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We present a case of disseminated congenital toxoplasmosis in a newborn born to a mother who had been immunized against toxoplasmosis before conception. The mother was reinfected, likely by ingestion of imported raw horse meat during pregnancy. This clinical presentation is exceptional in France and raised the possibility of infection by a highly virulent Toxoplasma strain. The strain responsible was isolated from the peripheral blood of the newborn, and when genotyped with microsatellite markers, it exhibited an atypical genotype, one which is very uncommon in Europe but had been described in South America. We tested the hypothesis of a reinfection with a different genotype by using an experimental mouse model, which confirmed that acquired immunity against European Toxoplasma strains may not protect against reinfection by atypical strains acquired during travel outside Europe or by eating imported meat.

Detection of Toxoplasma gondii oocysts in environmental samples is a great challenge for researchers as this coccidian parasite can be responsible for severe infections in humans and in animals via ingestion of a single oocyst from contaminated water, soil, fruits or vegetables. Despite field investigations, oocysts have been rarely recovered from the environment due to the lack of sensitive methods. Immunomagnetic separation, fluorescence-activated cell sorting, and polymerase chain reaction have recently shown promising use in detection of protozoa from complex matrices. Such procedures could be applied to T. gondii detection, if studies on the antigenic and biochemical composition of the oocyst wall are completed. Using such methods, it will be possible to assess the occurrence, prevalence, viability and virulence of T. gondii oocysts in environmental matrices and specify sources of human and animal contamination.

ABSTRACTToxoplasma gondiioocysts 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.

Giardia duodenalis, Cryptosporidium spp. and Toxoplasma gondii are protozoan parasites that have been highlighted as emerging foodborne pathogens by the Food and Agriculture Organization of the United Nations and the World Health Organization. According to the European Food Safety Authority, 4786 foodborne and waterborne outbreaks were reported in Europe in 2016, of which 0.4% were attributed to parasites including Cryptosporidium, Giardia and Trichinella. Until 2016, no standardized methods were available to detect Giardia, Cryptosporidium and Toxoplasma (oo)cysts in food. Therefore, no regulation exists regarding these biohazards. Nevertheless, considering their low infective dose, ingestion of foodstuffs contaminated by low quantities of these three parasites can lead to human infection. To evaluate the risk of protozoan parasites in food, efforts must be made towards exposure assessment to estimate the contamination along the food chain, from raw products to consumers. This requires determining: (i) the occurrence of infective protozoan (oo)cysts in foods, and (ii) the efficacy of control measures to eliminate this contamination. In order to conduct such assessments, methods for identification of viable (i.e. live) and infective parasites are required. This review describes the methods currently available to evaluate infectivity and viability of G. duodenalis cysts, Cryptosporidium spp. and T. gondii oocysts, and their potential for application in exposure assessment to determine the presence of the infective protozoa and/or to characterize the efficacy of control measures. Advantages and limits of each method are highlighted and an analytical strategy is proposed to assess exposure to these protozoa.

A multiplex PCR assay was designed for multilocus strain typing of Toxoplasma gondii based on length polymorphism of five microsatellite markers. Eight T. gondii strains already sequenced at these five markers were used as control isolates. This method is simple, rapid, reproducible, and adapted to a large set of isolates.

The ability of microorganisms to survive under extreme conditions is closely related to the physicochemical properties of their wall. In the ubiquitous protozoan parasite Toxoplasma gondii, the oocyst stage possesses a bilayered wall that protects the dormant but potentially infective parasites from harsh environmental conditions until their ingestion by the host. None of the common disinfectants are effective in killing the parasite because the oocyst wall acts as a primary barrier to physical and chemical attacks. Here, we address the structure and chemistry of the wall of the T. gondii oocyst by combining wall surface treatments, fluorescence imaging, EM, and measurements of its mechanical characteristics by using atomic force microscopy. Elasticity and indentation measurements indicated that the oocyst wall resembles common plastic materials, based on the Young moduli, E, evaluated by atomic force microscopy. Our study demonstrates that the inner layer is as robust as the bilayered wall itself. Besides wall mechanics, our results suggest important differences regarding the nonspecific adhesive properties of each layer. All together, these findings suggest a key biological role for the oocyst wall mechanics in maintaining the integrity of the T. gondii oocysts in the environment or after exposure to disinfectants, and therefore their potential infectivity to humans and animals.oocyst structure | oocyst integrity | oocyst resistance | Toxoplasma infectivity | transmission