European Association of Zoo- and Wildlife Veterinarians (EAZWV)

th
6 scientific meeting, May 24 - 28 - 2006. Budapest, Hungary

CHLAMYDOPHILA INFECTION IN DIFFERENT SPECIES OF REPTILES

M.J.L. KIK

AFFILIATION:
Department of Pathobiology, Division Pathology, Faculty of Veterinary Medicine
Utrecht University, Yalelaan 1, 3584 CL Utrecht, The Netherlands;

Abstract
13 different species of reptiles were necropsied; tissues were collected from all major organs,
processed for light microscopy, modified Machiavelli’s (Stamp) stain, and direct immuno-
fluorescence assay for chlamydia. Histologic examination demonstrated histiocytic granulomas
in the esophagus, the liver, the pancreas, and the lungs of the different animals. The direct
immunofluorescence assay was positive. From cloacal swabs of living contact animals and a
number of sick snakes the immunofluorescence assay was also positive.

Introduction
The order of Chlamydiales consists of the families Chlamydiaceae, Parachlamydiaceae,
Simkaniaceae, and Waddliaceae. The family Chlamydiaceae is divided in the genera
Chlamydophila and Chlamydia. The genus Chlamydophila comprises Chlamydophila psittaci,
Chlamydophila caviae, Chlamydophila pecorum, Chlamydophila felis, Chlamydophila abortus,
and Chlamydophila pneumoniae. The genus Chlamydia assimilates Chlamydia trachomatis,
Chlamydia muridarium, and Chlamydia suis (Everett et al., 1999). “Chlamydia and chlamydial
infections” in this paper refer to members of the family Chlamydiaceae.
The Chlamydia are obligate intracellular bacteria, and have a characteristic biphasic lifecycle in
the host.
During the last years knowledge about the host range has considerably extended. Chlamydial
infections have been described in amphibian, in numerous avian and mammal species, including
humans (Bodetti et al., 2002, Kik et al., 1997; Reed et al., 2000; Schachter, 1999). In general,
the infection can lead to a variety of clinical signs, ranging from localized conjunctivitis to
systemic disease. Relatively few chlamydial infections in reptiles have been reported (Homer et
al., 1994; Jacobson et al., 2002, 2004; Lock et al., 2003; Mohan et al., 2005; Soldati et al. 2004).
Different types of lesions are described. Granulomatous inflammation in a flap-necked
chameleon (Chameleo dilepis), necrotizing enteritis in green iguanas (Iguana iguana),
necrotizing myocarditis in green turtles (Chelonia mydas), histiocytic granulomas in different
tissues and lymphocytic and plasmacytic lesions throughout the gastrointestinal tract in emerald
tree boa (Corallus caninus) (Jacobson et al., 2004). Different species of chlamydiae are
described e.g. C. pneumoniae, C. felis, C. abortus, C. felis, and C. psittaci.
Apart from a few well documented cases, very often the detection of the presence of chlamydia
or chlamydia-like agents seems to be coincidental (Hotzel et al. 2005). However, the general
prevalence and pathogenic importance is not known yet. In this study we investigated tissues of
reptiles offered for autopsy at our department for the presence of Chlamydophila. Next to this
cloacal swabs from a number of live reptiles were examined with a direct immunofluorescence
assay for Chlamydia antigen.
Materials and methods
13 reptiles, from different species from private owners and one zoo, were offered for necropsy.
The necropsies were performed using a standard protocol. Tissue samples were fixed in 4%
phosphate-buffered formalin, embedded in paraffin, cut at 4 µm sections and stained with
hematoxylin and eosin (H&E). Duplicate impression smears from liver, spleen, lung and
intestinal content were stained with the Stamp stain (Machiavelli’s technique); frozen in acetone,
and subjected to a direct immunofluorescence assay (IFA) with fluorescence-isothiocyanate
conjugated anti-chlamydial monoclonal antibodies (ImagenTM Chlamydia, Dako Cytomation, UK).
Based on the positive IFA from an animal, living contact animals were screened for the presence
and shedding of Chlamydophila. A bearded dragon (Pogona vitticeps) and 2 tree boas (Corallus
hortulana) were presented with respiratory distress and nasal discharge. Cloacal swabs were
investigated with the IFA.
Transmission electron microscopy, following standard techniques, was done on liver tissue
samples from 1 corn snake. From a limited number of IFA positive snakes, culture of the
chlamydophila and subsequent PCR was performed using standard procedures. (Tempelmans
Plat et al., 2000; Mohan et al., 2005)
Bacteriological examination was performed on liver and intestinal contents using standard
techniques.

Results
The results of the pathologic examination of the different species of reptiles that tested positive
in the IFA are shown and summarized in table 1. On gross pathology all the snakes were in
moderate to bad body condition based on fat reserves. The different lizards were mostly in a
good body condition. From the turtles no specific remarks were made concerning the body
condition. In all animals more or less pathological changes of the liver were seen. 5 of the
animals showed pathological changes in the lung(s) consistent with pneumonia. A variety of
intestinal nematodes were seen. Different bacteria species, a.o. Salmonella spp. and
Pseudomonas spp. were cultured from the gut. The Stamp stain was positive in variable organs
of 7 of the animals. The IFA was positive in all animals Fig. 1); but not in all organs chlamydia
could be detected.

Figure 1. Lung of a skink (Tiliqua rugosa).

Note the intracytoplasmic inclusions (arrow)
characteristic of chlamydophila spp.
Immunofluorescence stain for chlamydia x
200.
Table 1. Patho-morphologic lesions, Stamp stain and IFA on chlamydia, from the different
necropsied reptile species.
Species Gross pathology Histopathological Stamp stain IFA Chlamydia
conclusion
Garter snake Moderate body Histiocytic Positive lung. Positive liver,
(Thamnophis condition. granulomas lung, spleen,
sp.) Exsudative pancreas, gut.
pneumonia, enteritis. necrotizing colitis,
Lungworms. pneumonia.
Corn snake Bad body condition. Non-suppurative Positive liver, Positive liver,
(Pantherophis Enlarged liver, gastro-enteritis. lung, spleen, lung, spleen,
guttatus) pancreas, pneumonia. Nefritis. gut. gut.
Pancreatitis. Positive in EM.
Ball python Moderate body Pyo- Positive liver, Positive liver,
(Python regius) condition, esophagitis, granulomatous lung, spleen, spleen, lung,
pale liver, colitis. esophagitis, gut. gut.
gastritis.
East Indian box Autolytic. Autolytic. Positive liver, Positive liver,
turtle (Cuora lung, spleen, spleen, gut.
amboniensis) gut.
Spiny turtle Endocarditis, gastritis, Necrotizing Negative. Positive liver,
(Heosemys enteritis, liver gastroenteritis. spleen, lung,
spinosa) necrosis. gut.
Anglehead lizard Dystocia, parathyroid Parathyroid Positive liver. Positive liver.
(Gonocephalus hyperplasia. Hyperplasia.
belli)
Mediterranean Pneumonia, Histiocytic Negative. Positive lung.
spur-thighed granulomatous granulomatous
tortoise (Testudo hepatitis, hepatitis,
graeca graeca) hemorrhages pneumonia.
Beals’s eyed Good body condition. Necro-purulent Negative. Positive
turtle Ascites, Liver inflammation in spleen, lung.
(Sacalia bealei) abscess, swollen liver. Metastatic
kidneys. calcification all
organs.
Corn snake Moderate body Esophagitis, Positive gut. Positive lung,
(Panterophis condition. pancreatitis, gut.
guttatus) Pneumonia, irregular pneumonia,
liver and kidneys. nephritis.
Mangrove Rat Bad body condition. Hepatitis, enteritis, Negative. Positive gut.
snake Nodules in liver. nephritis.
(Gonyosoma Inflammation coelomic
oxycephala) cavity.
Green iguana Good body condition Nephrosis. Negative. Positive gut.
(Iguana iguana) Hepatomegaly.
Preovulatory follicle
stasis.
Mangrove Bad body condition. Liver necrosis. Negative. Positive gut.
ratsnake Hepatomegaly. Gastritis.
(Gonyosoma Enteritis, ameba. Necrotizing
oxycephala) enteritis
Centralian blue- Good body condition. Tracheitis, Positive liver, Positive liver,
tongued lizard Pale heart, enlarged pneumonia, lung, spleen, spleen, lung,
(Tiliqua pale liver, pneumonia. gastroenteritis, gut. gut.
multifasciata) splenitis, nefritis.
Table 2 shows the results of the screening of the clinically healthy contact animals and the other
sick animals. From 30 garter snakes, 20 animals were positive in the IFA. All 9 tested blue-
tongued skinks were positive. 2 contact animals from one dead corn snake were both positive.
From the other collection of snakes, 3 out of 5 contact corn snakes were positive. The bearded
dragon and the tree boas all tested positive.

Table 2. Results of the immunofluorescence assay of different (contact) animals and sick
animals. N = number of animals tested.
Species N Cloacal swab IFA
Garter snake (Thamnophis sp. 30 Positive 20
Shingle back lizard (Tiliqua 2 Positive 2
rugosa)
Western blue-tongued lizard 3 Positive 3
(Tiliqua occipitalis)
Blotched blue-tongued lizard 4 Positive 4
(Tiliqua nigrolutea)
Corn snake (Pantherophis 2 Positive 2
guttatus)
Corn snake( Panterophis 5 Positive 3
guttatus) sick animal
Tree boa (Corallus hortulana) 2 Positive 2
sick animal
Bearded dragon (Pogona 1 Positive 1
vitticeps) sick animal

In the TEM investigation, intracytoplasmic inclusions, consistent with chlamydial particles were
detected in the liver of one of the corn snakes. After treatment with doxycyclin (VibravetR , Pfizer,
Capelle a/d IJssel, the Netherlands, 10 mg per kg bodymass during 10 days) of the skinks, the
corn snakes, the bearded dragon, and the tree boas, the animals tested negative in the IFA. The
culture and PCR of the Chlamydophila isolated from a garter snake and a corn snake revealed
Chlamydophila closely related to C. psittaci.

Discussion
In this present limited study, most of the involved snakes showed a moderate to bad body
condition. The body condition of the different chelonians was not accessed. The lizards all
proved to be in a very good body condition. In all the animals the liver was more or less involved
in the disease process. Furthermore, tracheitis, esophagitis, gastro-enteritis could be detected.
The contact animals showed different results in the IFA on cloacal swabs. This doesn’t mean
that they were really negative for chlamydia, but may not be shedding the chlamydia at the time
of the investigation.The development of systemic disease depends on the virulence of the
Chlamydophila strain, the species of animal, and the immune status of the animal. Given the fact
that in other animals (birds and mammals) chlamydia may be very pathogenic, part of the
disease of the reptiles may be explained by the presence of the chlamydia. The snakes bad to
moderate body condition may be attributed to a chronic disease, partly induced by the
chlamydia. In the lizards acute septicemia due to chlamydia is likely. Maybe due to diminished
resistance based on dystocia and pre ovulatory follicle stasis, present chlamydia may have
evolved in systemic disease. But in the skink no other signs of disease were monitored. All the
contact animals turned out positive in the IFA. Meaning that chlamydia were spread within this
collection of skinks. The diagnosis chlamydiosis is generally made by histological investigation of
affected tissues and electron microscopy. In our laboratory additional stains, Stamp and IFA are
routinely applied in the necropsy of psittacine bird, reptiles and amphibians. But in our cases,
only a small number of animals showed typical histiocytic granulomas, whereas the other cases
showed nonspecific lesions. Not all stamp stains were positive. Therefore other methods of
discerning chlamydia in tissues; the IFA and immunoperoxidase staining, electron microscopy,
in-situ hybridization, and PCR amplification of the 23S rRNA gene might be more appropriate
tools (Jacobson et al., 2004). The zoonotic capacity of reptile strains of chlamydia is not yet
known, but keeping in mind the pathogenic potential of chlamydia in other hosts, efficient
diagnostic tools should be applied for diagnosing chlamydial infection in reptiles.

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Msc thesis, university of utrecht, the netherlands.

Acknowledgement
Thanks to Mr. R. Kisjes, Department of Pathobiology, Division Pathology and Mr. A de Graaf,
Centre for Cell Imaging, Department of Biochemistry and Cell Biology, Faculty of Veterinary
medicine, Utrecht University, for assistance in preparing of the photograph.

mail@kikdierenarts.nl