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.

Validation of the Use of Dried Blood Samples for the Detection of Toxoplasma gondii Antibodies in Stray Cats (Felis s. catus).

If validated beforehand, the analysis of dried blood on blotting paper (BP samples) is very useful for monitoring free-ranging animals. We aimed to validate this method for the detection of antibodies against Toxoplasma gondii in stray cats. We used the modified agglutination test (MAT) in 199 sample pairs of sera and BP samples from 54, 39, 56, and 50 cats trapped during four periods in five dairy farms. Screening was at 1:6, 1:12, and 1:24 dilutions. The cut-off value was at MAT titre ≥ 24, but MAT titre ≥ 12 was also considered for BP samples that often have a higher dilution level. Depending on the period, sample type, and cut-off value, sensitivity of the analysis of the BP sample vs. serum varied from 87.1% to 100% and specificity ranged from 72.22% to 100%. The concordance values and Kappa coefficient showed a substantial to excellent agreement between the results of the two methods, whatever the cut-off value. These findings quantifiably validate the use of MAT on BP samples for the detection of antibodies to T. gondii in stray cats, but we recommend expressing results from BP samples with several cut-off values as the MAT titres tend to be lower than those of sera.

Exposure of pelagic seabirds to Toxoplasma gondii in the Western Indian Ocean points to an open sea dispersal of this terrestrial parasite.

Toxoplasma gondii is a protozoan parasite that uses felids as definitive hosts and warm-blooded animals as intermediate hosts. While the dispersal of T. gondii infectious oocysts from land to coastal waters has been well documented, transmission routes to pelagic species remain puzzling. We used the modified agglutination test (MAT titre ≥ 10) to detect antibodies against T. gondii in sera collected from 1014 pelagic seabirds belonging to 10 species. Sampling was carried out on eight islands of the Western Indian Ocean: Reunion and Juan de Nova (colonized by cats), Cousin, Cousine, Aride, Bird, Europa and Tromelin islands (cat-free). Antibodies against T. gondii were found in all islands and all species but the great frigatebird. The overall seroprevalence was 16.8% [95% CI: 14.5%-19.1%] but significantly varied according to species, islands and age-classes. The low antibody levels (MAT titres = 10 or 25) detected in one shearwater and three red-footed booby chicks most likely resulted from maternal antibody transfer. In adults, exposure to soils contaminated by locally deposited oocysts may explain the detection of antibodies in both wedge-tailed shearwaters on Reunion Island and sooty terns on Juan de Nova. However, 144 adults breeding on cat-free islands also tested positive. In the Seychelles, there was a significant decrease in T. gondii prevalence associated with greater distances to cat populations for species that sometimes rest on the shore, i.e. terns and noddies. This suggests that oocysts carried by marine currents could be deposited on shore tens of kilometres from their initial deposition point and that the number of deposited oocysts decreases with distance from the nearest cat population. The consumption of fishes from the families Mullidae, Carangidae, Clupeidae and Engraulidae, previously described as T. gondii oocyst-carriers (i.e. paratenic hosts), could also explain the exposure of terns, noddies, boobies and tropicbirds to T. gondii. Our detection of antibodies against T. gondii in seabirds that fish in the high sea, have no contact with locally contaminated soils but frequent the shores and/or consume paratenic hosts supports the hypothesis of an open-sea dispersal of T. gondii oocysts by oceanic currents and/or fish.

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.

A multi-event capture-recapture analysis of Toxoplasma gondii seroconversion dynamics in farm cats.

BACKGROUND: Domestic cats play a key role in the epidemiology of the parasite Toxoplasma gondii by excreting environmentally-resistant oocysts that may infect humans and other warm-blooded animals. The dynamics of Toxoplasma gondii seroconversion, used as a proxy for primo-infection dynamics, was investigated in five cat populations living on farms. METHODS: Serological tests on blood samples from cats were performed every three months over a period of two years, for a total of 400 serological tests performed on 130 cats. Variations in seroconversion rates and associated factors were investigated using a multi-event capture-recapture modelling approach that explicitly accounted for uncertainties in cat age and serological status. RESULTS: Seroprevalence varied between farms, from 15 to 73%, suggesting differential exposure of cats to T. gondii. In farms with high exposure, cats could become infected before reaching the age of six months. Seroconversion rates varied from 0.42 to 0.96 seroconversions per cat per year and were higher in autumn and winter than in spring and summer. CONCLUSION: Our results suggest inter-farm and seasonal variations in the risks of exposure to T. gondii oocysts for humans and livestock living on farms. The paper also discusses the role of young cats in the maintenance of environmental contamination by T. gondii oocysts on farms.

Toxoplasmosis in the Caribbean islands: literature review, seroprevalence in pregnant women in ten countries, isolation of viable Toxoplasma gondii from dogs from St. Kitts, West Indies with report of new T. gondii genetic types.

Little is currently known of clinical toxoplasmosis in humans and animals in the Caribbean. We investigated the prevalence of IgG and IgM antibodies in 437 pregnant women from 10 English speaking Caribbean countries. Overall, antibodies (IgG) to Toxoplasma gondii (modified agglutination test, MAT, cut-off 1:6) were found in 174 (39.8 %) of 437 human sera; specifically 12 of 38 from Antigua-Barbuda, 26 of 52 from Belize, 9 of 50 from Bermuda, 29 of 49 from Dominica, 18 of 49 from Grenada, 16 of 47 from Jamaica, 5 of 15 from Montserrat, 8 of 44 from St. Kitts/Nevis, 24 of 45 from St. Lucia, and 27 of 50 from St. Vincent/Grenadines were seropositive. All IgG-positive sera were tested for IgM antibodies using the immunocapture method; all sera were negative for IgM antibodies. Additionally, tissues and sera of 45 dogs from St. Kitts were examined for T. gondii infection. Antibodies (IgG, MAT, 1:≥25) were found in 19 (42.2 %) of 45 dogs. Muscle samples (tongue, leg) of 19 seropositive dogs were digested in pepsin, and homogenates were bioassayed in mice. Viable T. gondii were isolated from 6 dogs. T. gondii isolates were further propagated in cell culture. PCR-RFLP genotyping of cell culture derived tachyzoites using 10 genetic markers, SAG1, SAG2 (5' and 3' SAG2, and alt.SAG2) SAG3, BTUB, GRA6, c22-8, c29-2, L358, PK1, and Apico revealed that 4 isolates were ToxoDB PCR-RFLP genotype #2, and 2 were new genotypes #264 and #265. Review of 22 viable T. gondii isolates from chickens, dogs, and cats from Grenada and St. Kitts revealed that 1 isolate was type II, 13 were type III, and 8 were atypical. Thus, type III strains were predominant. Overall, the study revealed high prevalence of T. gondii in the Caribbean islands.

Species or local environment, what determines the infection of rodents by Toxoplasma gondii?

Toxoplasmosis is largely present in rural areas but its spatial distribution in this environment remains poorly known. In particular, it is unclear if areas of high density of cats, the only hosts excreting Toxoplasma gondii, constitute foci of high prevalence. To improve our understanding of the spatial distribution of T. gondii in rural areas, we performed a serological survey in rodents from two villages in France. We trapped 710 rodents including commensal rats and meadow or forest voles and mice. The presence of T. gondii was examined using PCR, mice inoculation and modified agglutination test for antibodies (MAT). We conducted multivariate and discriminant analyses to identify biological, ecological or spatial variables that could explain T. gondii serology in rodents. We then used a logistic regression to assess the relative influence of each explanatory variable. Overall seroprevalence was 4.1%. Commensal-rats were more infected (12.5%) than non-commensal species (3.7%). However, the major determinant of the risk of infection was the distance to the nearest farm (OR = 0.75 for 100 m), which explained the risk in all species or non-commensal species only. We contrast the role of species characteristics and that of the local environment, and discuss the risk of environmental contamination for humans.

Quantitative estimation of the viability of Toxoplasma gondii oocysts in soil.

Toxoplasma gondii oocysts spread in the environment are an important source of toxoplasmosis for humans and animal species. Although the life expectancy of oocysts has been studied through the infectivity of inoculated soil samples, the survival dynamics of oocysts in the environment are poorly documented. The aim of this study was to quantify oocyst viability in soil over time under two rain conditions. Oocysts were placed in 54 sentinel chambers containing soil and 18 sealed water tubes, all settled in two containers filled with soil. Containers were watered to simulate rain levels of arid and wet climates and kept at stable temperature for 21.5 months. At nine sampling dates during this period, we sampled six chambers and two water tubes. Three methods were used to measure oocyst viability: microscopic counting, quantitative PCR (qPCR), and mouse inoculation. In parallel, oocysts were kept refrigerated during the same period to analyze their detectability over time. Microscopic counting, qPCR, and mouse inoculation all showed decreasing values over time and highly significant differences between the decreases under dry and damp conditions. The proportion of oocysts surviving after 100 days was estimated to be 7.4% (95% confidence interval [95% CI] = 5.1, 10.8) under dry conditions and 43.7% (5% CI = 35.6, 53.5) under damp conditions. The detectability of oocysts by qPCR over time decreased by 0.5 cycle threshold per 100 days. Finally, a strong correlation between qPCR results and the dose infecting 50% of mice was found; thus, qPCR results may be used as an estimate of the infectivity of soil samples.

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.

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.