We developed an easy-to-use method for genotyping Toxoplasma gondii isolates in a single multiplex PCR assay with 15 microsatellite markers. This method was validated by testing 26 reference isolates that had been characterized with other sets of markers.The commonly used multilocus methods for genotyping Toxoplasma gondii strains employ PCR-restriction fragment length polymorphism (PCR-RFLP) analysis at 10 markers (16), sequencing at 8 introns of 5 loci (14), and length polymorphism analysis of 5 microsatellite (MS) markers (4). Advantages and disadvantages of these different markers have been reviewed elsewhere (16). Based on our experience within the Toxoplasma Biological Resource Center (BRC ToxoBS) and the National Reference Center for Toxoplasmosis, it appears that screening of a large number of clinical isolates for T. gondii strain typing should rely on an easy-to-use method with two different levels of discrimination. The first step of discrimination is the performed at the typing level: that is, employing the ability of markers to distinguish the major clonal lineages from atypical strains. In areas with a marked clonal population structure such as Europe, the genetic screening of clinical isolates should rapidly identify basic type II or III strains, which represent the majority of isolates, and atypical strains, which are the exception to the rule (3, 11). In this context, the identification of atypical strains is of clinical and epidemiological importance because atypical strains are usually associated with severe disease outcomes and contamination of individuals by non-European strains either during residence abroad or after consumption of imported meat (5, 9, 10). The second level of discrimination is the fingerprinting level, representing a high degree of discriminatory power for differentiating closely related strains belonging to the same lineage. This high-resolution analysis is required for identifying laboratory contaminations for diagnosis issues and for establishing a common source of infection among different infected individuals in an outbreak (7,8).Microsatellite (MS) sequences are tandem repeats of short (1 to 6 bp) DNA motifs that are ubiquitous in eukaryotic genomes and undergo length changes due to insertion or deletion of one or multiple repeat units. The most commonly proposed mutation mechanism for MS sequences is strand slippage, occurring predominantly during replication (13). The numbers of repeat motifs differ in a population, thereby creating multiple alleles at an MS locus. MS loci are amplified by PCR using fluorescently labeled forward and unlabeled reverse primers. The dye-labeled products are separated by size using automated electrophoresis and identified by fluorescence detection. We previously designed a multiplex PCR assay with 5 MS markers that were able to reach the typing level but not the fingerprinting level of discrimination between strains (4). Here we developed an easy-to-use and rapid genotyping method which aimed to ensure both levels of genetic discrimina...

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Experimental infections of Galba truncatula with Fasciola hepatica, Fascioloides magna, or Paramphistomum daubneyi were carried out at 20 degrees C to determine if the use of 14-cm petri dishes for breeding lettuce-fed snails enhanced the characteristics of snail infections. Compared to infected snails raised in boxes up to day 30 post-exposure and later in individual 35-mm dishes, the survival of G. truncatula kept in 14-cm dishes and the shell height of cercariae-shedding snails during the first 45 days were higher, whatever the digenean species is. The consequence of such enhanced characteristics was a greater production of metacercariae in the case of F. hepatica (1.7 to 5.6 times higher) and P. daubneyi (2.3 times). In contrast, metacercariae of F. magna were few in number, whatever the method of snail breeding is, and this might be explained by a still incomplete adaptation between the parasite of Czech origin and the French population of G. truncatula. The use of these 14-cm dishes reduced the time necessary for snail maintenance and metacercaria collection, thus allowing a decrease in the cost price of these larvae for commercial production.

An update on the redial generations of Fasciola hepatica was carried out to highlight the different developmental patterns of rediae, the effects of some factors on these generations, and the consequences of such developmental patterns on cercarial productivity. The development of generations is dependent on the behaviour of the first mother redia of the first generation. If this redia remains alive throughout snail infection, it produces most second-generation rediae. In contrast, if it dies during the first weeks, daughter redia formation is ensured by a substitute redia (the second mother redia of the first generation, or the first redia of the second generation). Environmental and biotic factors do not modify the succession of redial generations, but most act by limiting the numbers of rediae, either in all generations, or in the second and/or third generations. An abnormal development of rediae reduces the number of cercariae and most are formed by the second cohort of the first generation. By contrast, most cercariae are produced by the first cohort of the second generation when redial development is normal. The mother rediae described by previous authors might correspond to the first generation and the second cohort of the second generation, while daughter rediae would be the second cohort of the second generation and the first cohort of the third generation. Under certain circumstances, daughter redia formation is ensured by the first two mother rediae or all first-generation rediae, thus demonstrating that the first mother redia is not the only larva to ensure daughter redia formation.

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A total of 59 natural watercress beds in the Limousin region (central France) was surveyed over a 15-year period (1990-2004) to detect the contamination of watercress by the metacercariae of Fasciola hepatica and to determine the presence of larval forms in the two species of lymnaeids which live in these waterholes in June and July. The number of beds contaminated with F. hepatica metacercariae varied over the years, and the burden of the larvae on plants was low: a mean of 2.6-6.3 per bed. The same variability was also noted for natural infections of Galba truncatula with F. hepatica, as the annual prevalences ranged from 1.2% to 2.4%. Natural infections of Omphiscola glabra with F. hepatica were only detected from 1996 and the annual prevalences subsequently increased up to 1.4-1.8% between 2001 and 2004. However, for both lymnaeids, the variations in these prevalences with year were insignificant. The contamination of these beds with F. hepatica over the past 15 years was similar to that recorded in the same sites between 1970 and 1986. The main changes were the appearance of another digenea, Paramphistomum daubneyi, in the beds, and the possibility for O. glabra to naturally sustain the larval development of F. hepatica.

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The severity of toxoplasmic infection depends mainly on the immune status of the host, but also on the Toxoplasma gondii strains, which differ by their virulence profile. The relationship between the human host and T. gondii has not yet been elucidated because few studies have been conducted on human models. The immune mechanisms involved in the persistence of T. gondii in the brains of immunocompetent subjects and during the reactivation of latent infections are still unclear. In this study, we analyzed the kinetics of immune mediators in human nervous cells in vitro, infected with two strains of T. gondii. Human neuroblast cell line (SH SY5Y), microglial (CMH5) and endothelial cells (Hbmec) were infected separately by RH (type I) or PRU (type II) strains for 8 h, 14 h, 24 h and 48 h (ratio 1 cell: 2 tachyzoites). Pro-inflammatory protein expression was different between the two strains and among different human nervous cells. The cytokines IL-6, IL-8 and the chemokines MCP-1 and GROα, and SERPIN E1 were significantly increased in CMH5 and SH SY5Y at 24 h pi. At this point of infection, the parasite burden declined in microglial cells and neurons, but remained high in endothelial cells. This differential effect on the early parasite multiplication may be correlated with a higher production of immune mediators by neurons and microglial cells compared to endothelial cells. Regarding strain differences, PRU strain, but not RH strain, stimulates all cells to produce pro-inflammatory growth factors, G-CSF and GM-CSF. These proteins could increase the inflammatory effect of this type II strain. These results suggest that the different protein expression profiles depend on the parasitic strain and on the human nervous cell type, and that this could be at the origin of diverse brain lesions caused by T. gondii.