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Veterinary Microbiology 103 (2004) 121–126

Short communication

Occurrence of Chlamydiaceae spp. in a wild boar (Sus scrofa L.)

population in Thuringia (Germany)

Helmut Hotzela, Angela Berndtb, Falk Melzera, Konrad Sachsea,*

aInstitute of Bacterial Infections and Zoonoses, Friedrich-Loeffler-Institut,

Naumburger Straße 96a, 07743 Jena, GermanybInstitute of Molecular Pathogenesis, Friedrich-Loeffler-Institut,

Naumburger Straße 96a, 07743 Jena, Germany

Received 16 January 2004; received in revised form 18 May 2004; accepted 7 June 2004

Abstract

Tissue samples from lungs, pulmonary lymph nodes, large intestine, and uteri of 14 wild boar bagged at a seasonal hunt were

examined for the presence of chlamydiae, mycobacteria and mycoplasmas. Nested PCR detected chlamydial DNA in 57.1% of

the animals, predominantly in the lung. DNA sequencing identified Chlamydophila psittaci as the predominant species, but

Chlamydophila abortus and Chlamydia suis were also encountered. Immunohistochemical staining of tissue sections confirmed

the presence of typical chlamydial inclusions in lungs and uteri. While the role of Chlamydiaceae as pathogens in wild boar has

yet to be established, the present findings revealed a possible wildlife reservoir of these bacteria.

# 2004 Elsevier B.V. All rights reserved.

Keywords: Wild boar; Chlamydia; Chlamydophila; PCR; DNA sequencing; Immunohistochemistry

1. Introduction

While the role of chlamydiae as pathogens in

humans, birds and domestic animals is well estab-

lished (Storz and Kaltenbock, 1993), there are only

very few sporadic data on the situation in wild mam-

mals, most of which are more than 15 years old and

based exclusively on serological findings (Schmatz et

al., 1977; Rehacek et al., 1985; Giovannini et al.,

* Corresponding author. Tel.: +49 3641 804 334;

fax: +49 3641 804 228.

E-mail address: k.sachse@jena.bfav.de (K. Sachse).

0378-1135/$ – see front matter # 2004 Elsevier B.V. All rights reserved

doi:10.1016/j.vetmic.2004.06.009

1988). This scarcity of data is at least partly due to

the general problems of chlamydial diagnosis result-

ing from difficulties in culturing these obligate intra-

cellular bacteria. Serological assays usually lack the

specificity to identify the chlamydial species involved.

The introduction of PCR detection methods in the

1990s, as well as the refinement of immunohistochem-

ical techniques, have significantly improved the pos-

sibilities to diagnose chlamydial infections.

Wild boar and other wildlife animals have been

suggested to represent reservoirs for brucellae (Gibbs,

1997), mycobacteria (Bollo et al., 2000; Machackova

et al., 2003), classical swine fever (Artois et al., 2002),

.

H. Hotzel et al. / Veterinary Microbiology 103 (2004) 121–126122

trichinellae (van der Giessen et al., 2001) and other

microbial pathogens, which may be transmitted to

domestic animals upon contact. In the case of chla-

mydiae, any evidence of their occurrence in wild boar

populations may help to assess potential risks, not

least because enzootic abortion in sheep (Longbot-

tom and Coulter, 2003), mastitis, abortion or arthritis

in cattle (Perez-Martinez and Storz, 1985) and

pigs (Wittenbrink et al., 1991; Busch et al., 2000;

Vanrompay et al., 2004) represent economically

important diseases. Additionally, the zoonotic poten-

tial of species like Chlamydophila (Cp.) psittaci

and Cp. abortus has to be considered. Hunters are

thought to be at risk (Deutz et al., 2003), and so could

be other persons handling carcasses and raw game

meat.

In the present study, tissue samples of lungs, lymph

nodes, intestine, and uteri from wild boar bagged at a

seasonal hunt were examined for the presence of

Chlamydiaceae by nested PCR and immunohistology.

2. Materials and methods

2.1. Origin of samples

The hunt took place in a forest area of eastern

Thuringia (Germany) in December 2002. To facilitate

this study, the hunters brought all bagged animals to a

central sample collection point, where specimens of

lungs, pulmonary lymph nodes, large intestine, and

uterus (from females) were taken and put into plastic

bags. A total of 46 organ tissue samples from 14 wild

boar were examined, among them 7 adult males, 6

adult females and 1 young animal aged less than a year

of unknown gender. The carcasses as well as the

organs that were sampled were examined for gross

pathology. The materials were stored at �20 8C until

laboratory examination.

Table 1

PCR primers for nested amplification

Primer designation

191CHOMP

CHOMP371

201CHOMP

CHOMP336s

2.2. PCR examination of tissue samples

DNAwas extracted from tissue using the High Pure

PCR Template Preparation Kit (Roche Diagnostics,

Mannheim, Germany) according to the instructions of

the manufacturer. The nested PCR assay using primer

pairs 191CHOMP/CHOMP371 and 201CHOMP/

CHOMP336s (Table 1), which amplifies a 437 to

455-bp segment in variable domains III and IV of

the ompA gene of all species of the genera Chlamydia

and Chlamydophila, was described previously (Sachse

and Hotzel, 2002). Amplification cycles were run

according to the following temperature-time profile:

denaturation at 95 8C for 30 s, primer annealing at

60 8C for 30 s, and primer extension at 72 8C for 30 s,

with 35 cycles in the first and 20 cycles in the second

round. All DNA extracts were also PCR tested for

mycoplasmas (Hotzel et al., 2002) and mycobacteria

(Kirschner and Bottger, 1998). PCR products were

separated by 1% agarose gel electrophoresis and

visualised by ethidium bromide staining under UV

light.

2.3. DNA sequencing

For nucleotide sequencing, positive bands were cut

out of the gel and DNA extracted using the QIAquick

Gel Extraction Kit (QIAGEN, Hilden, Germany).

These extracts were subjected to cycle sequencing

using the BigDyeTM Terminator Cycle Sequencing

Ready Reaction Kit (Applied Biosystems, Darmstadt,

Germany) and processed by the ABI PRISM 310

Genetic Analyzer (Applied Biosystems). The species

identity based on the partial ompA sequence was

established through BLAST search (http://

www.ncbi.nlm.nih.gov/blast/). Sequence alignments

and analysis were conducted using the Vector NTI

Suite 8.0 software package (Informax Inc., Oxford,

UK).

Nucleotide sequence (50–30)

GCI YTI TGG GAR TGY GGI TGY GCI AC

TTA GAA ICK GAA TTG IGC RTT IAY GTG IGC IGC

GGI GCW GMI TTC CAA TAY GCI CAR TC

CCR CAA GMT TTT CTR GAY TTC AWY TTG TTR AT

H. Hotzel et al. / Veterinary Microbiology 103 (2004) 121–126 123

2.4. Immunohistochemistry

Samples from two PCR-positive animals were

examined by immunohistochemistry to determine

the localisation of chlamydial bodies in tissue. Cryo-

stat sections of lungs and uteri of 7 mm thickness were

prepared, and detection of Chlamydiaceae was done

using a primary monoclonal antibody against chlamy-

dial LPS (Chemicon, Hofheim, Germany) and a com-

mercially available staining kit (APAAP, ChemMate

Detection Kit, alkaline phosphatase/red, rabbit/mouse,

DakoCytomation, Hamburg, Germany). For negative

control, slides were incubated with pre-immune

mouse serum (dilution 1:500) instead of the anti-

Chlamydia antibody. Sections were counterstained

with haematoxylin and mounted with Canada balsam

(Riedel de Haen AG, Seelze-Hannover, Germany).

3. Results

The results of PCR examination and species identi-

fication by nucleotide sequencing are presented in

Table 2. Chlamydial DNAwas detected in eight animals

(57.1%), i.e. five females (rate of 83.3% among females)

and three males (rate of 42.9%). Sequencing of the

amplified ompA segments from organ tissue samples

revealed three different chlamydial species: Cp. psittaci

(10 positive samples/4 animals), Cp. abortus (4/2), and

Chlamydia (C.) suis (3/2). Among all organs, the lung

was most frequently found to be infected, i.e. in seven of

the eight chlamydia-positive animals. In three females,

the uterus samples (including placenta in one case)

tested positive. Other affected organs included pulmon-

Table 2

Chlamydiaceae species identified by nested PCR and DNA sequencing in

Boars Gilts an

Animal designation L LN I S Animal

1 CPa – CPa nd 2

5 – CABd – nd 3

6 – nd – nd 4

7 – – – - 9

8 – nd – nd 12

10 – – – nd 13

14 CPb – – nd

11

L = lung; LN = pulmonary lymph node; I = intestine; S = spleen; U = uterus;

= not determined. GenBank accession numbers of sequences: aAY601753

ary lymph nodes with five positives and large intestine

with two positives.

Immunohistochemical examination of samples

from PCR-positive animals 2 and 3 revealed the

presence of chlamydial inclusions in uteri and lungs.

In uterus samples, chlamydial cells were found exclu-

sively in myometrium. Typically, a few positively

stained spots, up to four spots per histological section,

appeared to be distributed throughout the whole myo-

metrium. Higher magnification revealed that the spots

represented clusters of a small number of infected

smooth muscle cells, or isolated single cells in some

instances (Fig. 1A). In lung samples, chlamydial

infection was seen in smooth muscle cells, alveolar

walls, interstitium and endothelium. Infected smooth

muscle cells located around some bronchioli were

most conspicuous and showed characteristic dot-

shaped staining (Fig. 1B). In a few instances of

alveolar walls being infected, destruction of alveolar

wall cells was additionally noticed (Fig. 1C). In

contrast, positively stained cells in interstitium and

endometrium were rarely encountered and staining

intensity appeared rather low.

The carcasses as well as the organs that were

accessible for sampling did not show macroscopical

signs of pathological changes.

No mycobacteria and mycoplasmas could be

detected by culture and PCR testing of the samples.

4. Discussion

Interestingly, the spectrum of Chlamydiaceae spe-

cies detected in the present panel of specimens is

Sus scrofa L.

d sows

designation L LN I U P

CPa CPa – CPa nd

CPa – – CPa nd

CSe CABc – CABc CABc

CSf CSf – – nd

– nd nd – nd

CPa CPa – – nd

Pig (sex not determined)

– – – nd nd

P = placenta; CP = Cp. psittaci; CAB = Cp. abortus; CS = C. suis; nd

, bAY601750, cAY601754, dAY601755, eAY601752, fAY601751.

H. Hotzel et al. / Veterinary Microbiology 103 (2004) 121–126124

Fig. 1. Immunohistochemical staining of wild boar tissue

samples infected with chlamydiae. Chlamydial cells appear in

red colour, host cell nuclei in blue. (A) Uterus section from animal

3. The arrow indicates an infected smooth muscle cell of the

myometrium. (B) Lung section from animal 3 showing an infected

bronchiolus. Single arrow: infected smooth muscle cells; arrowhead:

bronchial epithelium; double arrow: bronchial lumen filled with

leukocytes. (C) Lung section from animal 3 showing an infected

alveolar wall. The arrow indicates the position of infected cells of

the alveolar wall.

identical to that in domestic pigs. Nevertheless, the

possibility that these boar acquired chlamydiae

through contact with domestic pigs appears very

unlikely in view of animal husbandry practices in

the region as free-range keeping systems are very rare.

There is no sufficient evidence to define each of

the three species as a pathogen or commensal, respec-

tively, in wild boar. Recent investigations in

slaughtered domestic pigs indicated that Cp. abortus

could possibly be associated with abortion (Schiller

et al., 1997; Thoma et al., 1997), and C. suis

was demonstrated to be capable of causing acute

pneumonia in experimental infection (Rogers et

al., 1996; Sachse et al., 2004), as well as conjuncti-

vitis (Rogers and Andersen, 1999) and intestinal

lesions (Rogers and Andersen, 2000). In the case

of Cp. psittaci, the agent is widespread among wild

birds (Kaleta, 2002; Taday, 1998), thus indicating a

potential route of transmission. Again, there is no

evidence on the role of this newly defined species

(Everett et al., 1999) as a pathogen in mammals, even

though mammalian isolates of Cp. psittaci seem to be

genetically highly homologous, if not identical, to

avian strains representing the causative agent of

psittacosis.

The low detection rate in samples from the large

intestine (2 positives out of 13) does not suggest the

intestinal tract functioning as a reservoir for chlamy-

dial bodies as was postulated for domestic pigs, cattle

and sheep (Storz and Kaltenbock, 1993).

Analysis in PCR products of a 369-bp segment of

the ompA gene revealed the following general fea-

tures: (i) the sequences of all amplicons from different

organs of the same animal proved identical, with the

exception of animal 4, which harboured two different

chlamydial species; (ii) partial ompA sequences of Cp.

psittaci were identical in 9/10 samples, only that of

animal 14 was distinct in a single base (1/369 = 0.3%);

(iii) sequences of Cp. abortus were identical in lymph

node, uterus and placenta samples of animal 4, but

distinct from the lymph node sample of animal 5 in

two positions; and (iv) in contrast, C. suis sequences

from animals 4 and 9 were clearly distinct (71/369 =

19.2%), which is in line with the generally high

genetic heterogeneity of this species (Everett et al.,

1999).

To our knowledge, this is the first report combining

molecular and histological evidence of the occurrence

H. Hotzel et al. / Veterinary Microbiology 103 (2004) 121–126 125

of Chlamydiaceae in wild boar. Although the general

prevalence of these agents cannot be assessed on the

basis of the present data, their potential role as patho-

gens in mammalian wildlife, as well as reservoirs for

cases of transmission to domestic animals and even

humans deserves more attention by researchers and

diagnosticians in the future.

Acknowledgements

The authors are grateful to Mr. H. Bottcher from the

Thuringian Forestry Administration for encourageing

this study. We also thank Byrgit Hofmann and Karola

Zmuda for excellent technical assistance.

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