Anatomical distribution of Toxoplasma gondii in naturally and experimentally infected lambs.

Consumption of raw or undercooked meat containing Toxoplasma gondii tissue cysts is one of the main sources of infection for humans worldwide. Among the various species intended for human consumption, sheep appear to be a high risk for human infection. The present study focused on the detailed anatomical distribution of Toxoplasma gondii in naturally and experimentally infected lambs using fresh and frozen samples of various pieces of meat, from a public health perspective. The first objective was to rank the edible parts intended for human consumption according to the detectable parasite burden by real-time PCR targeting the 529-bp repeated element. The second objective was to evaluate the impact of freezing by comparing the detection efficiency of the quantitative PCR between fresh and frozen tissues, as imports of lamb carcasses/cuts may arrive frozen or chilled. The highest estimated parasite loads were observed in skeletal muscles, and more particularly in edible portions such as quadriceps femoris muscle, intercostal muscles, deltoid muscle and diaphragm, with a significant difference in detectable parasite burden between fresh and frozen samples (p < 0.0001) or natural and experimental infection (p < 0.0001). Thoracic and pelvic limbs (3278-1048 parasites/g muscle) were ranked at the top of the list. Toxoplasma gondii DNA was detected in all the edible parts of lamb studied. These results suggest that lamb meat represents a risk for consumers. Further investigations are needed in order to confirm these differences in larger numbers of animals and in different breeds.

Title: Distribution anatomique de Toxoplasma gondii chez des agneaux infectés naturellement et expérimentalement. Abstract: La consommation de viande crue ou insuffisamment cuite contenant des kystes tissulaires de Toxoplasma gondii est l'une des principales sources d'infection pour l'homme dans le monde. Parmi les différentes espèces destinées à la consommation humaine, le mouton apparaît à haut risque d'infection humaine. La présente étude s'est concentrée sur une distribution anatomique détaillée de Toxoplasma gondii chez des agneaux infectés naturellement et expérimentalement à l'aide d'échantillons frais et congelés de divers morceaux de viande, du point de vue de la santé publique. Classer les parties comestibles destinées à la consommation humaine, selon la charge parasitaire détectable par une PCR en temps réel ciblant l'élément répété de 529 pb était un premier objectif. Un second objectif était d'évaluer l'impact de la congélation en comparant l'efficacité de détection de la PCR quantitative entre les tissus frais et congelés, car les importations de carcasses/coupes d'agneau peuvent arriver congelées ou réfrigérées. Les charges parasitaires estimées les plus élevées ont été observées dans les muscles squelettiques et plus particulièrement dans les parties comestibles telles que le quadriceps fémoral, les muscles intercostaux, le deltoïde et le diaphragme avec une différence significative de charge parasitaire détectable entre les échantillons frais et congelés (p < 0,0001) ou l'infection naturelle et expérimentale (p < 0,0001). Les membres thoraciques et pelviens (3278 à 1048 parasites/g de muscle) ont été classés en tête de liste. L'ADN de T. gondii a été détecté dans toutes les parties comestibles étudiées de l'agneau. Ces résultats suggèrent que l'agneau représente un risque pour les consommateurs. Des investigations supplémentaires doivent être effectuées afin de confirmer les différences mentionnées ci-dessus chez plus d'animaux et dans différentes races.

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.

New strategy for the survey of Toxoplasma gondii in meat for human consumption.

Monitoring of Toxoplasma infection in animals destined for human consumption is a great challenge for human toxoplasmosis prevention. This study aimed to compare results obtained from a naturally infected population of sheep using different tests and targeting an original matrix: meat samples and muscle fluids collected at the slaughterhouse. A commercial ELISA test was performed on diaphragm fluids from 419 ovine carcasses collected at the slaughterhouse. A MAT (modified agglutination test) was performed on heart fluids obtained from the same animals. In addition, all hearts were bioassayed in mice. Serological test agreement, the relative sensitivity of ELISA MAT and mouse bioassay as well as a correlation between titres and parasite isolation probability were statistically evaluated. The overall agreement (kappa coefficient=0.64) of ELISA on diaphragm fluids and MAT on heart fluids is substantial and subsequently both tests can be used for epidemiological studies. Relative sensitivity was higher for MAT performed on cardiac fluids (90%) than ELISA on diaphragm fluid (61%). For both serological tests, relative sensitivity is lower in lambs younger than 12 months. Relative sensitivity of mouse inoculation was 42%. A significant correlation was obtained between increasing MAT titres and probability to isolate live parasite from the heart. When the fluid titre was higher than 1:16, parasites were isolated in 65% of cases. When it was lower, isolation failed in 95% of the cases. According to our results, cardiac fluids appear to be a relevant matrix for toxoplasmosis survey in meat.

Primary in vitro culture of porcine tracheal epithelial cells in an air-liquid interface as a model to study airway epithelium and Aspergillus fumigatus interactions.

Since the airway epithelium is the first tissue encountered by airborne fungal spores, specific models are needed to study this interaction. We developed such a model using primary porcine tracheal epithelial cells (PTEC) as a possible alternative to the use of primary human cells. PTEC were obtained from pigs and were cultivated in an air-liquid interface. Fluorescent brightener was employed to quantify the internalization of Aspergillus fumigatus conidia. Potential differences (Vt) and transepithelial resistances (Rt) after challenge with the mycotoxin, verruculogen, were studied. Primers for porcine inflammatory mediator genes IL-8, TNF-alpha, and GM-CSF were designed for a quantitative real-time PCR procedure to study cellular responses to challenges with A. fumigatus conidia. TEM showed the differentiation of ciliated cells and the PTEC ability to internalize conidia. The internalization rate was 21.9 ± 1.4% after 8 h of incubation. Verruculogen (10(-6) M) significantly increased Vt without having an effect on the Rt. Exposure of PTEC to live A. fumigatus conidia for 24 h induced a 10- to 40-fold increase in the mRNA levels of inflammatory mediator genes. PTEC behave similarly to human cells and are therefore a suitable alternative to human cells for studying interaction between airway epithelium and A. fumigatus.

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

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 (<12months) 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% (11.6-31.5%) for lambs and 89% (73.5-100%) for adults (P<0.0001). No significant 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% (3-7.5%) (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.

Establishment and characterization of continuous hematopoietic progenitors-derived pig normal mast cell lines.

Mast cells (MCs) are tissue resident, hematopoietic stem cells-derived elements, distributed throughout the body. They are the pivotal mediating cells of allergic reactions. In addition, in mice, MCs play a critical role in the defense against several pathogens, such as bacteria, parasites and viruses. Whereas the biology of rodent and human MCs has been extensively studied using in vitro derived populations, the role of MCs in pigs has not yet been evaluated, given the very low availability of pure porcine MCs populations. In the present report, we describe an original method to obtain continuous factor-dependent normal pig MCs (PMC) lines from fetal hematopoietic progenitors. These Stem Cell Factor (SCF) and Interleukin-3- (IL-3)-dependent PMC lines retain their capacity to growth after conventional freezing methods and exhibit most of the morphological and biochemical properties of normal, although immature, MCs, including metachromatic granules containing sulfated polysaccharides, the expression of c-kit and high-affinity IgE receptors (FcepsilonRI), and the ability to store histamine that is released upon cross-linking of FcepsilonRI. In vitro derived PMC lines might thus be valuable tools to further investigate the reactivity of these elements towards several parasites frequently encountered in pig, such as, but not limited to, Ascaris suum, Trichinella spiralis or Trichuris suis, or towards antigens derived from these pathogens.

Bartonella chomelii sp. nov., isolated from French domestic cattle (Bos taurus).

Two strains of bacteria isolated from the blood of French domestic cows were found to be similar to Bartonella species on the basis of phenotypic characteristics. Genotypic analysis based on sequence comparison of the 16S rRNA and citrate synthase (gltA) genes and on DNA-DNA hybridization showed that the two isolates represent a distinct and new species of Bartonella. Moreover, the phylogenetic analysis inferred from comparison of 16S rRNA and gltA sequences demonstrated that the new Bartonella species is related to other ruminant-derived Bartonella species. The name Bartonella chomelii is proposed for the new species. The type strain of Bartonella chomelii sp. nov. is A828T (=CIP 107869T=CCUG47497T).