Veterinary Parasitology 142 (2006) 376–379

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Short communication
Toxoplasma gondii infection in sheep from Haute-Vienne,
France: Seroprevalence and isolate genotyping by
microsatellite analysis
Aurélien Dumètre, Daniel Ajzenberg, Luc Rozette,
Aurélien Mercier, Marie-Laure Dardé *
EA3174, Neuroparasitologie et Neuroépidémiologie Tropicale, Faculté de Médecine,
2 Rue du Dr Marcland, F-87025 Limoges, France
Received 3 May 2006; received in revised form 23 June 2006; accepted 6 July 2006

Abstract
Ingesting meat of free-range livestock, mainly sheep, is associated with human toxoplasmosis in European countries. Data on
Toxoplasma gondii infection in French ovine livestock are relatively scarce. Sera from 164 lambs and 93 ewes slaughtered in Haute-
Vienne district, France, were tested by a direct agglutination test. Antibodies to T. gondii were found in 36 (22.0%) lambs and in 61
(65.6%) ewes. In addition, to attempt parasite isolation for genotyping, hearts from 50 other ewes were obtained from a local
slaughterhouse, and were screened by a direct agglutination test. T. gondii was isolated in 8 of 30 seropositive hearts bioassayed in mice.
All isolates were type II by genetic characterization at five microsatellite loci (TUB2, TgM-A, W35, B17, B18). These results indicate that
ovines slaughtered in France may be highly infected by T. gondii with a potential risk of parasite transmission to humans by consumption
of undercooked meat. Multilocus microsatellite analysis shows the predominance of type II in sheep as previously described in humans.
# 2006 Elsevier B.V. All rights reserved.

Keywords: Toxoplasma gondii; Sheep; Seroprevalence; Meat; Multilocus genotyping; Microsatellite

1. Introduction In this paper, the prevalence of T. gondii antibodies

Toxoplasma gondii is a widely prevalent protozoan
of warm-blooded animals including humans, livestock
and marine mammals (Dubey and Beattie, 1988). In
France, ingesting meat of free-range livestock such as
sheep is associated with T. gondii acquisition by humans
(Baril et al., 1999). However, little is known about T.
gondii prevalence and genotype distribution in French
ovines, especially in slaughtered animals, which will be
later processed for food (AFSSA, 2005).
* Corresponding author at: Laboratoire de Parasitologie-Mycologie,
CHU Dupuytren, 2 Avenue Martin Luther King, 87042 Limoges,
France. Tel.: +33 555 056 160; fax: +33 555 056 177.
E-mail address: darde@unilim.fr (M.-L. Dardé).
was studied in lambs and ewes from Haute-Vienne
district, France, and parasite isolation was attempted
from slaughtered ewes. Isolates were genetically
characterized by a multilocus microsatellite analysis
(Ajzenberg et al., 2005).
2. Material and methods
2.1. Sheep
A total of 164 lambs and 93 ewes were serologically
tested between December 2004 and September 2005.
The sample size was calculated for an expected
seroprevalence of 10% for lambs and 50% for ewes
with an absolute precision of 10% (Toma et al., 2001).

0304-4017/$ – see front matter # 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.vetpar.2006.07.005
 A. Dumètre et al. / Veterinary Parasitology 142 (2006) 376–379
Animals originated from two major slaughterhouses
(Bellac and Limoges) of Haute-Vienne, a French central
district with an important production of ovine livestock.
Most of the lambs (3–11 months old) and ewes (>1-
year-old) were raised in extensive farms located in a 30–
40 km area around each slaughterhouse. Blood samples
were collected from the assembly line from one out of
10 consecutive lambs and one out of five consecutive
ewes. This method was designed to reduce the over-
representation of animals raised together.
In addition, between June 2005 and March 2006, a
butcher provided us with 50 hearts, in 10 batches of one
to seven samples each, from ewes slaughtered in
Limoges. Intracardiac blood was collected for serologic
examination. Hearts of seropositive ewes were bioas-
sayed in mice to isolate T. gondii.
2.2. Serologic examination for T. gondii
Serum samples from freshly slaughtered animals
were screened at 1:20, 1:40, 1:80 and 1:400 dilutions
with a direct agglutination test using whole formalin-
preserved tachyzoites as the antigen (Desmonts and
Remington, 1980). Serum samples from ewe hearts
were obtained from the blood clot. They were diluted
two-fold starting at a 1:20 dilution.
2.3. Bioassay for T. gondii in mice
Hearts of seropositive (serum dilution  1:20) ewes
were bioassayed in mice after digestion in pepsin as
described previously (Dubey, 1998). Briefly, heart
tissue (50 g) was ground in five volumes (w/v) of
aqueous 0.9% NaCl (saline), mixed with five volumes
of acidic pepsin and this mixture was incubated in
Table 1
Prevalence of Toxoplasma gondii antibodies in lambs and ewes from two slaughterhouses in Haute-Vienne district, France
Animals Number tested
a
Bellac slaughterhouse
Lambs 93
Ewes 60
Limogesb slaughterhouse
Lambs 71
Ewes 33
Overall
Lambs 164
Ewes 93
a
Latitude 468070 0000 N, longitude 018030 0000 E.
b
Latitude 458510 0000 N, longitude 018150 0000 E.
377
shaker water bath for 1 h at 37 8C. The digest was
centrifuged, neutralized, mixed with gentamycin, and
finally inoculated intraperitonealy (i.p.) into three to
five Swiss Webster mice. Mice were examined for T.
gondii infection and survivors were bled 4 weeks
postinoculation (pi). Their serum was tested for T.
gondii antibodies with the direct agglutination test
starting at a 1:20 dilution (Desmonts and Remington,
1980).
2.4. Genotyping of T. gondii isolates
DNA from brains of infected mice was extracted
using the QIAamp1 DNA MiniKit (Qiagen, Courta-
boeuf, France). Genetic characterization was done at
five microsatellite loci in a multiplex PCR assay
(TUB2, TgM-A, W35, B17, B18) (Ajzenberg et al.,
2005). Briefly, we used the QIAGEN1 Multiplex PCR
kit (Qiagen, Courtaboeuf, France) and amplifications
were carried out in a GeneAmp1 PCRSystem 2700
thermalcycler (Applied Biosystems, Courtaboeuf,
France): 15 min at 95 8C (initial activation step),
followed by 35 cycles consisting of 94 8C for 30 s,
63 8C for 3 min, and 72 8C for 60 s. The last extension
step was at 60 8C for 30 min. PCR products were
visualized on a 2% agarose gel stained with ethidium
bromide in order to confirm DNA amplification.
Finally, PCR products were run on a polyacrylamide
gel POP4 (Applied Biosystems, Courtaboeuf, France)
for genetic analysis. Signals were read with an
automatic sequencer (Abiprism 310 collection 1.0,
Applied Biosystems, Courtabœuf, France) and the data
were stored and analyzed with GeneScanTM analysis
software (version 2.1, Applied Biosystems, Courta-
bœuf, France).
Number positive (%) Agglutination titer
20 40 80 400
18 (19.4) 5 2 0 11
42 (70.0) 0 15 3 24
18 (25.4) 5 6 2 5
19 (57.6) 0 1 2 16
36 (22.0) 10 8 2 16
61 (65.6) 0 16 5 40
378 A. Dumètre et al. / Veterinary Parasitology 142 (2006) 376–379

Table 2
Multilocus microsatellite (MS) genotyping of Toxoplasma gondii isolated in ewes from Limoges slaughterhouse, France
Ewe number Direct agglutination Tissue No. of positive mice/no. Isolate namea MS
titer tested of inoculated mice genotype
5 160 Heart 1/4 Fr2-2005-Ovi ari 01 II
6 80 Heart 3/4 Fr2-2005-Ovi ari 02 II
13 160 Heart 2/5 Fr2-2005-Ovi ari 03 II
14 160 Heart 3/5 Fr2-2005-Ovi ari 04 II
16 160 Heart 3/5 Fr2-2005-Ovi ari 05 II
27 160 Heart 3/3 Fr2-2005-Ovi ari 06 II
33 160 Heart 3/3 Fr2-2006-Ovi ari 01 II
38 160 Heart 3/3 Fr2-2006-Ovi ari 02 II
a
Fr: France; 2: Limoges; year of isolation; abbreviation of latin name of the species (Ovis aries).

3. Results et al., 2002a). In France, type II largely predominates in

Antibodies to T. gondii were found in 36 of 164
(22.0%) lambs and in 61 of 93 (65.6%) ewes
slaughtered in Haute-Vienne district (Table 1). High
agglutination titers (1:400) were more often found in
ewes from both slaughterhouses. In an attempt to
isolate T. gondii from sheep tissue, we found antibodies
in 40 of 50 (80.0%) hearts from ewes with unknown
serologic status slaughtered in Limoges. T. gondii was
isolated from 8 of 30 seropositive hearts bioassayed in
mice (Table 2). All isolates were avirulent for mice
and exhibited a type II genotype at five micro-
satellite markers (TUB2, TgM-A, W35, B17, B18)
identical to Me49, a reference type II strain (Ajzenberg
et al., 2005).
4. Discussion
Ovine meat is considered as an important source of T.
gondii infection by humans in France and other
European countries (Baril et al., 1999; Tenter et al.,
2000). It is one of the main risk factor for Toxoplasma
infection during pregnancy. However, data on toxo-
plasmosis in French ovine livestock are scarce. Between
1960 and 1997, eight investigations have reported a 15–
92% seroprevalence in asymptomatic animals (AFSSA,
2005), and a higher seroprevalence in ewes than in
lambs as described previously (see Dubey and Beattie,
1988, for review). Our results in slaughtered lambs
(22.0%) and ewes (65.6%) are in agreement with these
data. The seroprevalence in lambs cannot be related to a
transfer of maternal antibodies through colostrum,
which occurs within 3 months after birth (Waldeland,
1977).
The vast majority of T. gondii isolates has been
classified into three genetic types (I–III) at least in
Europe and USA (Howe and Sibley, 1995; Ajzenberg
human congenital toxoplasmosis (73 of 86 isolates)
whatever the clinical findings (Ajzenberg et al., 2002b).
Here, isolates from ewes are genotype II as reported
previously in asymptomatic or aborted lambs from
France (Ajzenberg et al., 2002a and unpublished data
from the BRC ToxoBS), United Kingdom (Owen and
Trees, 1999) and Denmark (Jungersen et al., 2002).
Other genotypes are probably rare in Europe (Aspinall
et al., 2002). In this latter study, four type I and two
mixed genotypes (I + II) were found in food samples
prepared with lamb meat.
According to our results in sheep and in other
domestic and wild animals (Ajzenberg et al., 2002a
and unpublished data from the BRC ToxoBS),
genotype II is probably predominant in animals and
their environment in France. Investigations are in
progress to refine T. gondii infection and genotype
distribution in meat-producing animals. Sampling in
slaughterhouses may be more relevant than in farms
since slaughtered animals are processed for human
food. By testing one out of five or ten consecutive
animals, it is possible to reduce the over-representa-
tion of infected animals and restrict the screening to
one to three animals per farm.
Acknowledgements
We thank the staffs of Bellac and Limoges
slaughterhouses for their technical assistance in
obtaining blood samples from sheep. We thank the
members of BRC ToxoBS group (French parasitologist
network for Toxoplasma isolate collection) for unpub-
lished data on genotyping. This work was supported by
Programme Régional d’aide à la Recherche Universi-
taire and Fonds de Croissance Recherche 2002, 2003
(Conseil Régional du Limousin) and by the French
Ministry of Research (grant no. 02 G 0418).
 A. Dumètre et al. / Veterinary Parasitology 142 (2006) 376–379
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