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Veterinary Parasitology 129 (2005) 259–266
Detection of Echinococcus multilocularis in wild boars in
France using PCR techniques against larval form
J.M. Boucher a, R. Hanosset b, D. Augot a, J.M. Bart c, M. Morand d,R. Piarroux c, F. Pozet-Bouhier d, B. Losson b, F. Cliquet a,*
a AFSSA Nancy, Laboratoire d’Etudes et de Recherches sur la Rage et la Pathologie des Animaux Sauvages,
Domaine de Pixerecourt-B.P. 9, Malzeville F 54220, Franceb Departement des Maladies Infectieuses et Parasitaires, Universite de Liege, Faculte de
Medecine Veterinaire, 20 b-43B-4000, Liege, Belgiquec Sante et Environnement Rural, Universite de Franche-Comte (SERF) EA 2276,
Hopital J. Minjoz-3, Bd Alexandre Fleming-25000, Besancon, Franced Laboratoire Departemental d’analyse du Jura, Boulevard Theodore Vernier-BP 376-39016 Lons le Saunier, Cedex France
Received 25 March 2004; received in revised form 7 September 2004; accepted 17 September 2004
Abstract
Recently, new data have been collected on the distribution and ecology of Echinococcus multilocularis in European
countries. Different ungulates species such as pig, goat, sheep, cattle and horse are known to host incomplete development of
larval E. multilocularis. We report a case of E. multilocularis portage in two wild boars from a high endemic area in France
(Department of Jura). Histological examination was performed and the DNAwas isolated from hepatic lesions then amplified by
using three PCR methods in two distinct institutes. Molecular characterisation of PCR products revealed 99% nucleotide
sequence homology with the specific sequence of the U1 sn RNA gene of E. multilocularis, 99 and 99.9% nucleotide sequence
homology with the specific sequence of the cytochrome oxydase gene of Echinococcus genus and 99.9% nucleotide sequence
homology with a genomic DNA sequence of Echinococcus genus for the first and the second wild boar, respectively.
# 2004 Elsevier B.V. All rights reserved.
Keywords: Echinococcus multilocularis; Wild boar; Polymerase chain reaction; DNA; Sequence
1. Introduction
Echinococcus multilocularis is the causative agent
of alveolar echinococcosis, a zoonotic parasitic
* Corresponding author. Tel.: +33 3 83 29 89 50;
fax: +33 3 83 29 89 58.
E-mail address: f.cliquet@afssa.fr (F. Cliquet).
0304-4017/$ – see front matter # 2004 Elsevier B.V. All rights reserved
doi:10.1016/j.vetpar.2004.09.021
disease, which may be fatal for man. Naturally, the
tapeworm inhabits the small intestine of different
carnivorous mammals species predominantly foxes as
definitive hosts and arvicolid grassland rodents as
intermediate host (Rausch, 1995) especially of the
genus Microtus.
In recent years, a variety of mammalian animal
species (Eckert et al., 2001) has been described in
.
J.M. Boucher et al. / Veterinary Parasitology 129 (2005) 259–266260
several western European countries and in Japan as
‘‘accidental’’ natural hosts of metacestodes of E.
multilocularis including domestic dogs (Losson and
Coignoul, 1997), domestic and wild pigs (Sakui et al.,
1984; Kamiya et al., 1987; Pfister et al., 1993; Sydler
et al., 1998), monkeys (Rietsch and Kimmig, 1994)
and the nutria (Myocastor coypus) (Worbes et al.,
1989; Eckert, 1996; Ohbayashi, 1996).
Kolarova (1999) has reported the distribution of
E. multilocularis developmental stages in animals and
humans in the central and eastern Europe. Unusual
hosts were cattle, sheep and pig.
In France, the first cases of human alveolar
echinococcosis have been diagnosed in 1889 (Boucher
et al., 2001). The geographical distribution of the
parasite is restricted to the eastern parts of the country
(Kern et al., 2003). However, recent data suggest an
increase of the prevalence in foxes in different areas
(Giraudoux et al., 2001). At present, the number of
human new cases occurring in France is estimated at
around 10 per year (Dorchies et al., 2002).
This paper reports the presence of E. multilocularis
in two wild boars living in a French endemic area.
Histological examination as well as PCR techniques
followed by nucleotide sequencing were used for
assessing the identification of hepatic lesions as well
as the degree of similarity of the DNA sequences with
the published ones.
2. Material and methods
2.1. Sample collection
Wild boar liver samples were collected in a hyper
endemic area for E. multilocularis of the north-east
of France by the Veterinary Departmental Labora-
tory of Jura region during the winter 2001. The
samples were collected from two carcasses after
hunting and were stored at �20 8C before analysis.
The first wild boar (No. N976, ONC 060680) was a
young (<1 year) and the second was an adult (>3
years) (No. N977, ONC 060681). At gross exam-
ination, the first liver showed approximately 50
nodules (5 mm diameter) localised in the parench-
yma and the second liver showed only one nodule
measuring 20 mm in diameter. Pieces of tissue were
embedded in paraffin wax, cut in 5 mm sections and
stained with haematoxylin and eosin coloration
technique.
2.2. Extraction of DNA from nodules samples
and coloration
For each animal, samples (up to 0.6 g) were taken
from the fresh nodular lesions. The first part was
used for the haematoxylin eosin safrin coloration
and the periodic-acid-Schiff coloration the second
part was divided into small pieces then digested in
the presence of 900 mg of proteinase K (Invitrogen)
at 56 8C during 12 h in 0.5 ml of 10 mM Tris–HCl
(pH 7.5), 10 mM Na2EDTA, 50 mM NaCl, 2%
sodium dodecyl sulfate, 10 mM dithiothreitol.
DNA was extracted with 1 ml of phenol–chloro-
form–isoamyl alcohol (25:24:1) and 1 ml of chloro-
form–Tris-EDTA (10:1). DNA was precipitated
with Precipitator (Q-BIOgene, France). After
vacuum drying, the precipitate was suspended in
50 ml of ultra pure autoclaved water and stored
at �80 8C.
2.3. DNA amplification
DNA amplification step used three primers pairs.
The first PCR for the amplification of 3 ml of DNA
extract was performed as described by Bretagne et al.
(1993) then modified by Monnier et al. (1996) using a
mastercycler gradient (Eppendorf, France). The
primers used were selected from the U1 sn RNA
gene of E. multilocularis.
The second primer pair used for the amplification
of 4 ml of DNA extract was selected from the
cytochrome oxydase gene of Echinococcus genus
(which includes four species: E. multilocularis,
E. granulosus, E. vogeli and E. oligarthrus) from
mitochondrial DNA sequence (Bowles et al., 1992).
The amplification step was performed for 40 cycles
(1 min 94 8C, 1 min 60 8C, and 1 min 72 8C) using a
mastercycler gradient (Eppendorf, France).
The third primer pair used for the amplification
of 4 ml DNA extract was selected from genomic
DNA sequence from three species of Echinococcus
genus: E. multilocularis, E. granulosus and E. vogeli
(Gottstein and Mowatt, 1991).
After the amplifications, the totality of the PCR
products were separated by electrophoresis on 1%
J.M. Boucher et al. / Veterinary Parasitology 129 (2005) 259–266 261
agarose gel and were visualised by ethidium bromide
staining and UV illumination.
Negative controls (distilled and autoclaved water)
and positive control (DNA extract from adult form of
E. multilocularis) were included in each PCR round.
In order to exclude any possibility of contamination
and to confirm all results, DNA amplifications
(each conducted with distinct primers) and products
sequencing were performed in two different institutes
with cross-sending of all samples (as an example, for a
DNA extract, amplification was performed using a set
of primers in AFSSA laboratory and sequencing in
SERF laboratory and vice versa with the second set of
primers).
2.4. DNA sequencing
The PCR products (392 bp with primers described
by Bretagne et al. (1993), 296 bp with the primers
described by Bowles et al. (1992) and 361 bp with the
primers described by Gottstein and Mowatt (1991)
were extracted from agarose gel using MiniElute Gel
Extraction kit (Qiagen, France) and PCR product
sequencings were performed by using Dye Terminator
Technology (Applied Biosystem) and analysed on
automatic DNA sequencer ABI Prism (Perkin-Elmer).
Fig. 1. Microscopic examination of the first wild boar. (a) Granulomatous
was composed by cystic structures filled with an amorphous material and
centre was surrounded by a granulomatous reaction. Individual nodular stru
eosin stain 40� magnification). (b) Non-cellular laminated layer irregularl
material (1000� magnification).
The assessment of nucleotide sequence homologies
was performed by comparison with sequences
already known by using GenBank databases (acces-
sion numbers M38199, AF408684, AB018440,
AF408686, M73768).
3. Results
Histologically, the lesions were diagnosed as
subacute to chronic multifocal hepatitis. The lesions
were constituted of three different parts; the center had
a variable structure and was edged by an inflammatory
infiltration, itself surrounded by a fibroconjonctive
capsule.
In the first wild boar (N976), most of the hepatic
parenchyma was normal, except the presence of focal
interstitial fibrosis (Fig. 1). All these lesions were
edged by a cell rich fibroconjonctive capsule,
delimited in the inner part by a subacute inflammatory
reaction mostly composed of lymphocyte and macro-
phage infiltration, with few multinucleated giant cells.
In most structures, the capsule and the inflammatory
cells surrounded a necrotic core with evidence of
dystrophic calcification bodies. Occasionally, the
center of nodules was composed by a cystic structure,
Echinococcus multilocularis lesion in liver. The centre of the nodule
necrotic core with evidence of dystrophic calcification bodies. The
cture may have merged to a polynodular structure (haematoxylin and
y folded, and periodic-acid-Schiff positive filled with an amorphous
J.M. Boucher et al. / Veterinary Parasitology 129 (2005) 259–266262
lined by a thin (10–15 mm) non-cellular laminated
layer irregularly folded. This layer was periodic-acid-
Schiff positive (Jubb et al., 1993; Haller et al., 1998).
The cyst was filled with an amorphous materiel.
Sometimes, the individual nodular structure may have
merged to a polynodular structure.
In the second wild boar (N977), most of the hepatic
parenchyma was constituted by 1–2-mm diameter
multifocal nodules (Fig. 2). The microscopic aspect
was very similar to the first case. Nevertheless, the
Fig. 2. Microscopic examination of the second wild boar. (a)
Hepatic cyst of Echinococcus multilocularis consisting of a non-
cellular laminated layer which included amorphous material. This
cyst was surrounded by a granulomatous reaction (hematoxylin and
eosin stain: 100� magnification). (b) Non-cellular laminated layer
irregulary folded and periodic-acid-Schiff positive (1000� magni-
fication).
lesions were more evolutive, and were characterised
on one hand by necrotic center and on the other hand
by necrotic center with dystrophic calcification
bodies. Finally, in some cases, the center of nodules
was composed by a cystic structure, lined by a thin
(10–15 mm) non-cellular laminated layer irregularly
folded and periodic-acid-Schiff positive. Such a
structure was probably considered as being germinal
syncytial layer (Jubb et al., 1993; Haller et al., 1998),
this cyst being filled with an amorphous material. All
the different lesions were limited by a subacute to
chronic inflammatory reaction.
In the two cases, although no protoscoleces were
identified, these histological findings may suggest a
metacestode stage of the tapeworm E. multilocularis.
However, cysts of E. granulosus with an atypical
polycystic structure have been confused with meta-
cestodes of E. multilocularis. Therefore, the diagnosis
has to be based on several reliable criteria such as PCR
and DNA sequencing techniques (Eckert et al., 2001).
Results of PCR product sequencing with the three
primers pair (Figs. 3–5) confirm the diagnosis: for the
first wild boar, comparisons of sequences obtained
with GenBank databases showed 99% of homology
for cytochrome oxydase sequence and U1 sn RNA
gene sequence, and 99.9% for BG 1/3 sequence. For
the second wild boar, these percentages of homologies
were 99.9% for genomic DNA sequences and 99% for
the U1 sn RNA gene sequence (Table 1).
4. Discussion
E. multilocularis infection in wild boar has been
reported for the first time by Pfister et al. (1993) in
endemic areas of E. multilocularis in Germany. The
spectrum of possible host spread in wild fauna seems
to be larger than principal known intermediate and
definitive hosts.
Previous papers indeed have reported spontaneous
infections of E. multilocularis in domestic pigs (Sakui
et al., 1984; Kawamoto et al., 1985; Senuma et al.,
1986; Kamiya et al., 1987; Sydler et al., 1998). In
Switzerland, a seroprevalence study in breeding sows
indicated that 2.9% of animals had specific E.
multilocularis serum antibodies.
In our study, the wild boars were originated from a
well-known endemic area in France (Department of
J.M. Boucher et al. / Veterinary Parasitology 129 (2005) 259–266 263
Fig. 3. Nucleotide sequences of a 292 bp fragment of the U1 sn RNA gene, for the two liver lesions analyzed (WB1 and WB2). The third
sequence (Em (M73768)) is the corresponding part of Echinococcus multilocularis U1 sn RNA gene published by Bretagne et al. (1993).
Jura). At our known, no cases of E. granulosus have
been reported so far in other intermediate host from
this area. The results of fields studies conducted in
Franche-Comte and Jura (France) between 1984–89
and 1996–98 indicate an increase of prevalence rate in
fox populations, suggesting a higher contamination of
the rural environment (Giraudoux et al., 2001).
Therefore, it is not surprising that wild boars living
in such high endemic area may be infected, in
Fig. 4. Nucleotide sequences of a 396 bp fragment of the mitochondrial CO
(Em (AB018440)) and fourth (Eg (AF408686)) sequences are the corr
granulosus fragment of the mitochondrial CO1 gene, respectively (Bowl
consideration of their dietary habits (feeding of roots,
carcasses and faeces).
The two wild boars had hepatic nodular lesions
(we did not receive other organs) and no proto-
scoleces were founded. These observations of lesions
in a hepatic nodule (histological section) and absence
of protoscoleces are consistent with previous reports
(Sakui et al., 1984; Kamiya et al., 1987; Pfister et al.,
1993) and could imply the wild boar to be an
1 gene, for the two liver lesions analyzed (WB1 and WB2). The third
esponding part of Echinococcus multilocularis and Echinococcus
es et al., 1992).
J.M. Boucher et al. / Veterinary Parasitology 129 (2005) 259–266264
Fig. 5. Nucleotide sequences of a 281 bp fragment of the BG 1/3 sequence, for the two liver lesions analyzed (WB1 and WB2). The third (Em
(M38199)) and fourth (Eg (AF408684)) sequences are the corresponding part of Echinococcus multilocularis and Echinococcus granulosus
published fragments of this sequence, respectively (Gottstein et al., 1992).
aberrant host not involved in the transmission of the
parasite.
In an unusual host, we may hypothesize that the
lesions observed in the liver would be the result of
E. granulosus or E. multilocularis infection. However,
E. granulosus showed few strains with specific
nature (Eckert, 1997), differences between strains
are morphologically, biochemically and genetically
important. Therefore, PCR methods amplifying
different specific segments of DNA sequence of
Echinococcus frequently used for diagnosis are a
valuable tool to determine the portage of the parasite
in carnivorous species (definitive host) and to identify
lesions from intermediate hosts (Gasser, 1999).
Table 1
Similarity homology of PCR products sequencing using different primers
% Similarity Primers
Cytochrome oxydase sequencea (%)
Wild boar 1 99
Wild boar 2 99.9
a Bowles et al. (1992).b Gottstein and Mowatt (1991).c Bretagne et al. (1993).
However, in view of the risk of false positive
responses, results produced by the use of such
techniques have to be confirmed either by hybridisa-
tion with a specific DNA probe or by sequencing in
order to identify origin of PCR products. The use of a
different set of primers in two different institutes and
the exchange of PCR products to characterise the
origin of parasitic lesions allow not to confuse
metacestodes of E. multilocularis with cysts of
E. granulosus with atypical polycystic structure.
Previous reports using molecular genetic
approaches to characterise the intermediate host for
E. multilocularis involving rodents, pigs, monkeys
and dogs (Mathis and Deplazes, 2002) are published.
with published DNA sequences
BG1/3 sequenceb (%) Bretagne sequencec (%)
99.9 99
99.9 99
J.M. Boucher et al. / Veterinary Parasitology 129 (2005) 259–266 265
To our knowledge, it is the first report of use of
different sets of primers to identify E. multilocularis
parasitic nodules.
In our hands, the use of two sets or more PCR
primers is the most accurate method for assessing
individual diagnosis in definitive host (such as foxes or
domestic animals) or intermediate host (such as rodent
species or aberrant host), allowing to discard
contamination risk due to the use of very sensitive
nested PCR techniques. This ‘‘multi-DNA-target’’
system permits also to exclude false positive results in
large epidemiological prevalence surveys of cat and
dog populations (study actually in course).
In a context where an increase of fox populations in
France and in several European countries is occurring
since few years (Wandeler et al., 2003), such unusual
infections may be indicators for higher environmental
contamination with E. multilocularis eggs and might
reveal an increase of the potential risk for humans.
Further investigations should be conducted on wild
and domestic ungulates, particularly in areas with
intensive domestic pig and outdoor husbandry, to
estimate the importance of the parasite portage as well
as the epidemiological role in the transmission of the
parasite.
Acknowledgments
We thank Dr. D. Cassart for her advices, S. Etienne
and G. Farre for their help.
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