hospital
on the 1st of January 2014 with its mother, after
collection from a tree on the Fraser Coast of Queens-
land, Australia. The adult female had a stained and
wet rump. Swabs of her urogenital sinus and urine
sediment swab tested positive for Chlamydiaceae
family-specific lipopolysaccharide (LPS) antigen us-
ing a rapid immunoassay (Clearview Chlamydia MF;
Alere, Waltham, Massachusetts, USA) and chla-
mydial cystitis was diagnosed. Antibiotic therapy
was commenced (chloramphenicol 60 mg/kg subcu-
taneously q24h) (BOVA Compounding, Caringbah,
New South Wales, Australia). The male joey ap-
peared healthy and was housed with its mother while
she was undergoing treatment.
Seven days post admission the joey was noted to be
coughing after eating. On clinical examination,
including auscultation of the chest, there was no evi-
dence of respiratory compromise or other abnormal-
ity. Fourteen days post admission the joey was
found coughing on the floor of his enclosure and
had audible respiratory sounds. Following anaes-
thesia and intubation, copious mucopurulent exudate
was observed oozing from the epiglottis/larynx. Cyto-
logical evaluation of this exudate revealed large
numbers of degenerate neutrophils. A swab of this
exudate tested positive for Chlamydiaceae family-
specific LPS antigen using the rapid immunoassay
described above. DNA extracts of a swab of this
exudate were subjected to a real-time polymerase
chain reaction (PCR) assay using 23s Chlamydia
genus-specific primers and ompB C. pecorum and C.
pneumoniae species-specific primers (Ehricht et al.,
2006; Govendir et al., 2012). Chlamydia genus and C.
pecorum assays were positive.
Radiographs revealed severe opacity of the entire
right lung field and more than half of the left lung
field, consistent with pneumonia. The joey was
treated with intravenous fluids and chloramphenicol
(BOVA Compounding) at a dose of 60 mg/kg subcu-
taneously q24h and nebulized daily with 20 mg
gentamicin and 0.25 ml salbutamol in 4 ml of saline
and oxygen therapy, but died 2 days later.
Post-mortem examination revealed a fibrinous
exudate over the surface of the right lung lobes with
pleural adhesions. The right lung lobes and left cra-
nial lung lobes were consolidated. On cut section,
foamy purulent exudate oozed from the lung paren-
Please cite this article in press as: Mackie JT, et al., Pneumonia due to Chlamydia pecorum in a Koala (Phascolarctos cinereus), Journal of
Comparative Pathology (2016), http://dx.doi.org/10.1016/j.jcpa.2016.07.011
chyma and was present along the length of the tra-
chea. There was a mild excess of slightly blood-
tinged fluid in the pericardial cavity. All other organs
appeared grossly normal. Bacterial culture of bron-
choalveolar lavage fluid collected at post-mortem ex-
amination yielded a very light mixed growth of upper
respiratory flora.
Histological examination revealed severe chronic
diffuse pyogranulomatous bronchopneumonia, with
proliferation of bronchiolar and alveolar epithelium
and interstitial fibrosis (Fig. 1A). Numerous hyper-
plastic bronchiolar epithelial cells contained large
cytoplasmic inclusions, which consisted of aggregates
of >50 small basophilic punctate organisms approxi-
mately 0.5e1.0 mm in diameter (Fig. 1B). A Gram
stain and periodic acideSchiff (PAS) stain did not
reveal bacteria or fungi and did not stain the organ-
isms in the inclusions. There were minor changes in
the liver (hepatocellular fatty change) and spleen
(lymphoid depletion) and no significant changes in
other tissues examined (including the kidney).
Formalin-fixed and paraffin wax-embedded lung
tissue were subjected to immunohistochemistry
(IHC) using a rabbit anti-chlamydial LPS antibody
(Cellabs, Brookvale, New South Wales, Australia)
and an immunoperoxidase detection system (Im-
mPRESSÔ; Vector Laboratories, Burlingame, Cali-
fornia, USA) (Higgins et al., 2005). The sections
showed strong labelling of chlamydial inclusions
within the cytoplasm of respiratory epithelial cells in
association with the marked inflammation
(Fig. 1C), consistent with the cytoplasmic inclusions
noted on histopathology. Additional sections, cut us-
ing a new, sterile, microtome blade, were subjected
to DNA extraction (Quickextract FFPE DNA Ex-
tracting Solution; EPICENTRE Biotechnologies,
Madison, Wisconsin, USA) for genus- and species-
specific real-time PCR, as described above. Chlamydia
genus and C. pecorum assays were positive.
Formalin-fixed lung tissue was fixed in 2.5%
glutaraldehyde, post-fixed in 1% osmium tetroxide
and prepared for transmission electron microscopy
(TEM). Ultrastructural examination revealed the
cytoplasmic inclusions in bronchiolar epithelial cells
containing different developmental stages of chla-
mydial organisms. Small, 250e350 nm in diameter,
round forms had an eccentric electron-dense core
and a granular cytoplasm surrounded by a cyto-
plasmic membrane and an outer envelope (elemen-
tary bodies) (Fig. 1D). Large, 600e800 nm in
diameter, oval to round forms had a granular cyto-
plasm enclosed by an inner cytoplasmic membrane
and an external outer envelope (reticulate bodies).
There were small numbers of oval to round interme-
diate forms, approximately 400 nm in diameter,
 Chlamydial Pneumonia in a Koala 3

Fig. 1. A. Pyogranulomatous bronchopneumonia with hyperplastic bronchiolar epithelial cells and interstitial fibrosis. HE. Bar, 120 mm.
B. Hyperplastic bronchiolar epithelial cells contain cytoplasmic aggregates of basophilic punctate organisms (arrows). HE. Bar,
40 mm. C. Cytoplasmic inclusions in bronchiolar epithelial cells exhibit strong immunohistochemical labelling of chlamydial lipo-
polysaccharide antigen. Bar, 30 mm. D. Microcolony of chlamydiae in a bronchiolar epithelial cell, composed of elementary bodies
(arrowhead), reticulate bodies (asterisk) and intermediate bodies (arrow). TEM. Bar, 500 nm.

with a centrally located electron-dense core and gran-
ular cytoplasm. In most chlamydial cells, the endo-
some membrane was not detected.
This report provides the best evidence to date of
chlamydial infection causing pneumonia in a koala,
and the first evidence that C. pecorum is capable of in-
fecting the bronchiolar epithelium of the koala.
It cannot be determined whether this koala suc-
cumbed due to treatment failure or simply an over-
whelming infection. A compounded formulation of
chloramphenicol was used due to lack of availability
of a recommended commercial formulation (CEVA
Animal Health, Glenorie, New South Wales,
Australia) (Govendir et al., 2012).
The size range of the individual organisms in
routine haematoxylin and eosin -stained sections re-
flected the presence of not only chlamydial elemen-
tary bodies, which are the smaller, infectious
particles, but also reticulate bodies, which are the
larger, metabolically active stage of the chlamydial
life cycle, as well as intermediate bodies. Elementary
bodies and reticulate bodies demonstrated by TEM
in urogenital epithelium of koalas have been reported
to measure 0.4e0.6 mm and 0.8e1.2 mm, respectively
(Ladds, 2009).
Confirmation that organisms invade cells in tissue
containing lesions is an important step in the elucida-
tion of pathogenesis. Subclinical chlamydial infection
is common in koalas (Polkinghorne et al., 2013). Cul-
ture, IHC or PCR may yield evidence of chlamydiae
in the absence of local colonization, due to transfer of
organisms from infected sites to non-infected sites in
peripheral blood mononuclear cells, which might be
present in any vascular tissues or inflammatory
exudate of an infected animal, in the absence of local
chlamydial infection (Bodetti and Timms, 2000;
Higgins et al., 2005).
In koalas, IHC has previously demonstrated chla-
mydial antigen associated with inflammation in the
reproductive tract (male and female), kidney and
bladder, with positive labelling of epithelial cells
and macrophages (Higgins et al., 2005; Burach
et al., 2014). Less frequently, chlamydial antigen
has been detected in splenic and hepatic
macrophages, variably associated with
inflammation. In lung tissue, chlamydial antigen
was detected in macrophages in one of 10 koalas
with interstitial pneumonia (Higgins et al., 2005).
In another study, chlamydial antigen was detected
in alveolar epithelial cells of one koala in the absence
of inflammation (Burach et al., 2014). In this latter
study, chlamydiae were not detected by IHC or
PCR in the single koala with interstitial pneumonia
(Burach et al., 2014).

Please cite this article in press as: Mackie JT, et al., Pneumonia due to Chlamydia pecorum in a Koala (Phascolarctos cinereus), Journal of
Comparative Pathology (2016), http://dx.doi.org/10.1016/j.jcpa.2016.07.011
4 J.T. Mackie et al.
Chlamydial organisms were isolated from the nasal
septum of a koala with rhinitis/pneumonia complex
and demonstrated by PCR in similarly affected koalas
(Brown and Grice, 1984; Jackson et al., 1999;
Wardrop et al., 1999). Limited transmission studies
have suggested an aetiological role of isolates of
koala origin in producing urogenital and respiratory
infections, but the role of such organisms in causing
pneumonia has not been confirmed (Brown et al.,
1987). In the present study, chlamydial organisms
were discernible on routine histopathology within hy-
perplastic bronchiolar epithelium, which was inti-
mately associated with inflammation, and were
confirmed by TEM and IHC. Real-time PCR identi-
fied these as C. pecorum.
In addition to causing disease in koalas, C. pecorum
has been described to cause a variety of syndromes
in cattle and sheep including pneumonia, but there
appear to be no previous reports of it causing pneu-
monia in the koala or other non-ruminant animal spe-
cies (Longbottom, 2004). This case indicates that C.
pecorum should be considered as a possible aetiological
agent of pneumonia in the koala.
Acknowledgments
The TEM was supported in part by the Queensland
Department of Environment and Heritage Protection
(grant number KRG005). The authors thank Dr A.
Casteriano (Koala Health Hub, The University of
Sydney) for assistance with IHC and PCR.
Conflict of Interest Statement
The authors declare no conflicts of interest with
respect to the publication of this article.
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½ Received,
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Please cite this article in press as: Mackie JT, et al., Pneumonia due to Chlamydia pecorum in a Koala (Phascolarctos cinereus), Journal of
Comparative Pathology (2016), http://dx.doi.org/10.1016/j.jcpa.2016.07.011