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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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