Echinococcus multilocularis to trace the history of expansion USING the genetics of Echinococcus multilocularis to trace the history of expansion from an endemic area G. Umhang1, J. Knapp2, V. Hormaz1, F. Raoul2, F. Boué1 1 ANSES, Nancy Rabies and Wildlife Laboratory, NRL Echinococcus spp., Malzéville, France 2 Chrono-Environnement UMR Université de Franche-Comté – CNRS 6249, Besançon, France Infect. Genet. Evol. (2014), http://dx.doi.org/10.1016/j.meegid.2014.01.018. I’m Gérald UMHANG from the anses Nancy NRL for Echinococcus spp. in France. I will present you the results of the study : using the genetics of Em to trace the history of expansion from an endemic area.

Westward spread of E. multilocularis in France Positive fox Negative fox (Combes et al. 2012, 2013) Em presence in 35/42 départements First report in 25 départements General expansion observed in Europe in the last 15 years. EmsB microsatellite analysis: Description of a mainland-island system of transmission of Em in Europe (Knapp et al. 2009) Recently in France, a westward spread of Em was observed. The presence of the parasite was described in 35 of the 42 départements investigated, with a first report of Em in 25 of theses departements. In Europe, a general expansion of the distribution range has been observed over the last fifteen years. In order to describe the transmission of Em, the EmsB microsatellite analysis was used to described a mainland-island system of transmission with a spread ruled by founder event across historical areas (Alpes??) and peripheral areas. The aims of our study was to describe the genetic diversity of Em in France in order to propose a spatio-temporal scenario of expansion. Aims : describe the genetic diversity in France propose a spatio-temporal scenario of the expansion

Collection of worms Five areas : east to west with fox prevalence from 42.8% to 11.5% Final panel: 383 worms from 128 foxes Areas Nb of worms Nb of foxes West 51 16 North 23 12 Champagne-Ardenne 45 14 Lorraine 79 27 Historical focus 185 59 Worms samples came from the fox intestines collected within the framework of the large-scale survey presented in the previous slide. Five areas (representing 17 departements) were sampled with fox prevalence ranged from about 12% in the North and West to about 40% in the East The final panel was constituted of 383 worms from 128 foxes and the number of worms per regions ranged from 23 to 185. We performed EmsB genotyping then a richness and diversity analysis. EmsB genotyping, Richness, diversity and clustering analysis

EmsB genotyping 22 different EmsB profiles 2 main profiles P04 and P17 (34% total worms), present in 5 areas (except P04 in North) 2 and 6 profiles are shared between 3 or 2 regions, respectively Dominant profiles only (>50% of the worms) in the North (P18) and West (P06) regions 5 profiles were previously identified in Europe (Knapp et al. 2009) P01 P03 P04 P05 P07 P02 P08 P10 P11 P06 P09 P12 P13 P14 P15 P21 P16 P17 P19 P22 P20 P18 Outgroup 100 87 91 88 95 99 82 94 96 97 75 98 92 77 (a) Looking at the results: we described 22 different EmsB profiles, with two main profiles P04 and P17 representing 34% otf the total worms collection. These two profiles are the only profiles present in the five regions (if we except absence of P04 in the North). There are profiles shared between regions : two profiles are present in 3 regions, and 6 profiles are present in two regions. Presence of a dominant profile only in the North (P18) and in the West (P06). Five profiles were previously identified in the European study.

Specific richness, genetic diversity, cluster analysis Areas Nb of worms Nb of observed profiles Nb of profiles estimated Genetic diversity West 51 6 7.96 2.50 North 23 5 6.91 2.77 Champagne-Ardenne 45 6.00 4.5 Lorraine 79 10 1.71 3.82 Historical focus 185 13 1.72 5.13 Gradient of genetic diversity Assemblages clustering together: Champagne Ardenne + North Historical focus + Lorraine West closer to historical focus +Lorraine When looking at the genetic diversity and specific richness, despite an opportunistic sampling we have a good estimation of the richness of profiles. Concerning the genetic diversity, a gradient can be observed from North and West to the historical focus. Based on the distance between profile assemblages, Champagne-Ardenne and North clustered together as do the historical focus and Lorraine. The west region is close to the historical focus and Lorraine and more distant from the two others regions.

Spatio-temporal scenario of Em expansion 3 2 1 1-2-3 Shift value of Em: ~3km/year (Takumi 2008, Denzin 2012) West and North separated from historical focus by several hundred km colonization occurred several decade ago in these two areas According to all these results we can propose a spatio-temporal scenario of expansion of Em in France with a spread from the historical focus to Lorraine, then Champagne-Ardenne, and to North. Although North and West are geographically close, they share only one common profile (P17, in blue) and clustering analysis showed that West is more close to historical focus which have 4 common profiles with the 6 present in the West. So there is no expansion from North to West but a second expansion from historical focus to West Since genetic diversity values are similar between West and North, low values. Both regions exhibited a dominant profile arguing for a recent colonization. We can conclude that these 2 phenomena of expansion seems to be contemporary Looking at the mathematic models, a shift value of Em estimated at 3km/year: North and West distant of several hundred kilometers from historical focus, so the colonization of North and West seems to have occurred several decades ago.

Conclusion Analysis of the genetic diversity of Em in France by EmsB microsatellite: The East part of France was confirmed as an historical endemic area. 2. Understanding of the spatial and temporal expansion in the country which occurred by two expansion phenomena. The westward spread of Em in France is not as recent as previously thought, highlighting the potential human risk due to non awareness of the disease. 4. These new elements strengthened the need of the monitoring of Em to explore its probable occurrence in many other “départements”. 5. Finally, this surveillance must be accompanied with information to the public and especially in the medical sector even in the areas were the parasite was today not endemic. Analysis of the genetic diversity of Em in France by EmsB microsatellite: The East part of France was confirmed as an historical endemic area. 2. Understanding of the spatial and temporal expansion in the country which occurred by two expansion phenomena. The westward spread of Em in France is not as recent as previously thought , highlighting the potential human risk due to non awareness of the disease. 4. These new elements strengthened the need of the monitoring of Em to explore its probable occurrence in many other “départements”. 5. Finally, this surveillance must be accompanied with information to the public and especially in the medical sector even in the areas were the parasite was today not endemic.

Thank you for your attention Acknowledgements Eliz for organisation of fox sampling Departmental veterinary laboratory for providing worms samples Thank you for your attention