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Lnag Halos a,*, Anne Thbault b, Dominique Aubert c,d, Myriam Thomas a, Catherine Perret a, Rgine Geers c,d, Annie Alliot a, Sandie Escotte-Binet c,d, Daniel Ajzenberg f,g, Marie-Laure Dard f,g, Benoit Durand e, Pascal Boireau a, Isabelle Villena b
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Article history: Received 30 April 2009 Received in revised form 15 June 2009 Accepted 17 June 2009 Available online xxxx Keywords: Toxoplasma gondii Ovine meat Prevalence Bioassay Zoonosis France

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Consumption of sheep meat presents a risk of human contamination by Toxoplasma gondii. A nationwide study was conducted in France to evaluate the prevalence of Toxoplasma in fresh ovine meat. A sampling procedure was established to guarantee the representativity of consumption. As is the case for meat consumed, half of the samples were from France and half were imported from other countries. Animals were selected according to their age, as lamb (<12 months) represents 90% of the meat consumed. Available data for French samples allowed the selection of 16 districts distributed in seven areas according to their density of production. Diaphragms and hearts from 433 sheep were collected. Diaphragms were collected from 398 imported carcasses. Fluids from hearts and diaphragms were tested serologically. All hearts were bioassayed in mice and parasite isolates were genotyped using PCR-restriction fragment length polymorphism and microsatellite markers. Prevalence estimates were calculated, taking into account uneven distribution of production and age. For French meat, the effect of area, age and their interactions was evaluated. The overall estimate of Toxoplasma seroprevalence was 17.7% (11.631.5%) for lambs and 89% (73.5100%) for adults (P < 0.0001). No signicant difference was observed between imported and French meat. In France, seroprevalence in lambs showed an increasing North-western to Southern gradient. The proportion of French carcasses carrying live parasites according to bioassay results was estimated at 5.4% (37.5%) (45 genotype II; one genotype III). This study offers an accurate drawing of the toxoplasmosis pattern amongst sheep consumed in France and a model for a zoonosis hazard control survey. 2009 Published by Elsevier Ltd. on behalf of Australian Society for Parasitology Inc.

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ENVA/AFSSA/INRA UMR BIPAR, National Reference Laboratory for Food-Borne Parasites, 23 Avenue du Gnral de Gaulle, 94 700 Maisons-Alfort, France AFSSA-DERNS, Maisons-Alfort, France Laboratoire de Parasitologie, EA3800, IFR53, CHU Reims, France d Centre National de Rfrence (CNR) Toxoplasmose/Toxoplasma Biological Resource Center (BRC), Reims, France e Unit dEpidmiologie, AFSSA LERPAZ, Maisons-Alfort, France f Laboratoire de Parasitologie-Mycologie, EA 3174-NETEC, Facult de Mdecine, Universit de Limoges, Limoges, France g CNR/Toxoplasma BRC, CHU Dupuytren 87025 Limoges, France

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An innovative survey underlining the signicant level of contamination by Toxoplasma gondii of ovine meat consumed in France

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1. Introduction

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Toxoplasmosis is a worldwide zoonosis, due to Toxoplasma gondii, a parasite of all warm-blooded animals including humans (Dubey and Beattie, 1988; Dubey, 1996; Tenter et al., 2000). In humans, infection is usually either asymptomatic or the cause of mild u-like symptoms. However, toxoplasmosis can be life threatening in immunocompromised individuals. Moreover, if acquired during pregnancy, toxoplasmosis can cause miscarriage or congenital malformations affecting the brain, eyes or other organs of the foetus. Infection is generally acquired by ingestion of viable tissue
* Corresponding author. Tel.: +33 1 43 96 72 15; fax: +33 1 43 96 71 90. E-mail address: lhalos@vet-alfort.fr (L. Halos).

cysts contained in meat or oocysts excreted by cats that contaminate the environment (Dubey, 1996). In Europe, recent studies have shown that seroprevalence in young women ranges between 35% and 60% (Cook et al., 2000; Tenter et al., 2000). Climatic conditions, as well as factors associated with lifestyle and diet, have been presented to explain the differences in the observed prevalence (Tenter et al., 2000). Consumption of undercooked infected meat is considered as a major risk factor, especially in Europe, where it has been attributed 3063% of infections (Cook et al., 2000; Tenter et al., 2000). In France, this risk factor is probably higher than in other countries due to a traditional habit of undercooked meat consumption. The most recent study revealed a seroprevalence of 43.8% amongst pregnant French women (Berger et al., 2007). The authors

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2.1. Sampling strategy

2.1.1. Sample size For an expected prevalence ranging from 15% to 92% as previously observed in different studies on sheep, a sample size of 800 animals enabled us to guarantee relative precision between 12% and 20% (Dohoo et al., 2003). National data on domestic sheep meat production and imports show that imported meat represents 46.6% of fresh sheep meat consumed in France (Ofce de llevage, 2007). Only fresh meat was targeted, as it has never been frozen and is thus at risk for T. gondii carriage. A proportional allocation of 50% was then given to imported ovine meat and French ovine meat in order to reect the ratio of meat consumed by the French population. 2.1.2. Samples of French origin The sampling strategy was established with the objective of being representative of sheep slaughtered in France. The sampling was planned to be conducted in 17 administrative districts with 25 samples collected in each district. Districts were stratied consid-

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2. Materials and methods

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suspected ovine meat, especially lamb meat, to be a risk factor for human infection. Amongst the infected meat, lamb meat is supposed to be a major source of toxoplasmosis worldwide (Tenter et al., 2000; Dumtre et al., 2006; Dubey and Jones, 2008; Kijlstra and Jongert, 2008). Moreover, a positive correlation between regional consumption of sheep meat and toxoplasmosis prevalence was demonstrated: geographical distribution of estimates of ovine meat consumption showed that in areas where ovine consumption is higher, a higher toxoplasmosis seroprevalence in humans is also observed (Berger et al., 2007). As accurate information on the prevalence of the parasite in ovine meat is lacking, no further investigation was possible. The seroprevalence of toxoplasmosis amongst sheep shows great variation from one study to another. As an example, in France, seroprevalence data obtained vary between 22% in lambs and 65% in adult sheep (Dumtre et al., 2006) and 92% in a general ovine population (Cabannes et al., 1997). Variations are driven by the country or the geographical area where the studies were performed, as well as the tests chosen, the size of the animal sample and the age of the animals sampled (Tenter et al., 2000; Kijlstra and Jongert, 2008). No nationwide studies have yet been undertaken in Europe (Tenter et al., 2000). Gaining a better understanding of the spatial distribution of parasitic diseases within endemic regions is increasingly recognised as being critical to the rational design and monitoring of parasitic control programs. There is a strong need to conduct largescale epidemiological studies in animals consumed by humans. Moreover, to guarantee an accurate evaluation of the true prevalence in meat consumed, data on meat production and consumption patterns should be taken into account in the methodology. No nationwide study has ever been conducted on toxoplasmosis contamination in livestock animals in France (Afssa, 2005) and no study taking into account meat production and consumption patterns has ever been conducted worldwide. We present herein, to our knowledge, the rst large-scale epidemiological study on toxoplasmosis in sheep in France as well as an innovative survey methodology based on nationwide data on ovine meat production and consumption. The aim of the study was to estimate seroprevalence of Toxoplasma infection in fresh sheep meat consumed in France, to study the geographical and age variations of this prevalence, as well as to search for live Toxoplasma parasites in meat and identify T. gondii genotypes circulating amongst sheep.

ering two variables: the geographical region (AREA) and the number of animals slaughtered in the district (CATEG). The rst level of stratication (AREA) was obtained by dividing France into seven areas, using a geographical information system (Arcview 8.3): (1) Western region: middle level of ovine breeding; (2) Mid-western region: high level of ovine breeding; (3) Pyrenees mountains region: high level of ovine breeding focused on meat production; (4) Mid-eastern region: low level of ovine breeding; (5) Central region: low level of ovine breeding; (6) Northern region: very low level of ovine breeding; (7) South-eastern region: high level of ovine breeding. The second level of stratication (CATEG) was dened considering the sheep meat production level of each district, expressed in tons (T) of carcasses (Fig. 1). Three meat production classes were dened. Limits were dened by analysis of frequency by optimal breaks or Jenks method implemented in Arcview 8.3 (Coulson, 1987). Districts with an annual sheep meat production >5,006,628 T were considered as districts of high production density, districts with production between 1,392,527 and 5,006,628 T were classied as districts of medium production density, and districts with production 61,392,526 T were classied as districts with low meat production. The SURVEYSELECT procedure of SAS (version 9.1) was used to select districts using unequal sampling probabilities based upon the annual number of animals slaughtered in each district. District selection was performed in order to have a minimum of two districts in each region (AREA) and a predened number of districts for each category of production density (CATEG). Using this selection procedure, all of the ve districts belonging to the category of high density, seven districts from the category of medium density and ve from the category of low density, were selected (Fig. 1). A nal stratication was applied according to the age of tested animals. Lamb meat represents 90% of ovine meat slaughtered and consumed in France. This led to the allocation of 6/25 samples from adult sheep and 19/25 samples from lambs in each district, considering the age of 12 months as the limit between lambs and adults. Adults were deliberately over-represented to guarantee similar precision in lamb and adult prevalence estimates, considering previous values of 65% for adults and 22% for lambs obtained in

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Please cite this article in press as: Halos, L., et al. An innovative survey underlining the signicant level of contamination by Toxoplasma gondii of ovine meat consumed in France. Int. J. Parasitol. (2009), doi:10.1016/j.ijpara.2009.06.009

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Fig. 1. Sampling plan. The seven different areas of production are numbered from 1 to 7 and delimited by a black line: (1) Western region; (2) Mid-western region; (3) Pyrenees mountains region; (4) Mid-eastern region; (5) Central region; (6) Northern region; (7) South-eastern region. Colored areas represent the density of ovine meat production in each district. Selected districts are presented with a black spot.

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an earlier study conducted in France (Dumtre et al., 2006). An indication of age was included both qualitatively (lamb/adult) and quantitatively (6 month intervals) in the questionnaire accompanying each meat sample.

2.4. Bioassay for T. gondii in mice After approval of the local Animal Research Ethical Committee, whole hearts were bioassayed in outbred female Swiss Webster mice (Charles River Laboratory, France). Briey, each whole heart was mixed and incubated at 37 C for 2.5 h with trypsin (nal concentration 0.25%). The suspension was then ltered, pelleted by centrifugation, washed in saline and resuspended in a saline solution containing penicillin G and streptomycin. This homogenate was inoculated i.p. into two mice (Villena et al., 2004; Afonso et al., 2007). Mice were bled 4 weeks post-inoculation and their serum was tested at 1:25 dilution for T. gondii antibodies with the MAT. Seropositive mice were killed 60 days post-inoculation and their brains were examined for tissue cysts. 2.5. Genotyping of T. gondii isolates Brain cysts from seropositive mice were isolated by percoll gradient centrifugation (Cornelissen et al., 1981). DNA was extracted using QIAamp DNA minikit (Qiagen, Courtaboeuf, France). Strain typing was performed using three single nucleotide polymorphisms (SNPs) studied by PCR-restriction fragment length polymorphism (RFLP) (SAG2, SAG1 and GRA7) and six microsatellite markers in a multiplex assay associating TUB2, W35, TgM-A, B18, B17 and M33 (Ajzenberg et al., 2005; Dubey et al., 2007). All strains isolated were cultivated and banked in the Toxoplasma Biological Resource Center (BRC, Limoges, France). 2.6. Statistical analyses 2.6.1. Denition of cases A previous study using MAT and ELISA in sheep has shown a very high degree of agreement between the two serological tests (Mainar-Jaime and Barbern, 2007). Here, correlation between the two serological tests was good (data not shown), allowing us to dene a positive case as a sample showing a positive result for at least one of the three tests performed (MAT, ELISA or bioassay in mice). For imported samples, only the ELISA test was conducted on diaphragm uids. A sample showing a positive result with ELISA was considered as positive. 2.6.2. Samples of French origin Proportions of positive results should not be interpreted as prevalence because of the unequal sampling procedure used to obtain the representative pattern of sheep meat consumed in France. Descriptive prevalence estimates were calculated using PROC SURVEYFREQ (SAS version 9.1) in order to take into account the unequal sampling procedure. Age was taken into account as a qualitative variable expressed as adults (>12 months) or lambs (<12 months). Statistical analysis of prevalence according to the age of animals, the region (AREA) and the meat production level of the region (CATEG: ordinal scale) were studied using a generalised linear model (logistic link) (R.2.7.0, MASS and lme4 package, 2008) (Faraway, 2006). Age was then treated as a quantitative variable by grouping animals in ve age levels of 6 months each: from 0 to 6 months (level 1); from 6 to 12 months (level 2), from 12 to 18 months (level 3), from 18 to 24 months (level 4) and more than 24 months (level 5). For the categorical AREA variable, the reference class was the region with the lowest prevalence in lambs. The absence of a slaughterhouse or district effect was veried using a mixed model, both variables being treated as nested random effects (Pinheiro and Bates, 2004). The shape of the ageprevalence relationship allows the determination of the age at which animals become infected. The model was used to predict prevalence variations according to age in two contrasted areas: the area

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2.2. Sampling protocol All samples were collected between February and July 2007.

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2.3. Serological tests

The modied agglutination test (MAT) for the detection of T. gondii-specic IgG antibodies was performed as previously described on all cardiac uids using an antigen prepared from formalin-xed whole RH strain tachyzoites (Dubey and Desmonts, 1987). Cardiac uids were collected by pipetting the exudate in the bottom of each plastic box. Each uid was serially twofold diluted. The threshold dilution was 1:6. The ELISA test, Elisa Pourquier (Institut Pourquier, France) was performed on diaphragm uids according to the manufacturers instructions except that uid dilution was 1:4 and not 1:20 due to the weaker concentrations in body uids compared with sera (Nckler et al., 2005). Meat juice was obtained from 25 g of diaphragms cut into small pieces and frozen overnight at 20 C in a plastic bag. After thawing at room temperature, the meat juice was collected with a pipette into a microtube as previously described (Nckler et al., 2005).

Please cite this article in press as: Halos, L., et al. An innovative survey underlining the signicant level of contamination by Toxoplasma gondii of ovine meat consumed in France. Int. J. Parasitol. (2009), doi:10.1016/j.ijpara.2009.06.009

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2.2.2. Imported samples Only fresh ovine carcasses from which internal organs (including the heart) have been removed are imported into France. For this reason, only diaphragms were available from imported carcasses collected in Rungis. Diaphragms were collected as previously described for animals of French origin.

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2.2.1. Samples of French origin Two districts were sampled per week. In each selected district, the main slaughterhouses were visited with a maximum of two slaughterhouses per district. In these slaughterhouses, the interval between two sampled carcasses was evaluated taking into account the total number of animals slaughtered on the same day in the slaughterhouse and the total number of animals to be sampled. This method was designed to reduce the over-representation of animals raised together (Cochran, 1977). Hearts were collected in individual plastic boxes and kept refrigerated (+4 C) for 12 days and transported to the Parasitology Laboratory of National Reference Centre for toxoplasmosis in Reims (France). Diaphragms were collected in plastic bags and kept frozen after transportation to the National Reference Laboratory for food-borne parasites in MaisonsAlfort (France). Plastic bags were used to collect diaphragms to facilitate storage as well as uid collection.

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2.1.3. Imported samples Each month, 80 diaphragms were sampled by the veterinary staff of the veterinary services amongst the imported fresh meat to reach a total of 400 samples. Sampling was performed in the international food market of Rungis, which is the main centre for importation of sheep meat according to the French Ministry of Agriculture. The four main countries of origin for imported sheep meat were represented (i.e. United Kingdom (UK), Ireland, The Netherlands and Spain). The limited duration of the sampling plan did not enable a representative allocation between these sources.

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Adults > 12 months

2.6.3. Imported samples The sampling was assumed to be representative of imports from each country. Crude prevalence estimates were calculated using the same procedure as that used for French meat production, allocated weights being based on the relative importance of each country in French imports. Results were analysed with PROC SURVEYFREQ. Because heart tissues were not available from imported carcasses, only serological ELISA tests on diaphragms uids were conducted. The corresponding bias for under-detection in imports was corrected using the formula TPij = (APij + FNij)/nij (Pouillot et al., 2004). TPij is the random variable denoting the true prevalence for country: i and age category: j. nij is the number of tested animals for country i and age category j. APij is a random variable following a binomial distribution, with nij trials and pij calculated on observations. FNij is the random variable denoting the number of false negative animals for country i and age category j. This variable follows a negative binomial distribution of parameters APij + 1.p is the relative sensitivity of detection for imported animals follows a beta distribution B(s, ns) (Pouillot et al., 2004) with s: number of animals positive in ELISA, and n: number of animals positive in ELISA, MAT or bioassay. The mean corrected prevalences for lambs and adults were calculated from 20,000 bootstrap samples (Davison and Hinkley, 1997). The model was written with R 2.7.0 software (R Development Core Team, 2005). Bootstrap sampling results were compared with statistical results for non-corrected prevalence. 2.6.4. Overall prevalence A bootstrap procedure was used to estimate an overall mean prevalence for meat from lambs and adult sheep consumed in France, taking into account the relative portions of imported and locally produced sheep meat in French consumption (20,000 samples, R 2.7.0 software) for both adults and lambs.

Genotype II Genotype II Genotype II + 1 lamb with genotype III Genotype II Genotype II Genotype II Genotype II 0.82 (0.461) 1 0.68 (0.360.99) 1 (0.81) 0.84 (0.641) 0.83 (0.41) 0.59 (0.360.81) 4: Mid-eastern area (2) 5: Central area (2) 6: Northern area (1) 7: South-eastern area (4) 50 (45; 5) 50 (37; 13) 25 (19; 6) 101 (75; 26) 0.14 0.12 0.21 0.24 (0.030.25) (0.0040.23) (0.010.41) (0.140.34) 2/49 (0/44; 2/5) 6/49 (1/36; 5/13) 2/14 (0/8; 2/6) 11/99 (2/74; 9/25) 1: Western area (2) 2: Mid-western area (3) 3: Pyrenees mountains area (2) 426 (343; 83) 0.15 (0.080.21) 0.81 (0.720.90) Mean prevalence detection in ovine meat:0.20 (0.140.27) CI: condence interval. Total Lambs < 12 months 50 (41; 9) 101 (90; 11) 49 (36; 13) 0.10 (00.23) 0.11 (0.040.17) 0.32 (0.150.48) 48/397 (14/315; 34/82) 0.02 (0.0070.037) 0.42 (0.270.57) Mean prevalence of detected living parasites in ovine meat:0.054 (0.030.075) Adults > 12 months 0.49 (0.0050.989) 0.41 (0.060.775) 0.42 (0.080.75) 0 0.04 (0.00.09) 0.106 (0.0050.21) 4/50 (0/41; 4/9) 11/88 (5/77; 6/11) 12/48 (6/35; 6/13) Lambs < 12 months 0 0.035 (00.11) 0 0.048 (00.1) 0.4 (01) 0.34 (0.020.66) 0.21 (00.875) 0.44 (0.210.67)

with the lowest prevalence rate in lambs, and the area with the highest prevalence rate.

Genotypes

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3. Results 3.1. Sampling plan

3.1.1. Samples of French origin Sampling was planned in 17 selected districts: Area 1 (Western region), Ille-et-Vilaine and Vende; Area 2 (Mid-western region), Vienne, Haute-Vienne and Charente; Area 3 (Pyrenees mountains region), Pyrnes-Atlantiques and Hautes-Pyrnes; Area 4 (Mid-eastern region), Rhne and Drome; Area 5 (Central region), Seine-et-Marne and Val-dOise; Area 6 (Northern region), Oise and Meuse; Area 7 (South-eastern region), Aveyron, Alpes de Haute-Provence, Lot and Vaucluse. One selected district (Meuse) from the Northern region could not be sampled because the slaughtering activity ended a month before starting the study. In two districts, no samples from adults were available. Twentyve samples were collected per district, except for three districts (Vienne, Haute-Vienne and Aveyron) where more than 25 samples were collected and kept in the statistical analysis. Calculations took this change into account. A total of 433 samples were collected. For seven samples, qualitative indications of age (lamb/adult) were not available, so a total of 426 samples were used for statistical analyses of prevalence (Table 1). Similarly, quantitative indications of age (6 month

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Please cite this article in press as: Halos, L., et al. An innovative survey underlining the signicant level of contamination by Toxoplasma gondii of ovine meat consumed in France. Int. J. Parasitol. (2009), doi:10.1016/j.ijpara.2009.06.009

Areas of production (number of districts sampled in the area)

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Mean prevalence (95% CI)

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classes) were not available for 47 samples: 379 samples were thus used to study the relation between age and prevalence. 3.1.2. Imported samples A total of 398 diaphragms were collected at the international market of Rungis of which 22 presented dehydration of the meat which did not allow uid collection. Age was missing for two further samples. A total of 374 animals were thus included in statistical analyses, originating from the UK, Spain, Ireland and The Netherlands (Table 2). 3.2. Prevalence analysis 3.2.1. Overall prevalence Taking into account unequal repartition of meat production and age as well as under-estimation due to a single Elisa test for imported meat, the overall prevalence of toxoplasmosis in sheep meat consumed in France was estimated at 17.7% (11.631.5%) for lamb meat, and at 89% (73.5100%) for adult meat. The relative sensitivity of ELISA tests alone was calculated taking as a reference the joint results of ELISA, MAT and mouse bioassays from French meat and estimated at 61.7% (Beta (71, 44)). 3.2.2. Samples of French origin Prevalence of T. gondii in sheep carcasses in French slaughterhouses was estimated at 15% (821%) for lambs and at 81% (72 90%) for adults (Table 1). According to the region, prevalence varied between 10% and 32% for lambs (Fig. 2), and between 59% and 100% for adults. Whatever the region, prevalence estimates were markedly lowers in lambs than in adults. The generalised linear model showed no signicant effect of sheep meat production levels (CATEG) on prevalence levels. Conversely, a signicant effect of the AREA variable was observed. The Western area (Area 1, prevalence in lambs: 10%) was dened as the reference class (i.e. area with the lowest prevalence in lambs). A signicant effect of the AREA variable was obtained for two southern regions (Table 3): Pyrenees mountains (Area 3, prevalence in lambs: 32%) and South-eastern area (Area 7, prevalence in lambs: 24%). In both regions, correlations between age and area also had a signicant effect on prevalence (Table 3). For the following analyses, those two areas (Area 3 and Area 7) were merged in a single Southern region. The joint age and area effect in lambs was further studied considering the relation between age and prevalence in the two most contrasted areas: Western area (Area 1) and Southern area (joined Areas 3 and 7). Results showed that the shape of the relationship between age and prevalence in the Southern region differs from the Western region (Fig. 3). In the Western region, seroprevalence in lambs is initially low (<20%) and shows a rapid increase between the age of 12 and 18 months to reach a seroprevalence over 80%. In the Southern region, seroprevalence in lambs is initially higher and shows a slower increase from more than 20% to less than 80% in the same age groups.

3.2.3. Imported samples The raw overall prevalence in imported meat was estimated at 15% (1119%) for lamb meat and at 79% (5893%) for adult meat (Table 2). This estimate was corrected to take into account the lower sensitivity of the detection in imported meat. Corrected prevalence (Table 2) was estimated at 23% (1433%) for lambs and at 97% (81100%) for adults. As for French animals, a strong effect of age was observed (P < 0.0001). No signicant difference between countries was observed. 3.3. Toxoplasma gondii isolation and genotyping All digestion products of 433 heart samples obtained from French carcasses were inoculated into mice. For 29 hearts, isolation of the parasite was not possible because of death of inoculated mice due to bacterial infection. Amongst the 404 remaining samples, age was missing for seven animals. A total of 397 animals were therefore included in the data analyses, of which 48 resulted in the development of toxoplasmosis in inoculated mice. Amongst these animals, 41 were positive with both the ELISA and the MAT tests, two were negative with both of the serological tests, ve were negative with the ELISA only and two were negative with the MAT only. This result corresponds to a prevalence estimate of 5.4% (37.5%) in all French meat production (Table 1). Prevalence estimates was again markedly higher in adults (42% (2757%)) than in lambs (2% (0.73.7%)). Living parasites were isolated from all seven areas of production (Table 1). Genotyping was not possible for two of the 48 seropositive mice. All of the genotypes strains belonged to T. gondii genotype II, except for one strain from the Pyrenees mountains area, which belonged to genotype III. 4. Discussion The present study is, to our knowledge, the rst survey of T. gondii prevalence in livestock using a sampling plan based on meat production data. We believe it is also the rst nationwide study on toxoplasmosis carriage amongst farm animals in France. The estimated prevalence obtained after statistical corrections taking the distribution of meat production into account can be considered as representative of the true seroprevalence of T. gondii amongst sheep carcasses consumed in France. The overall estimate of seroprevalence of T. gondii in sheep carcasses was 17.7% (11.631.5%) for lambs younger than 12 months of age and 89% (73.5100%) for adults older than 12 months of age. Our results thus show that approximately 1/5 of all animals consumed in France have been in contact with, or carry T. gondii. A strong age effect is observed (P < 0.0001). Differences in prevalence between lambs and adults may be related to the oral route of contamination, older animals being more exposed to the parasite and for a longer time than lambs (Waldeland, 1977; Lunden et al., 1994). Those results are in accordance with seroprevalences obtained in different studies (Tenter et al., 2000; Klun et al., 2006; Dumtre et al., 2006).

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Table 2 Toxoplasma gondii seroprevalence results for sheep meat imported from different countries. Mean seroprevalences were obtained with classical statistical analysis and re-evaluated values were obtained after correction for under-estimation. Sample size(number of lambs; number of adults) Mean seroprevalence (95% CI) Lambs < 12 months Total imports UK Spain Ireland Netherlands 374 (276; 98) 177 (167; 10) 97 (20; 77) 60 (52; 8) 40 (37; 3) 0.15 0.19 0.05 0.11 0.16 (0.110.19) (0.120.25) (0.000.15) (0.020.21) (0.040.29) Adults > 12 months 0.79 0.80 0.42 0.88 0.33 (0.580.93) (0.501.00) (0.310.54) (0.581.00) (0.001.00) Total 0.22 0.22 0.35 0.21 0.18 (0.180.28) (0.150.28) (0.250.45) (0.110.32) (0.050.29) Reevaluated seroprevalence mean and (95% CI) by bootstrap Lambs < 12 months 0.23 (0.140.33) 0.28 (0.180.39) 0.1 (00.35) 0.17 (0.060.36) 0.24 (0.080-48) Adults > 12 months 0.97 (0.811) 1 (0.81) 0.74 (0.51) 1 (0.8751) 0.66 (01)

UK, United Kingdom; CI, condence interval.

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Fig. 2. Map of the mean seroprevalence of Toxoplasma gondii-positive samples for lambs. The seven different areas of production are numbered from 1 to 7 (see Fig. 1). Colored areas represent the stratication of seroprevalence in the different geographical areas.

Fig. 3. Predictive probability of acquisition of Toxoplasma gondii infection by sheep during the rst 36 months of their life time in two contrasted regions: Western region and Southern region, areas 3 and 7 (see Fig. 1) being merged in a single Southern region. Ages were clustered by six month clusters after 18 months of age. Results were extrapolated using R 2.7.0 software (R Development Core Team, 2005).

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No signicant differences were observed between seroprevalence calculated for French and imported samples, nor within imported samples from the four exporter countries. However, further analyses would be needed to accurately conrm this result due to a lack of available data on imported meat. For French samples, the sampling plan enabled us to analyse the effect of different parameters: geographical origin, age of the animal and sheep meat production level in the area where the sample was collected. Other parameters such as seasonal variations or herd characteristics could not be taken into account because of the timeframe (6 month period) and because samples were collected in slaughterhouses. Nevertheless, statistical analyses reveal that two major aspects are of importance to explain variations in the prevalences observed: the age of animals, as previously discussed, and the geographic origin of the meat. At rst, signicant differences were observed in prevalence between areas with an increasing gradient from the North-western region to the Southern region. As only one district could be sampled in the Northern region (Area 6), the results obtained from this area were difcult to take into account. In addition, an interesting and unexpected result

of the study was the difference in the evolution of the seroprevalence according to the age of animals from different geographic regions. This result suggests that toxoplasmosis infection kinetics over the lifetime of animals vary according to the area. This phenomenon was observed in two contrasted areas. In the Western region, where sheep density is low, overall prevalence estimates were also low, and predicted age-specic prevalence started from a very low value in <6-month-old lambs but increased sharply around 18 months. In the Southern region, where sheep density is higher, prevalence estimates were also higher, and predicted age-specic prevalence started from a relatively high value in <6month-old animals, with a slow increase with age. Differences in breeding management and production structures probably explain these results (Kijlstra and Jongert, 2008). These could also be due to the particular genetic susceptibility to T. gondii of certain genetic lines of sheep as previously described (Buxton et al., 2007). Further investigations are needed to analyse these differences more precisely.

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Table 3 Generalised linear model of prevalences according to the area, the age of animals and the age-area interaction. Parameter estimate 3.82 1.17 Reference 1.9 2.7 4.14 2.03 1.52 2.23 1.17 0.73 5.02 0.49 0.19 0.73 Standard error 0.96 0.38 Reference 1.86 1.12 326 1.70 1.27 1.02 0.84 0.45 326 0.58 0.56 0.41 P-value: Walds test 7.1 e05 0.0021 Reference 0.31 0.01 0.98 0.23 0.23 0.03 0.16 0.10 0.98 0.39 0.72 0.07

Intercept Age Area 1 (Western) Area Area Area Area Area Area Area Area Area Area Area Area 2 3 4 5 6 7

(Mid-western) (Pyrenees mountains) (Mid-eastern) (Central area) (Northern Area) (South-eastern Area)

2/Age 3/Age 4/Age 5/Age 6/Age 7/Age

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Our study also showed that selected districts for which seroprevalence was higher in lambs corresponded to those for which seroprevalence was also high in humans (Berger et al., 2007). The direct link between lamb infection and human contamination at the area level was difcult to assess because meat is not always consumed in the same area that animals are slaughtered. Nevertheless, this higher seroprevalence of the parasite in several species could be related to an intense circulation of the parasite in those areas, perhaps due to specic environmental conditions (felids densities, climatic specicities, etc.). Those areas may be considered as risky areas for toxoplasmosis infection and specic investigations should be conducted. According to our results, an estimated 5.4% of the ovine carcasses consumed in France harbour live parasites. Bioassays in mice presented a lack of sensitivity (Dubey and Beattie, 1988), especially in cases where the whole heart digest could not be fully inoculated. These estimates probably under-evaluate the effective parasitological prevalence in ovine meat. The very high isolation rate in adult meat is noteworthy but as adult meat is seldom eaten raw, the risk is lower than with lamb meat. On the other hand, the presence of live parasites in the heart does not assert their presence in pieces of carcasses that will be consumed, such as skeletal muscles. Nevertheless, several previous studies showed a persistence of viable tissues cysts in different skeletal muscles as well as in the heart after experimental infection of sheep (Dubey, 1984; Esteban-Redondo et al., 1999). Knowledge of the distribution of the parasite within the body of livestock animals remains lacking (Esteban-Redondo et al., 1999). Finally, those results assume that risk linked to ovine consumption is signicant for toxoplasmosis infection in humans in France. This assertion is corroborated by specic studies in humans (Berger et al., 2007). Special care should be given to lamb consumption, which is likely to be eaten undercooked and thus represents a real risk for toxoplasmosis acquisition in humans. Genotype II was largely predominant amongst isolates found in this study. It is also the most frequently identied genotype in humans in France, as well as in animals and humans throughout Europe (Dard, 2008). Genotyping of 53 T. gondii isolates from United States (US) lambs revealed 57 strains with 15 genotypes (Dubey et al., 2008) indicating high genetic diversity of T. gondii in lambs in the US, which is different from the French situation where genotype II is obviously wide-spread over the country as shown in the present study. Interestingly, one sample was infected with a genotype III parasite, which is rare in France but seems to be more frequent in Portugal or Spain (De Sousa et al., 2006). The lamb harbouring this isolate was from the Spanish border in the Pyrenees mountains area. We believe this is the rst non-type II genotype found in sheep in Europe since all published strains isolated in Europe from sheep or lambs were genotype II: UK (Owen and Trees, 1999), Denmark (Jungersen et al., 2002) and France (Dumtre et al., 2006). It should be noted that data from the UK and Denmark were obtained from aborted lambs and not from sheep slaughtered for human consumption. No survey on slaughtered sheep was undertaken in these countries. The present study offers new and reliable facts about toxoplasmosis infection in sheep meat in France. New questions emerge from the obtained results, concerning the risky areas as well as the evolution of parasite carriage over the course of the lifetime of animals. Further investigations are needed to better understand those new aspects of T. gondii ecology. This study offers a new mapping of a toxoplasmosis pattern amongst sheep consumed in France. This type of survey should also be undertaken for other production animals such as beef, pigs and chickens and comparative studies should be conducted in other countries, in order to provide a wider knowledge on the animal sources of T. gondii for human contamination.

Finally, we propose here an innovative model for a foodborne zoonosis hazard control survey, which can be used as a basis for other investigations.

Acknowledgements We acknowledge the Direction Gnrale de lAlimentation (DGAl) of the French Ministry for Agriculture for nancial (Grant PS TOXO 2007) and informative support. We gratefully thank Pr. S. Bretagne from University Paris XII for great discussion and advice and Dr. Maria Mavris and Malachi ORourke for critical reading of the manuscript. We thank the French members in charge of the report on Toxoplasmosis (AFSSA, 2005), who all have to be associated to this work for their recommendations for investigation of meat as a source of human contamination (F. Derouin, Coordinator of working group, University Paris VII; Ph. Dorchies, Ecole Nationale Vtrinaire, Toulouse; V. Goulet, Institut Veille Sanitaire, Saint Maurice; F. Peyron, University Lyon 1; P. Thulliez, Institut Puriculture, Paris; C. Bultel; S. Tenailleau, Direction Gnrale de la Sant, Paris and S. Roze from AFSSA).

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