1. New insight in lymnaeids snails as intermediate hosts of Fasciola hepatica in Ecuador Caron Yannick – ycaron@ulg.ac.be 16 -20 August - Liverpool

2. Introduction Overview - Trematoda / Digenea - 2 hosts - Economically importance: -Daily weight gain - Fertility - Milk - Liver → 52 million € per year in Switzerland (Schweizer et al, 2005)

3. Egg Miracidium Intermediate host : - Galba truncatula, … Cercaria Definitive host : - Ovine, Caprine - Cattle - Man - Rodent, … Metacercaria Life cycle

4. Definitive host in South America –Scarce data in man Altiplano (Bolivia): up to 68% of an Andean community were copropositive Ecuador: 6% were seropositive –Scarce data in livestock Atílio (Argentina) abattoir: 28% of bovine livers were condemned Machachi (Ecuador) abattoir: 12% → 50 million US$ in Peru (liver condemnation) Introduction

5. Intermediate host in South America –Correct identification of Lymnaeidae based on morphology is very difficult Environmental plasticity in shell shape Extremly homogenous anatomical traits –8 neotropical species are valid (Correa et al. 2011) –… and others: Galba sp. = G. schirazensis Galba truncatulaG. viatrix G. cubensisLymnaea diaphana G. cousiniL. rupestris G. neotropicaPseudosuccinea columella

6. Aims of the study Assess analytic reliability of DNA extraction and multiplex PCR versus microscopic dissection Provide information on lymnaeid snails in Ecuador Monitor lymnaeids infection status during a three month longitudinal survey

7. Materials & Methods –Farm with a historic of fasciolosis –Sampling campaign during 3 months –Snail sampling > 4 mm During 30 min

8. Materials & Methods –Infection status Snail crushing Multiplex PCR –Snail identification Morphology Molecular (ITS-2)

9. Results Farm « La Fontana » –184 Hosltein Jersey cattle –Historic of fasciolosis Coproscopy : 50% (4/8) –Treated every three months with nitroxynil –2 Biotopes were sampled

11. Biotope 1

13. Biotope 2

15. Results Sampling campaign –From April to July 2013 Biotope 1: 1055 snails Biotope 2: 427 snails Infection status –Crushing Global prevalence –1.75% (26/1482) No positive snail in biotope 2 Biotope 1 global prevalence: 2.46% (26/1055)

16. Results Infection status –Multiplex PCR F. hepatica –BLASTn: Cox1 : 99% (GQ121276) Global prevalence –6% (89/1482) Biotope 1 –8.15% (86/1055) Biotope 2 –0.7% (3/427) ITS-2 Fasciola sp. M 1 2 3 4 5 6 7 8 9 10 C- C+

17. Results Infection status –Microscope versus PCR Relative sensitivity = 25.8% –A lot of false negative with microscopy Relative specificity = 99.7% –Very few false positive with microscopy (3/26)

18. Results Snail identification –Based on morphology A priori only one taxon Morphologically undistinguishable from: –G. truncatula –G. cubensis –G. neotropica –G. viator –G. schirazensis

19. Results Snail identification –Based on ITS-2 (24 random snails) All the sequence were identical BLASTn : ITS2 : 100% G. schirazensis (JF272602)

20. Discussion/Conclusion Reliable protocol –DNA extraction (Chelex®) –PCR multiplex (internal control) Sampling campaign too short –Longer longitudinal study –Season effect ? Snail generation number ? Complementarity crushing and PCR –Fasciola DNA and alive larvae

21. Discussion/Conclusion G. schirazensis –Intermediate host ? No ! (Bargues et al, 2011) –Natural infection (n= 8752) from 8 countries = 0 –Experimental infection (n=338) = 0 But this study: –Endemic area –Only this species was found –6% harbour Fasciola spp. DNA and 99% alive larvae –Same situation in Venezuela and Colombia with only G. shirazensis in highly endemic area

22. Discussion/Conclusion Three alternative developmental pathways of F. hepatica in the absence of G. truncatula were identified (Rondelaud et al, 2014): 1/ Infection in the first week of life Strong mortality, low prevalence, low cercarial shedding 2/ Sequential infection (Calicophoron daubneyi then F. hepatica) Low prevalence, low cercarial shedding

23. Discussion/Conclusion Three alternative developmental pathway of F. hepatica when G. truncatula is absent were identified (Rondelaud et al, 2014): 3/ Infection of successive generations of snails Progressive increase in prevalence GenerationLymnaea fuscusRadix balthica F1-11.52 F24.122.2 F318.527.2 → F434.4 →48.3 → → Cercarial shedding

24. Discussion/Conclusion G. schirazensis is not totally refractory: –Previous contact with F. hepatica Need higher selection pressure (n>5) –Co-infection with another digenea –Allopatric infection versus sympatric

25. Acknowledgment Huynen Foundation –Prof. Pascal Leroy –Monique Leclerc Zoonosis International Center in Ecuador –Prof. Washington Benítez-Ortis –Maritza Celi-Erazo –Angel Mosquera simbaña –Elizabeth Minda

26. Thank you for your attention !