New tools for new challenges – Echinococcus epidemiology on the move Thomas Romig Universität Hohenheim Stuttgart, Germany

‚Epidemiology‘ geographical records host records prevalence data temporal and spatial changes ‚risk factors‘ (humans, animals)

‚Tools for epidemiology‘ parasite identification / characterization (incl. intraspecific diversity) host identification survey methods / diagnosis of infection associative studies - models

‚Tools for epidemiology‘ parasite identification / characterization (incl. intraspecific diversity) host identification survey methods / diagnosis of infection associative studies - models

Parasite identification / characterization

Parasite identification / characterization E. multilocularis: focus on intraspecific variation E. granulosus (sensu lato): species identification still important intraspecific variation

E. multilocularis: numerous PCRs, immunodiagnostic systems research shift from records / prevalence data to other questions: expansion routes, introduction events origin of newly discovered foci role of host species ....

E. multilocularis: Intraspecific ‚microdiversity‘ of genetic markers microsatellites: EmsB (Knapp et al., 2008, 2009; Umhang et al., 2014) mt markers (various; 366-3558 bp) (Bowles et al., 1992; Bagrade et al., 2008; Nakao et al., 2009; Jenkins et al., 2012; Konyaev et al., 2012; Schurer et al., 2013)

E. multilocularis: Intraspecific ‚microdiversity‘ of genetic markers: world distribution...

N N A O A E E A N Mitochondrial haplotype clades (Nakao et al., 2009; Jenkins et al., 2012)

E. multilocularis: Intraspecific ‚microdiversity‘ of genetic markers ...biogeographical hypotheses...

Europe: Mainland-island hypothesis (Knapp et al., 2009)

E. multilocularis: Intraspecific ‚microdiversity‘ of genetic markers ...introduction / translocation events...

Origin of introduced E. multilocularis on Svalbard (Knapp et al

Origin of introduced E. multilocularis on Svalbard (Knapp et al

E. multilocularis: Intraspecific ‚microdiversity‘ of genetic markers ...persistence of isolated populations...

Origin of E. multilocularis in northern Italy (Casulli et al., 2009)

E. multilocularis: Intraspecific ‚microdiversity‘ of genetic markers ...expansion routes...

? Expansion of E. multilocularis in France (Umhang et al., 2014)

E. multilocularis: Intraspecific ‚microdiversity‘ of genetic markers ...human infections

Europe: are certain mt haplotypes overrepresented in humans? 12 / 16 isolates from human AE patients from southern Germany conform to the most frequent haplotypes in animals in Europe (= E5 of Nakao et al., 2009) (Ebi et al., unpublished)

Parasite identification / characterization E. multilocularis: focus on intraspecific variation E. granulosus (sensu lato): species identification still important intraspecific variation

CE Echinococcus classification E. granulosus s.l. E. granulosus s.s. G1 sheep strain G2 Tasmanian sheep strain G3 buffalo strain E. equinus (G4 - horse strain) E. ortleppi (G5 - cattle strain) E. canadensis G6/7 camel-pig strain G8 ‚American‘ cervid strain G9 variant pig strain G10 ‚European‘ cervid strain E. felidis lion strain E. multilocularis E. shiquicus E. oligarthra E. vogeli CE E. granulosus s.l.

Techniques for species identification Numerous PCR based methods for species diagnosis PCR (Bowles et al., 1992) Nested PCR (Dinkel et al., 2004) RFLP (Hüttner et al., 2009) Multiplex PCR (Boubaker et al., 2013 LAMP (Wassermann et al., 2014)

Applications (limitless....) Identity of human parasites (Dinkel et al., 2004; Casulli et al., 2010; Schneider et al., 2010; Alvarez Rojas et al., 2013) Pathogenicity (Schneider et al., 2010, Sadjjadi et al., 2013) Host spectrum, geography (Konyaev et al., 2012; Nakao et al., 2013) Wildlife studies (Hüttner et al., 2009; Simsek & Eroksuz, 2009; Guerra et al., 2012; Schurer et al., 2013)

Characterization of intraspecific diversity Mitochondrial markers: partial cox1, nad1 (Bowles et al., 1992; Bowles & McManus, 1993; Casulli et al., 2008; Hailemariam et al., 2012; Sharma et al., 2013) complete cox1 (Yanagida et al. 2012; Konyaev, 2013) complete mt genome, nuclear markers (.....)

Characterization of intraspecific diversity Application: Phylogeny / taxonomy of E. canadensis (Nakao et al., 2013) dispersal history / epidemiology of E. granulosus s.s.

Echinococcus granulosus s.s. Conventional subdivision: G1, G2, G3 (Bowles et al., 1992) Numerous microvariants (haplotypes) described, based on cox1 sequences of various lengths (Yanagida et al. ,2012, Casulli et al., 2012; Hailemariam et al. 2012, Sharma et al., 2013; Nakao et al., 2013)

Haplotype networks in Iran and China E. granulosus s.s.: Haplotype networks in Iran and China Yanagida et al., 2012

E. granulosus s.s.: Diversity decreasing from eastern Europe towards Italy Casulli et al., 2012

E. granulosus s.s.: Comparability between studies problematic (different sequence lengths...) Here: entire cox1 (1609 bp) Haplotype networks (TCS – Clement et al., 2002)

Armenia, Iran, Jordan, China, Peru, Russia E. granulosus s.s.: cox1 haplotypes Armenia, Iran, Jordan, China, Peru, Russia Ebi et al., unpubl.

Applications: ... biogeography subpopulations in different host species (incl. humans)? Discrete sympatric life cycles (domestic – wildlife)? ...

2) Coprodiagnosis of host species

2) Coprodiagnosis of host species Wild definitve hosts: faecal samples from the environment Conventional identification: ‚field signs‘ (size, shape, smell, location...) Uncertainties, skill dependent

Kenya: field signs vs. mt marker (Kagendo et al., unpublished) 98 faecal samples from Kenyan conservation areas RFLP-PCR of cob sequence (311 bp) Hph1 digestion (Hüttner et al., 2009, Kagendo et al., unpublished)

Only 60% of samples were correctly identified by experienced field workers field signs mt marker lion 82 58 (25 hyena, 1 leopard) hyena 9 3 (4 lion, 2 leopard) wild dog 5 0 (3 leopard, 1 hyena, 1 lion) jackal 2 0 (1 leopard, 1 hyena)

Nonaka et al., 2009 multiplex PCR, Japanese carnivores Hüttner et al., 2009 RFLP, African felids Dinkel et al., 2011 multiplex PCR, Tibetan canids and Echinococcus Kagendo et al. (..) RFLP, African felids and canids

3) Imaging Ultrasonography: established survey method for human CE (Macpherson et al., 1987) previous studies with animals discouraging

Imaging Ultrasonography of sheep / goats: Sage et al., 1998 54% / 98% Guarnera et al., 2001 3 positive sheep Lahmar et al., 2007 89 / 248 liver cysts Livestock data still largely rely on post mortem

New method from Sardinia (Dore et al., in prep.): High sensitivity Upright position, no shaving Application on the farm: contribution to education Liver cysts only (few animals with only lung cysts) Valuable addition to the toolbox for surveillance of CE

Thank you! Antonio Varcasia Dorothy Kagendo Hasmik Gevorgyan Marion Wassermann Anke Dinkel Sandra Schroer Dennis Ebi Sarah Wagner