Toxoplasma gondii in beef consumed in France: regional variation in seroprevalence and parasite isolation.

In France, the consumption of cattle and sheep meat appears to be a risk factor for infection of pregnant women with Toxoplasma gondii. Several nation-wide surveys in France have investigated the prevalence of T. gondii in sheep and pig meat, but little is known at present about the prevalence of the parasite in beef. The main objective of the present cross-sectional survey was to estimate the seroprevalence of T. gondii infection in beef consumed in France. A secondary objective was to attempt to isolate T. gondii from cattle tissues and to study the geographical and age variations of this seroprevalence. The overall estimate of seroprevalence of T. gondii in bovine carcasses (n = 2912), for a threshold of 1:6 was 17.38%. A strong age effect was observed (p < 0.0001) with a seroprevalence of 5.34% for calves (<8 months) and 23.12% for adults (>8 months). Seroprevalence estimates given by area of birth and area of slaughtering for adults showed that the areas with the highest seroprevalence were not the same between these two variables. Only two strains, corresponding to genotype II, were isolated from heart samples, indicating that there is a limited risk of human infection with T. gondii, which needs to be correlated with the food habit of consuming raw or undercook (bleu or saignant) beef. However, new questions have emerged, especially concerning the isolation of parasites from beef and the precise role of bovines, generally described as poor hosts for T. gondii, in human infection.

TITLE: Toxoplasma gondii dans la viande bovine consommée en France : variation régionale de la séroprévalence et isolement de parasites. ABSTRACT: En France, la consommation de viande bovine et ovine apparaît comme un facteur de risque pour la contamination des femmes enceintes par Toxoplasma gondii. Plusieurs enquêtes nationales ont été réalisées afin de déterminer le niveau de contamination par T. gondii de la viande ovine et porcine, en France, mais très peu est encore connu quant à la prévalence du parasite dans la viande bovine. La présente enquête transversale avait pour objectif principal d'estimer la séroprévalence de l'infection à T. gondii dans la viande bovine consommée en France, ainsi que d'isoler T. gondii à partir de tissus de bovins et d'étudier, à titre d'objectif secondaire, les variations géographiques et d'âge de cette prévalence. L'estimation globale de la séroprévalence de T. gondii dans les carcasses de bovins (n = 2912) était de 17,38 % (pour un seuil de dilution à 1:6). Un effet significatif de l'âge a été observé (p < 0,0001) avec une séroprévalence de 5,34 % pour les veaux (<8 mois) et de 23,12 % pour les adultes (>8 mois). Les estimations de séroprévalence données par zone de naissance et par zone d'abattage pour les adultes montrent que les zones de séroprévalence les plus élevées n'étaient pas les mêmes pour ces deux variables. Seulement deux souches, de génotype II, ont été isolées à partir d'échantillons de cœurs, soulignant que le risque d'infection humaine est limité, mais doit être corrélé avec les habitudes de consommation alimentaire de la viande bovine peu/pas cuite (bleu ou saignante). Cependant, de nouvelles questions se posent, notamment en ce qui concerne l'isolement du parasite à partir de la viande bovine, ainsi que le rôle précis des bovins, généralement décrits comme des hôtes médiocres pour T. gondii, dans la contamination humaine.

Optimization of the cryopreservation of biological resources, Toxoplasma gondii tachyzoites, using flow cytometry.

The conservation of Toxoplasma gondii strains isolated from humans and animals is essential for conducting studies on Toxoplasma. Conservation is the main function of the French Biological Toxoplasma Resource Centre (BRC Toxoplasma, France, http://www.toxocrb.com/). In this study, we have determined the suitability of a standard cryopreservation methodology for different Toxoplasma strains using the viability of tachyzoites assayed by flow cytometry with dual fluorescent labelling (calcein acetoxymethyl ester and propidium iodide) of tachyzoites. This method provides a comparative quantitative assessment of viability after thawing. The results helped to define and refine quality criteria before tachyzoite cryopreservation and optimization of the cryopreservation parameters. The optimized cryopreservation method uses a volume of 1.0 mL containing 8 × 10(6) tachyzoites, in Iscove's Modified Dulbecco's Medium (IMDM) containing 10% foetal calf serum (FCS). The cryoprotectant additive is 10% v/v Me2SO without incubation. A cooling rate of ∼1 °C/min to -80 °C followed, after 48 h, by storage in liquid nitrogen. Thawing was performed using a 37 °C water bath that produced a warming rate of ∼100 °C/min, and samples were then diluted 1:5 in IMDM with 5% FCS, and centrifuged and resuspended for viability assessment.

Evaluation of a strategy for Toxoplasma gondii oocyst detection in water.

Several recent outbreaks of toxoplasmosis were related to drinking water. We propose a strategy for Toxoplasma oocyst detection as part of an approach to detecting multiple waterborne parasites, including Giardia and Cryptosporidium spp., by the U.S. Environmental Protection Agency method with the same sample. Water samples are filtered to recover Toxoplasma oocysts and purified on a sucrose density gradient. Detection is based on PCR and mouse inoculation (bioassay) to determine the presence and infectivity of recovered oocysts. In an experimental seeding assay with 100 liters of deionized water, a parasite density of 1 oocyst/liter was successfully detected by PCR in 60% of cases and a density of 10 oocysts/liter was detected in 100% of cases. The sensitivity of the PCR assay varied from less than 10 to more than 1000 oocysts/liter, depending on the sample source. PCR was always more sensitive than mouse inoculation. This detection strategy was then applied to 139 environmental water samples collected over a 20-month period. Fifty-three samples contained PCR inhibitors, which were overcome in 39 cases by bovine serum albumin addition. Among 125 interpretable samples, we detected Toxoplasma DNA in 10 cases (8%). None of the samples were positive by mouse inoculation. This strategy efficiently detects Toxoplasma oocysts in water and may be suitable as a public health sentinel method.