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