Vet Pathol 41:388–397 (2004)

Detection of Mycobacteria and Chlamydiae in Granulomatous

Inflammation of Reptiles: A Retrospective Study
G. SOLDATI, Z. H. LU, L. VAUGHAN, A. POLKINGHORNE, D. R. ZIMMERMANN, J. B. HUDER,
AND A. POSPISCHIL

Institute of Veterinary Pathology (GS, ZHL, LV, A. Pospischil) and Swiss National Center for Retroviruses (JBH),
University of Zurich, Switzerland; Center for Molecular Biotechnology, School of Life Sciences, Queensland University
of Technology, Brisbane, Australia (A. Polkinghorne); and Institute of Clinical Pathology, University Hospital, Zurich,
Switzerland (DRZ)

Abstract. A retrospective study on reptile tissues presenting with granulomatous inflammation was per-
formed to detect the possible presence of mycobacteria and chlamydiae in these lesions. Ninety cases including
48 snakes, 27 chelonians, and 15 lizards were selected. Mycobacteria were detected by Ziehl–Neelsen (ZN)
staining and a broad-range polymerase chain reaction (PCR) followed by DNA sequencing. To detect chla-
mydiae, immunohistochemistry with monoclonal antibodies against chlamydial lipopolysaccharide (LPS) and a
Chlamydiales order–specific PCR and sequencing were applied. Acid-fast bacilli were found in 14 cases (15.6%)
by ZN staining and in 23 cases (25.6%) by PCR. Sequence analysis revealed the presence of Mycobacteria
other than Mycobacterium tuberculosis complex (MOTT). Chlamydial LPS antigen was observed within gran-
ulomas from five samples (5.6%), whereas the PCR screen revealed 58 positive cases (64.4%). Of these, 9
cases (10%) showed 98–99% similarity to Chlamydophila (Cp.) pneumoniae and 49 cases (54.4%) displayed a
high similarity (88–97%) to the newly described ‘‘Chlamydia-like’’ microorganisms Parachlamydia acanth-
amoebae and Simkania negevensis. Results from this study confirm, on the one hand, that MOTT are probably
the most important infectious etiology for granulomatous inflammation in reptiles. On the other hand, they
indicate that chlamydia infects reptiles and that Cp. pneumoniae should be considered an etiological agent of
granulomatous lesions of reptiles. Because both MOTT and Cp. pneumoniae are human pathogens, the potential
of zoonotic transmission from reptiles to humans has to be considered. In contrast, the significance of Chla-
mydia-like isolates remains completely open, and further studies are needed to evaluate their role.

Key words: ‘‘Chlamydia-like’’ isolates; Chlamydophila pneumoniae; granulomatous inflammation; immu-

nohistochemistry; mycobacteria other than Mycobacterium tuberculosis complex; polymerase chain reaction
and sequencing; reptiles; Ziehl–Neelsen.

Infectious organisms, including bacterial, fungal,
parasitic, and algal agents, are major cause of disease
and mortality in reptiles.18 Reptiles react to many of
these infections by developing granulomatous inflam-
mations that may be classified as heterophilic or his-
tiocytic, depending on the etiology and the host’s cel-
lular response.19,43 The heterophilic granuloma, usually
induced by extracellular pathogens, is characterized by
an accumulation of heterophils, whose degranulation
and central necrosis appear to act as foreign bodies
and stimulate a strong macrophage response. The his-
tiocytic granuloma, caused by intracellular bacteria,
appears to have a different pathogenesis. Early lesions
are formed from organized collections of large foamy
macrophages, with the centrally located macrophages
often undergoing necrosis.19,43 In reptiles surviving the
acute infection stage, both types develop into chronic
granulomas, in which epitheloid cells, lymphocytes,
plasma cells, and eventually multinucleated giant cells
surround the central lesion. Older granulomas are ad-
388
ditionally walled-off within a fibrous connective tis-
sue.43
Mycobacteria are classical etiologic agents of gran-
ulomatous reactions in human and animal hosts.5 The
genus Mycobacterium (M.) comprises about 85 spe-
cies, all sharing the characteristic morphologic features
of gram-positive, aerobic, acid-alcohol fast bacteria.22
The pathogenicity of different mycobacteria varies sig-
nificantly, and for practical purposes they have been
differentiated into two groups: 1) the M. tuberculosis
complex (MTC), which includes M. tuberculosis, M.
bovis, M. africanum, M. microti and M. canetti, and
2) Mycobacteria other than the M. tuberculosis com-
plex (MOTT), comprising M. avium intracellulare, M.
avium ssp. paratuberculosis, M. leprae, M. leprae-
murium and atypical mycobacteria.46
Spontaneous mycobacterial infections have been re-
ported frequently in a wide variety of reptiles, includ-
ing snakes, turtles, lizards, and crocodiles.10,11,18,54 It is
generally believed that mycobacteria are contracted
Vet Pathol 41:4, 2004 Mycobacteria and Chlamydiae in Reptile Granulomas
through defects in the integument or by ingestion. Af-
ter necropsy, grayish-white nodules may be observed
in many organs and in the subcutis; histopathologic
examination shows typical granulomatous inflamma-
tions with multinucleated giant cells, common features
of these lesions. Unlike mammalian tubercles, calcifi-
cation has not been observed. Mycobacterial diagnosis
was performed by the acid-fast staining technique and,
where possible, the species was identified by bacteri-
ologic examination and included M. avium, M. che-
lonae, M. fortuitum, M. intracellulare, M. marinum,
M. phlei, M. smegmatis, and M. ulcerans.3,4,16,44,45,48
Polymerase chain reaction (PCR) has been used more
recently to confirm diagnosis of mycobacteriosis in
young freshwater crocodiles and in a royal python.3,24
A second obligate intracellular microorganism, chla-
mydia, sporadically infects reptiles, sometimes induc-
ing histiocytic granulomas.31,32
Chlamydiae have a unique biphasic developmental
cycle consisting of the infectious elementary body
(EB) and the noninfectious reticulate body (RB).52
Until 1999, four species were recognized within the
genus, namely Chlamydia psittaci, Chlamydia peco-
rum, Chlamydia trachomatis, and Chlamydia pneu-
moniae.52 They have recently been reclassified to allow
for a more accurate identification of the bacteria in-
fecting animals. The family Chlamydiaceae was divid-
ed into two genera, Chlamydia (C.) and Chlamydo-
phila (Cp.), with a total of nine species. Furthermore,
three new families, the Parachlamydiaceae, Simkani-
aceae, and Waddliaceae, comprehending the lately dis-
covered ‘‘Chlamydia-like’’ isolates were pro-
posed.15,27,50
Chlamydiae are widespread in warm-blooded hosts
including humans, birds, cats, dogs, sheep, cattle, pigs,
and koalas, causing a variety of diseases.53 Only spo-
radic cases of chlamydiosis have been recorded in rep-
tiles (C. psittaci at the time), including chameleons,
turtles, tortoises, crocodiles, snakes, and iguanas. Nec-
ropsy findings of chlamydial infections in reptiles in-
clude lesions in the heart, liver, lung, spleen, and small
intestine. Light microscopy examination revealed se-
vere inflammatory changes in a variety of tissues, of-
ten characterized by formation of well-defined granu-
lomas. Colonies of intracytoplasmic organisms mor-
phologically compatible with chlamydia were present
in sections stained either by the modified Gimenez’s
or by the modified Macchiavello’s method. Under
electron microscopic examination, inclusions within
the cytoplasm of macrophages composed of a popu-
lation of pleomorphic organisms compatible with the
various developmental stages of Chlamydia were ob-
served. In some cases, Chlamydia could be isolated by
inoculating specific pathogen-free chicken eggs with
389
the reptilian samples.25,30,32,33,55 Recently, T. Bodetti and
colleagues8 collected reptile tissues where the presence
of chlamydiae was analyzed by immunohistochemistry
(IHC), direct immunofluorescence, and by PCR and
DNA sequencing. Several chlamydial species includ-
ing Chlamydophila (Cp.) pneumoniae, Cp. abortus,
Cp. felis and Neochlamydia were found to be present
in these hosts.
The aim of this study was to detect the presence of
mycobacteria and chlamydiae in granulomatous in-
flammations of reptiles in a 10-year retrospective
study.
Materials and Methods
Selection of cases
Archived formalin-fixed and paraffin-embedded tissue
specimens from 90 reptiles (reptile Nos. 1–90; 48 snakes,
27 chelonians, and 15 lizards) necropsied at the Institute of
Veterinary Pathology, University of Zurich between 1990
and 1999 and presenting granulomatous inflammation were
investigated (Fig. 1). Of these, 67 were pets from private
owners, 21 had been housed in zoologic collections, 1 was
from a pet shop, and 1 was kept in a school.
Histopathology
Histologic sections of all selected cases were stained with
the hematoxylin and eosin (HE) technique and reviewed his-
tologically to verify the presence of granulomatous inflam-
mation and to distinguish the type of granulomatous lesion.
Screening for mycobacteria by Ziehl–Neelsen staining
Sections (2 ␮m) from all tissues collected were investi-
gated for the presence of mycobacteria using the Ziehl–Neel-
sen (ZN) stain. Sections of intestinal tissue from a deer with
Johne’s disease were used as the positive control.
IHC screening for chlamydiae
Paraffin sections were investigated for the presence of
chlamydial antigen using Chlamydiaceae family–specific
mouse monoclonal antibodies (Abs) directed against the
chlamydial lipopolysaccharide (cLPS; Clone ACI-P, Progen,
Heidelberg, Germany) and the EnVision Kit (Dako
ChemMate娂, Glostrup, Denmark) according to the manu-
facturer’s instructions. Briefly, paraffin sections were depar-
affinated in xylene, rehydrated through graded ethanol to wa-
ter, and pretreated by microwave heating (750 W for 10 min-
utes) for antigen retrieval. To inhibit the endogenous per-
oxidase activity, they were immersed in peroxidase-blocking
solution for 8 minutes at room temperature (RT) and then
incubated with the primary Ab at a working dilution of 1 :
200 in Ab diluent with background-reducing components for
30 minutes at RT. The sections were incubated for 30 min-
utes at RT with the link Ab, developed in peroxidase/3-ami-
no-g-ethylcarbazole (AEC) substrate solution for 5 minutes
at RT, counterstained with hematoxylin, and coverslipped.
A negative control of each section was performed using
the Ab diluent instead of the primary Ab. Intestinal tissues
390 Soldati, Lu, Vaughan, Polkinghorne, Zimmermann, Huder, and Pospischil Vet Pathol 41:4, 2004

Fig. 1. Lung; snake, case No. 5. Multiple gray nodules are visible within the lung.

from gnotobiotic piglets experimentally infected with por-
cine Chlamydia suis strain S45 were used as positive con-
trols.21
DNA extraction for PCR screening
Sections (30 ␮m ) were cut from each paraffin block and
placed in a sterile microcentrifuge tube. Paraffin was re-
moved by extraction with 1.2 ml of xylene. After centrifu-
gation at 13,000 ⫻ g for 5 minutes, residual xylene was
removed from the tissue pellet by twice extracting with 1.2
ml absolute ethanol. The sample was centrifuged (13,000 ⫻
g, 5 minutes), and ethanol was carefully removed. DNA for
PCR analysis was extracted from the tissue pellet using a
commercial DNA extraction kit (DNeasy娂 Tissue kit, Qia-
gen, Hilden, Germany).
PCR detection of mycobacterial DNA
DNA extracted from formalin-fixed and paraffin-embed-
ded tissue samples was screened for the presence of myco-
bacterial DNA using a broad-range PCR assay especially
developed for the detection of small DNA fragments in rou-
tine biopsies. Primers M65-Myco for (5⬘-GG(G/C/T)CT(G/
C)AA(A/G)CG(C/T)GGCATCG-3⬘) and M65-Myco rev (5⬘-
AGGCT(A/G)CCGAT(G/C)G(A/T)CTGGTC-3⬘) amplify a
137 bp fragment of the 65 kDa heat shock protein (hsp65)
gene.58 This gene, common to all mycobacteria, is more var-
iable than other target genes (e.g., 16S ribosomal RNA
(rRNA) gene) and is therefore adequate for the identification
of genetically related species.49 PCR reactions were per-
formed as described by Kipar et al.37
PCR detection of chlamydial DNA
A similar strategy was developed for the detection of chla-
mydial DNA. The primer pair of 23SAPF2 (5⬘-GAACCT-
GAAACCA(AG)TAGC-3⬘) and 23SAPR (5⬘-CTGGCTC-
ATCATGCAAAAGG-3⬘) was designed to amplify a small
92-bp fragment of the 23S rRNA gene to account for pos-
sible DNA degradation in the formalin-fixed tissue samples
while nevertheless amplifying diagnostically informative se-
quences for identification of members of the order Chla-
mydiales.
One and 10 ␮l of extracted DNA were added to a PCR
mixture containing 1⫻ PCR reaction buffer (2.5 mM MgCl2,
50 mM KCl and 10 mM Tris–HCl, pH 8.7; Qiagen), 200

→
Fig. 2. Small intestine; chelonian, case No. 12. Granulomatous enteritis. Fig. 2a. An accumulation of degranulated
heterophils can be seen within the submucosa. HE. Bar ⫽ 100 ␮m. Fig. 2b. Section adjacent to 2a showing large numbers
of acid-fast bacilli within the heterophilic infiltrate. ZN. Bar ⫽ 50 ␮m.
Fig. 3. Lung; snake, case No. 27. Granulomatous pneumonia. Fig 3a. Histiocytic granuloma within the lung parenchy-
ma. HE. Bar ⫽ 100 ␮m. Fig. 3b. Section adjacent to 3a: a positive staining can be seen. Chlamydial LPS IHC; AEC/
peroxidase method, hematoxylin counterstain. Bar ⫽ 100 ␮m.
Vet Pathol 41:4, 2004 Mycobacteria and Chlamydiae in Reptile Granulomas 391

Fig. 4. Lung; snake, case No. 3. Granulomatous pneumonia. Fig. 4a. The lung parenchyma is multifocally affected by
histiocytic granulomas. HE. Bar ⫽ 200 ␮m. Fig. 4b. Higher magnification of a granuloma revealing several acid-fast bacilli.
ZN. Bar ⫽ 20 ␮m.
Fig. 5. Pericardium; snake, case No. 3. Granulomatous pericarditis. Fig. 5a. Multiple granulomatous lesions displaying
a necrotic core surrounded by macrophages are present. HE. Bar ⫽ 100 ␮m. Fig. 5b. Section adjacent to 5a showing a
positive reaction both in the necrotic center and within macrophages. Chlamydial LPS IHC; AEC/peroxidase method,
hematoxylin counterstain. Bar ⫽ 50 ␮m.
392 Soldati, Lu, Vaughan, Polkinghorne, Zimmermann, Huder, and Pospischil
␮M of each deoxiribonucleotides (Roche, Basel, Switzer-
land), 1 ␮M of each primer (Microsynth, Balgach, Switzer-
land), 1⫻ Q-Solution (Qiagen), and 1 U HotStarTaq DNA-
Polymerase (Qiagen).
PCR cycling conditions consisted of an initial denatur-
ation step at 95 C for 15 minutes, followed by 45 cycles of
denaturation at 94 C for 30 seconds, primer annealing at 50
C for 30 seconds and extension at 72 C for 30 seconds, with
a final extension for 5 minutes at 72 C.
Negative controls were performed using a reaction mix-
ture with H2O substituting the template DNA. Template
DNA for positive controls was obtained from formalin-fixed,
paraffin-embedded tissues from gnotobiotics piglets that
were infected with C. suis strain S45.21
Sequencing
All PCR reactions were performed on a TGradient ther-
mocycler (Biometra GmbH, Göttingen, Germany). Ten mi-
croliters of each PCR product was electrophoresed in a 3.0%
TAE (40 mM Tris, 11.4% acetic acid, 1 mM ethylenedi-
aminetetraacetic acid) agarose gel (Agarose electrophoresis
grade, Gibco, Life Technologies, Carlsbad, CA) stained with
ethidium bromide and observed under UV illumination. The
desired fragments were excised, purified with the Qiagen’s
MinElute Gel Extraction Kit, and directly sequenced using
the dye terminator protocol with an automated sequencer
(Applied Biosystem 373A, Foster City, CA). The obtained
sequences were compared with the sequences available in
GenBank using the BLAST server from the National Center
for Biotechnology Information.1
Results
Histopathologic findings
In HE-stained histologic sections, heterophilic gran-
ulomas featuring masses of heterophilic granulocytes
partially infiltrated with macrophages were seen in 14
cases (Fig. 2a). Histiocytic granulomas were observed
in 17 cases, with lesions being characterized by ac-
cumulation of macrophages in various stages of acti-
vation and occasional giant cells (Figs. 3a, 4a). In 58
cases, however, granulomas consisted of a necrotic
center surrounded by macrophages, occasionally giant
cells, lymphocytes, plasma cells, and spindle-shaped
histiocytes, and were sometimes enclosed in fibrous
connective tissue. Because it was impossible to discern
whether macrophages or heterophils gave rise to the
necrotic area, they were classified as chronic granu-
lomas. Reptile No. 3 showed histiocytic granulomas
within the lung and a chronic granulomatous pericar-
ditis (Figs. 4a, 5a).
ZN and PCR detection of mycobacteria
In 14 cases (reptile Nos. 1–14), the ZN stain re-
vealed a variable number of acid-fast organisms in the
granulomatous lesions. They were present both intra-
cellularly within the cytoplasm of macrophages and
extracellularly in the necrotic centers (Figs. 2b, 4b).
Vet Pathol 41:4, 2004
Mycobacteria were found in the alimentary, respira-
tory, and genitourinary tracts, heart, spleen, peritoneal
surface, and central nervous system.
All 14 ZN-positive specimens and 12 cases without
demonstrable acid-fast bacilli (reptile Nos. 15–26)
yielded DNA fragments of the expected size by the
broad-range mycobacteria PCR.
In an attempt to further identify infecting mycobac-
terial strains, PCR products of hsp65 were directly se-
quenced. With the exception of three cases (reptile
Nos. 24–26), which were ZN negative and generated
sequences belonging to microorganisms other than
mycobacteria, the remaining 23 specimens showed
highest similarities with hsp65 sequences of MOTT, at
the amino acid level (44 amino acid residues). An un-
ambiguous classification at the species level was de-
termined in nine cases; sequence analysis of the re-
maining 14 specimens displayed 93–100% similarities
with corresponding sequences of several MOTT spe-
cies.
ZN stain, PCR, and sequencing results are summa-
rized in Table 1.
IHC and PCR detection of chlamydiae
Immunohistochemically, a positive staining for the
cLPS antigen was observed within granulomatous tis-
sue samples from lung, pericardium, kidney, and liver
derived from five reptiles (reptile Nos. 3, 19, 27–29).
Except for reptile No. 28, where in only a few cells
chlamydia could be detected, strong staining of chla-
mydial inclusions were observed, principally localized
within macrophages and in necrotic areas (Figs. 3b,
5b).
All immunohistochemically positive tissue samples
were later confirmed as positive using the Chlamydi-
ales order–specific PCR assay. Direct sequencing of
the 23S rRNA PCR products of all five cases revealed
sequences with 99% identity to Cp. pneumoniae. An
additional 53 immunohistochemically negative speci-
mens were found positive after PCR screening for
chlamydial DNA, and their identity was determined by
direct DNA sequencing. Four samples were found pos-
itive for Cp. pneumoniae (98–99% identical). Surpris-
ingly, comparative sequence analysis of the remaining
49 PCR-positive specimens showed high similarity
values (88–97%) with 23S rRNA sequences of mem-
bers of the newly described Chlamydia-like families
Parachlamydiaceae and Simkaniaceae. In particular,
on-line BLAST searches of GenBank for similar se-
quences revealed that these 49 isolates were 88–97%
similar with either Parachlamydia acanthamoebae or
Simkania negevensis. Because a definitive identifica-
tion was not possible, these isolates are referred to as
Chlamydia-like. Immunohistologic, PCR, and se-
quencing results are summarized in Tables 2, 3.
Vet Pathol 41:4, 2004 Mycobacteria and Chlamydiae in Reptile Granulomas 393

Table 1. Positive results of the mycobacterial screen by ZN stain, PCR, and sequence analysis.*
Mycobacterial Screening
Granuloma
Case No. Reptile Owner Type ZN PCR Sequencing
1 Snake Private Chronic ⫹ ⫹ MOTT
2, 3 Snake Private Histiocytic ⫹ ⫹ MOTT
4 Snake Private Chronic ⫹ ⫹ 97% M. confluentis
5, 6, 7 Snake Zoo Chronic ⫹ ⫹ MOTT
8 Snake Zoo Histiocytic ⫹ ⫹ 100% M. chelonae
9, 10 Chelonian Private Chronic ⫹ ⫹ MOTT
11 Chelonian School Histiocytic ⫹ ⫹ 100% M. haemophilum
12 Chelonian Shop Heterophilic ⫹ ⫹ MOTT
13, 14 Lizard Zoo Histiocytic ⫹ ⫹ MOTT
15 Snake Private Chronic ⫺ ⫹ 100% M. haemophilum
16 Snake Private Histiocytic ⫺ ⫹ 95% M. hiberniae
17 Snake Private Chronic ⫺ ⫹ MOTT
18 Snake Private Chronic ⫺ ⫹ 100% M. neoarum
19 Snake Private Histiocytic ⫺ ⫹ 100% M. confluentis
20 Snake Zoo Chronic ⫺ ⫹ MOTT
21 Chelonian Private Chronic ⫺ ⫹ MOTT
22 Chelonian Private Chronic ⫺ ⫹ 100% M. nonchromogenicum
23 Lizard Private Histiocytic ⫺ ⫹ 97% M. agri
* MOTT: Mycobacteria other than Mycobacterium tuberculosis compex.

Mixed and dual infections
In two cases (reptile Nos. 1 and 19), both Cp. pneu-
moniae and MOTT were detected either by staining
methods and PCR or by PCR alone. Reptile No. 3,
however, presented a dual infection: the granulomatous
pneumonia was caused by MOTT, whereas Cp. pneu-
moniae induced the pericardial granulomatous lesions
(Table 4, Figs. 4, 5).
In a total of 27 cases (reptile Nos. 25, 65–90) neither
mycobacteria nor chlamydiae could be detected.
Discussion
In this study, we investigated the presence of my-
cobacteria and chlamydiae in granulomatous inflam-
mation of reptiles. For the detection of these two in-
tracellular microorganisms a similar methodology was
developed and applied: first a staining procedure (ZN
and IHC, respectively) followed by broad-range PCR
assays and gene sequencing. This proved to be suc-
cessful.
The histologic classification of granulomatous in-
flammation was rather unproductive because in only
31 cases a clear distinction between heterophilic and
histiocytic granulomas was possible and represented
only the first step for etiologic diagnosis. Moreover,
the detection of acid-fast bacilli within heterophilic
granulomas (reptile No. 12) demonstrates that group-
ing these etiologic agents (i.e., extracellular and intra-
cellular bacteria with heterophilic and histiocytic gran-
ulomas, respectively) by lesion type is not always cor-
rect. In contrast, chronic granulomas involve the wide
spectrum of etiologies. In both cases, the necessity of
specific investigations is clearly required.
The microscopic identification of acid-fast bacilli
and chlamydiae by ZN and IHC, respectively, was less
sensitive than PCR, because it requires a large number

Table 2. Positive results of the chlamydial screen by cLPS IHC, PCR, and sequence analysis related to Chlamydophila
pneumoniae.
Chlamydial Screening
Granuloma
Case No. Reptile Owner Type IHC PCR Sequencing
3, 19, 27 Snake Private Histiocytic ⫹ ⫹ 99% Cp. pneumoniae
28 Snake Private Chronic ⫹ ⫹ 99% Cp. pneumoniae
29 Chelonian Private Chronic ⫹ ⫹ 99% Cp. pneumoniae
1, 31 Snake Private Chronic ⫺ ⫹ 99% Cp. pneumoniae
30 Snake Private Histiocytic ⫺ ⫹ 99% Cp. pneumoniae
32 Chelonian Private Chronic ⫺ ⫹ 98% Cp. pneumoniae
394 Soldati, Lu, Vaughan, Polkinghorne, Zimmermann, Huder, and Pospischil Vet Pathol 41:4, 2004

Table 3. Positive results of the chlamydial screen by cLPS IHC, PCR, and sequence analysis related to ‘‘Chlamydia-
like’’ isolates.
Chlamydial Screening
Case No. Reptile Owner IHC PCR Sequencing
11 Chelonian School ⫺ ⫹ ‘‘Chlamydia-like’’ isolate
12 Chelonian Shop ⫺ ⫹ ‘‘Chlamydia-like’’ isolate
14 Lizard Zoo ⫺ ⫹ ‘‘Chlamydia-like’’ isolate
23 Lizard Private ⫺ ⫹ ‘‘Chlamydia-like’’ isolate
2, 15–18, 24, 33–43 Snake Private ⫺ ⫹ ‘‘Chlamydia-like’’ isolate
5, 7, 8, 20, 44–48 Snake Zoo ⫺ ⫹ ‘‘Chlamydia-like’’ isolate
9, 22, 49–57 Chelonian Private ⫺ ⫹ ‘‘Chlamydia-like’’ isolate
58 Chelonian Zoo ⫺ ⫹ ‘‘Chlamydia-like’’ isolate
59–62 Lizard Private ⫺ ⫹ ‘‘Chlamydia-like’’ isolate
26, 63, 64 Lizard Zoo ⫺ ⫹ ‘‘Chlamydia-like’’ isolate

of organisms in the sample.5 In addition, a nonhomo-
geneous distribution of microorganisms within the
granulomatous inflammation must be taken into ac-
count.12,41 Regarding the chlamydial IHC, a third rea-
son could be a possible reduction in steady-state levels
of outer membrane constituents, including LPS in per-
sistent chlamydial infections.40 In contrast, the non-
detection of the 49 Chlamydia-like cases was an ex-
pected result because Chlamydia-like organisms ap-
pear not to be recognized by monoclonal Abs specific
for the LPS of the Chlamydiaceae.15
Although formalin-fixed and paraffin-embedded tis-
sues represent an invaluable resource for research, a
major problem is the physical and chemical alteration
of DNA during tissue processing, which limits the
length of PCR products that can be amplified.7 There-
fore, for a study aiming to maximally detect the pres-
ence of microorganisms, we suggest the use or the
development of PCR assays that amplify relatively
short DNA fragments.
The disadvantages of such a strategy for the my-
cobacteria were the difficulty in clearly distinguishing
between members of the MOTT and the amplification
of DNA belonging to organisms other than mycobac-
teria. For the chlamydiae, the limited length of the am-
plified 23S rRNA gene fragment did not allow com-
parative studies and phylogenetic analysis with corre-
sponding Cp. pneumoniae sequences detected in hu-
man, horse, koala, amphibian, and reptile hosts.
Within these limits, we could demonstrate the pres-
ence of MOTT in a total of 23 out of 90 reptiles
(25.56%). Therefore, although considered an uncom-
mon and sporadic disease of reptiles by some authors,
they represent probably the most important infectious
etiology for granulomatous inflammation.10,24,44
All 90 reptiles investigated involved either zoologic
collections or private owners. These captive reptiles
are frequently mismanaged and maintained in subop-
timal environmental conditions, circumstances that
may repress the immune system and favor the invasion
of opportunistic pathogens. MOTT normally live in
soil and aquatic habitats, and most of them are sap-
rophytes. However, some species are opportunistic
pathogens and can infect human and animal hosts with
underlying immunodeficiencies.29 Contaminated soil,
water, and food are the most likely sources of infection
for immunosuppressed reptiles, and infected reptiles
themselves could represent an additional reservoir for
humans.
The results of the chlamydial screen can be subdi-
vided into two parts: the detection of Chlamydia-re-
lated 23S rRNA sequences in 49 reptile samples and
the 9 cases of Cp. pneumoniae infection. The high
prevalence of Chlamydia-like organisms found in rep-
tile samples most likely reflects opportunistic infection
by an environmental pathogen.
Chlamydia-like organisms are obligate intracellular
bacteria with developmental stages similar to RBs and

Table 4. Dual and mixed infections.*

Chlamydial Screening Mycobacterial Screening
Granuloma
Case No. in/Type IHC Chlam-PCR ZN Myco-PCR
1 Lung/chronic ⫺ 99% Cp. pneumoniae ⫹ MOTT
19 Lung/histiocytic ⫹ 99% Cp. pneumoniae ⫺ 100% M. confluentis
3 Lung/histiocytic ⫺ — ⫹ MOTT
3 Pericardium/chronic ⫹ 99% Cp. pneumoniae ⫺ —
* MOTT: Mycobacteria other than Mycobacterium tuberculosis complex.
Vet Pathol 41:4, 2004 Mycobacteria and Chlamydiae in Reptile Granulomas
EBs of the known Chlamydiaceae, but they are mor-
phologically and antigenetically different.34–36 The or-
ganisms are distributed widely across diverse environ-
ments.13,26 Ossewaarde and Maijer detected more than
100 Chlamydia-like sequences in human, animal, and
environmental specimens, including distilled water,
phosphate-buffered saline, DNA isolation, and PCR
controls (results presented at the International Chla-
mydia Conference at Antalya, Turkey in June 2002).
Although the first described Chlamydia-like organ-
isms, S. negevensis and P. acanthamoebae, were con-
sidered environmental contaminants, serologic studies
and the molecular detection of Chlamydia-like rRNA
gene sequences in clinical samples suggest their pos-
sible association with respiratory tract diseases and
even arteriosclerosis.2,14,20,35,42 Whether Chlamydia-like
organisms are involved in the granuloma formation of
reptiles remains unknown. The use of a specific stain-
ing procedure would be of great value to determine
their localization within tissue specimens.17 Neverthe-
less, further studies are needed to clarify their potential
role as newly emerging pathogens.
Apart from these environmental Chlamydia-like or-
ganisms, the primary aim of this study was the detec-
tion of pathogenic Chlamydia species in granuloma-
tous inflammations of reptiles. From a veterinary pa-
thology perspective, the nine cases of Cp. pneumoniae
clearly demonstrate that chlamydial infection occurs in
reptiles and its prevalence may be greater than indi-
cated in the literature. In addition, the immunohisto-
chemical association of cLPS antigen to specific le-
sions such as granulomas confirms the pathogenicity
of Cp. pneumoniae for reptile hosts. This is a signifi-
cant finding for our understanding of reptiles diseases
and for the extension of the differential diagnosis of
granulomatous inflammation to include also Cp. pneu-
moniae. The source of infection remains unknown.
Chlamydiosis in reptiles and amphibians was frequent-
ly presumed to be caused by C. psittaci and conse-
quently a transmission from feral birds was pro-
posed.25,32,33 The application of PCR and gene sequenc-
ing to detect and identify the chlamydial species in-
fecting poikilothermic animals demonstrated the
presence of Cp. pneumoniae, a result that raises inter-
esting questions regarding their origin.8 There could
exist either an independent feral reservoir or a separate
biovar of Cp. pneumoniae; it is also possible that hu-
mans could be the reservoir of infection. An additional
interesting finding was our detection of both MOTT
and Cp. pneumoniae within the same reptile and in
two cases within the same tissue, indicating the pres-
ence of dual and mixed infections. We believe that the
pericardial granuloma in reptile No. 3, induced by Cp.
pneumoniae, was older than those observed in the
lung, suggesting that the chlamydial infection was pri-
395
mary, lowering the snake’s defense mechanisms and
facilitating the mycobacterial infection. In contrast, in
reptile Nos. 1 and 19, it was difficult do predict the
progression of infection.
The presence of Cp. pneumoniae isolates in reptiles
is also noteworthy from a purely microbiologic view.
Originally, the host range of Cp. pneumoniae was be-
lieved to be limited to humans.38 Our results, along
with the detection of Cp. pneumoniae in horse, koalas,
amphibians, and other reptiles, provide convincing ev-
idence for a wide host range.6,8,9,28,47,51,56
The presence of Cp. pneumoniae strains in reptiles
is important from a human perspective because Cp.
pneumoniae is a common cause of pneumonia and
bronchitis throughout the world and has been recently
associated with several chronic diseases, including cor-
onary heart disease, Alzheimer’s disease, and multiple
sclerosis.23,38,57 In our study and other studies the ani-
mal isolates of Cp. pneumoniae revealed few differ-
ences at the DNA level compared with the human
biovar.6,28,47,51,56 Although transmission between hu-
mans is believed to occur from person to person
through respiratory secretions,39 until more is known
about the epidemiology, pathogenicity, and transmis-
sion of the Cp. pneumoniae strains recovered from
captive and wild populations of cold-blooded animals,
their zoonotic potential must be considered. Moreover,
because all nine reptiles infected with Cp. pneumoniae
belonged to private owners and because of the increas-
ing ownership of reptiles as pets, caution must be ex-
ercised in the direct handling of these animals, espe-
cially by immunecompromised individuals.
Acknowledgements
We are grateful to the laboratory technical staff of the
Institute of Veterinary Pathology, University of Zurich, for
technical assistance. This work represents part of the require-
ments to obtain the degree of Dr. Med. Vet. from the Vet-
erinary Faculty of the University of Zurich (G. Soldati).
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