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

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

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

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

5. Parasite identification / characterization

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

7. 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
....

8. E. multilocularis:
Intraspecific ‚microdiversity‘ of genetic markers
microsatellites: EmsB
(Knapp et al., 2008, 2009; Umhang et al., 2014)
mt markers (various; 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)

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

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

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

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

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

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

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

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

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

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

19. ?
Expansion of E. multilocularis in France
(Umhang et al., 2014)

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

21. 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)

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

23. 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.

24. 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)

25. 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)

26. 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 (.....)

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

28. 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)

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

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

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

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

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

37. 2) Coprodiagnosis of host species

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

39. 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)

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

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

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

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

45. 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

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