Jean-Mathieu Bart

Practical Approach for Typing Strains of Leishmania infantum by Microsatellite Analysis

ABSTRACT Currently the universally accepted standard procedure for characterizing and identifying strains of Leishmania is isoenzyme analysis. However, in the Mediterranean area, despite their very wide geographical distribution, most Leishmania infantum strains belong to zymodeme MON-1. In order to increase our understanding of polymorphism in strains of L. infantum, we developed PCR assays amplifying 10 microsatellites and sequenced PCR products. The discriminative power of microsatellite analysis was tested by using a panel of 50 L. infantum strains collected from patients and dogs from Spain, France, and Israel, including 32 strains belonging to zymodeme MON-1, 8 strains belonging to zymodemes MON-24, MON-29, MON-33, MON-34, or MON-80, and 10 untyped strains. Five of the microsatellites were polymorphic, revealing 22 genotypes, whereas the five remaining microsatellites were not variable. In particular, MON-1 strains could be separated into 13 different closely related genotypes. MON-33 and MON-34 strains also gave two additional genotypes closely related to MON-1, while MON-29, MON-24, and MON 80 strains exhibited more divergent genotypes. Among the foci examined, the Catalonian focus displayed a high polymorphism, probably reflecting isoenzyme polymorphism, while the Israeli focus exhibited a low polymorphism that could be consistent with the recent reemergence and rapid spread of canine leishmaniasis in northern and central Israel. The strains originating from the south of France and the Madrid, Spain, area displayed significant microsatellite polymorphism even though they were monomorphic by isoenzyme analysis. In conclusion, microsatellite polymorphism exhibits a high discriminative power and appears to be suitable for characterization of closely related strains of L. infantum in epidemiological studies.

Genotyping of human cystic echinococcosis in Xinjiang, PR China

The Xinjiang Uygur Autonomous Region, multi-ethnic province in northwestern China, is one of the most important foci of human cystic echinococcosis (CE) in the world. Two Echinococcus granulosus genotypes (G1 and G6) are known to infect the intermediate hosts in this area but, to date, the source of the human infection remains unclear. The current study aimed to genetically analyse 67 hydatid cysts removed from 47 CE patients for which epidemiological, clinical and serological data were also recorded. Mitochondrial cox 1 gene sequencing suggested that the E. granulosus G1 genotype is the major source of infection (45/47 CE patients). Nevertheless, for the first time in China, 2 patients were found with hydatid cysts of the G6 genotype. In addition, 45 E. granulosus gravid tapeworms, isolated from 13 dogs, were genotyped. The majority of adult worms (42/45) exhibited the G1 genotype, whereas 3 adult tapeworms with the G6 genotype were found in one dog, that also harboured E. granulosus tapeworms of the G1 genotype. This sympatric occurrence of G1 and G6 genotypes of E. granulosus, not only in the same area but also in the same definitive host, raises the interesting question of putative genetic recombination between these E. granulosus genotypes.

Taxonomic position and geographical distribution of the common sheep G1 and camel G6 strains of Echinococcus granulosus in three African countries

The taxonomic and phylogenetic status of Echinococcus granulosus strains are still controversial and under discussion. In the present study, we investigated the genetic polymorphism of E. granulosus isolates originating from three countries of Africa, including a region of Algeria, where the common G1 sheep and the camel G6 strains coexist sympatrically. Seventy-one hydatid cysts were collected from sheep, cattle, camels, and humans. Two mitochondrial markers (cox1 and nad1) were used for strain identification. Two nuclear markers (actII and hbx2) were used to study the possible occurrence of cross-fertilization. Despite the heterogeneity observed among the G1 isolates, they were all localized within one robust cluster. A second strong cluster was also observed containing all of the G6 isolates. Both strains appeared as two distinct groups, and no cases of interbreeding were found. Thus, the attribution of a species rank can be suggested. We also found the Tasmanian sheep G2 strain for the first time in Africa. Because of the slight variations observed between the common sheep and the Tasmanian sheep strains, further studies should be carried out to elucidate the epidemiological relevance of this genetic discrimination.

Genetic diversity of the cestode Echinococcus multilocularis in red foxes at a continental scale in Europe

Background Alveolar echinococcosis (AE) is a severe helminth disease affecting humans, which is caused by the fox tapeworm Echinococcus multilocularis. AE represents a serious public health issue in larger regions of China, Siberia, and other regions in Asia. In Europe, a significant increase in prevalence since the 1990s is not only affecting the historically documented endemic area north of the Alps but more recently also neighbouring regions previously not known to be endemic. The genetic diversity of the parasite population and respective distribution in Europe have now been investigated in view of generating a fine-tuned map of parasite variants occurring in Europe. This approach may serve as a model to study the parasite at a worldwide level. Methodology/Principal Findings The genetic diversity of E. multilocularis was assessed based upon the tandemly repeated microsatellite marker EmsB in association with matching fox host geographical positions. Our study demonstrated a higher genetic diversity in the endemic areas north of the Alps when compared to other areas. Conclusions/Significance The study of the spatial distribution of E. multilocularis in Europe, based on 32 genetic clusters, suggests that Europe can be considered as a unique global focus of E. multilocularis, which can be schematically drawn as a central core located in Switzerland and Jura Swabe flanked by neighbouring regions where the parasite exhibits a lower genetic diversity. The transmission of the parasite into peripheral regions is governed by a “mainland–island” system. Moreover, the presence of similar genetic profiles in both zones indicated a founder event.

Assessment of Use of Microsatellite Polymorphism Analysis for Improving Spatial Distribution Tracking of Echinococcus multilocularis

ABSTRACT Alveolar echinococcosis (AE)—caused by the cestode Echinococcus multilocularis—is a severe zoonotic disease found in temperate and arctic regions of the northern hemisphere. Even though the transmission patterns observed in different geographical areas are heterogeneous, the nuclear and mitochondrial targets usually used for the genotyping of E. multilocularis have shown only a marked genetic homogeneity in this species. We used microsatellite sequences, because of their high typing resolution, to explore the genetic diversity of E. multilocularis. Four microsatellite targets (EmsJ, EmsK, and EmsB, which were designed in our laboratory, and NAK1, selected from the literature) were tested on a panel of 76 E. multilocularis samples (larval and adult stages) obtained from Alaska, Canada, Europe, and Asia. Genetic diversity for each target was assessed by size polymorphism analysis. With the EmsJ and EmsK targets, two alleles were found for each locus, yielding two and three genotypes, respectively, discriminating European isolates from the other groups. With NAK1, five alleles were found, yielding seven genotypes, including those specific to Tibetan and Alaskan isolates. The EmsB target, a tandem repeated multilocus microsatellite, found 17 alleles showing a complex pattern. Hierarchical clustering analyses were performed with the EmsB findings, and 29 genotypes were identified. Due to its higher genetic polymorphism, EmsB exhibited a higher discriminatory power than the other targets. The complex EmsB pattern was able to discriminate isolates on a regional and sectoral level, while avoiding overdistinction. EmsB will be used to assess the putative emergence of E. multilocularis in Europe.

Genetic diversity of Echinococcus multilocularis on a local scale

Echinococcusmultilocularis is the causative agent of human Alveolar Echinococcosis (AE), and it is one of the most lethal zoonotic infections in the Northern Hemisphere. In France, the eastern and central regions are endemic areas; Franche-Comté, Lorraine and Auvergne are particularly contaminated. Recently, several human cases were recorded in the French Ardennes area, a region adjacent to the western border of the E. multilocularis range in France. A previous study in this focus described a prevalence of over 50% of the parasite in red foxes. The present study investigated the genetic diversity of adult worms collected from foxes in a 900km(2) area in the Ardennes. Instead of a conventional mitochondrial target (ATP6), two microsatellite targets (EmsB and NAK1) were used. A total of 140 adult worms isolated from 25 red foxes were genotyped. After hierarchical clustering analyses, the EmsB target enabled us to distinguish two main assemblages, each divided into sub-groups, yielding the differentiation of six clusters or assemblage profiles. Thirteen foxes (52% of the foxes) each harbored worms from at least two different assemblage profiles, suggesting they had become infected by several sources. Using the NAK1 target, we identified 3 alleles, two found in association with the two EmsB assemblages. With the NAK1 target, we investigated the parasite breeding system and the possible causes of genetic diversification. Only one fox harbored hybrid worms, indicative of a possible (and rare) occurrence of recombination, although multiple infections have been observed in foxes. These results confirm the usefulness of microsatellite targets for assessing genetic polymorphism in a geographically restricted local range.

Multiple-Locus Variable-Number Tandem-Repeat Analysis for Rapid Typing of Candida glabrata

ABSTRACT A multiple-locus variable-number tandem-repeat analysis (MLVA) using six microsatellite markers was assessed in 127 Candida glabrata isolates. Thirty-seven different genotypes, stable both in vitro and in vivo, were observed. The highest discriminatory power (D = 0.902) was reached by using only four markers. MLVA seems to be relevant for C. glabrata typing.

Assessment of Echinococcus granulosus polymorphism in Qinghai Province, People’s Republic of China

Cystic echinococcosis (CE) is highly endemic in the Chinese province of Qinghai, located on the Tibetan Plateau. The Echinococcus granulosus sheep strain has already been reported in this focus. To improve our understanding of the role the parasite plays in the high prevalence observed in humans, we assessed the genetic polymorphism of 55 E. granulosus samples (37 from humans) using three discriminative mitochondrial markers: coxI, nadI and atp6. We obtained a total of 13 distinct genotypes which were all related to the common sheep G1 strain. Six of these genotypes have already been reported in China and other foci around the world. The remaining seven genotypes were new variants of the strain. The parasite population which was studied in the present work did not differ substantially from those observed in other foci of CE. Environmental conditions and human behaviour could explain the high incidence of the parasitic disease, particularly in the Tibetan population in the south of Qinghai, most of whom are livestock farmers.

Echinococcus multilocularis in Svalbard, Norway: Microsatellite genotyping to investigate the origin of a highly focal contamination

Echinococcus multilocularis is a threatening cestode involved in the human alveolar echinococcosis. The parasite, mainly described in temperate regions of the Northern hemisphere was described for the first time in 1999 in the High Arctic Svalbard archipelago, Norway. The origin of this contamination could be due to an anthropogenic introduction from mainland Europe by domestic dogs or with the introduction of the sibling vole, perhaps from mainland Russia (St. Petersburg area), or with roaming Arctic foxes, known as the main definitive host of the parasite in Arctic regions. The genetic diversity of E. multilocularis in Svalbard was investigated here for the first time by genotyping using EmsB microsatellite and compared to other genotyped populations in the main worldwide endemic areas. We found low polymorphism amongst the 27 metacestode isolates from sibling voles trapped in the core of the distribution area of the vole on Svalbard. E. mutilocularis Arctic populations, using the Arctic fox as the definitive host, were genetically separated from European temperate populations that use the red fox, but closely related to St. Lawrence Island samples from Alaska. The result is inconsistent with the hypothesis of an anthropogenic introduction from mainland Europe, but can be seen as consistent with the hypothesis that Arctic foxes introduced E. multilocularis to Svalbard.

Echinococcus vogeli Infection in a Hunter, French Guiana

Echinococcus vogeli infection in a hunter from the rain forest of French Guiana was confirmed by imaging and mitochondrial DNA sequence analysis. Serologic examination showed typical patterns for both alveolar and cystic echinococcosis. Polycystic echinococcis caused by E. vogeli may be an emerging parasitic disease in Central and South America.