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New insight in lymnaeids snails as intermediate hosts of Fasciola
hepatica in Ecuador
Caron Yannick – [email protected]
16 -20 August - Liverpool
Introduction• Overview
- Trematoda / Digenea- 2 hosts
- Economically importance:-Daily weight gain- Fertility- Milk- Liver→ 52 million € per year in Switzerland (Schweizer et
al, 2005)
Egg
Miracidium
Intermediate host :- Galba truncatula, …
Cercaria
Definitive host :- Ovine, Caprine- Cattle - Man- Rodent, …
Metacercaria
Life cycle
• Definitive host in South America– Scarce data in man
• Altiplano (Bolivia): up to 68% of an Andeancommunity 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
Introduction
• 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 truncatula G. viatrix
G. cubensis Lymnaea diaphana
G. cousini L. rupestris
G. neotropica Pseudosuccinea columella
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
Materials & Methods– Farm with a historic of fasciolosis– Sampling campaign during 3 months– Snail sampling
• > 4 mm• During 30 min
Materials & Methods
– Infection status• Snail crushing• Multiplex PCR
– Snail identification• Morphology• Molecular (ITS-2)
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
Biotope 1
Biotope 1
Biotope 2
Biotope 2
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)
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+
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)
Results
• Snail identification– Based on morphology
• A priori only one taxon • Morphologically undistinguishable from:
– G. truncatula– G. cubensis– G. neotropica– G. viator– G. schirazensis
Results
• Snail identification– Based on ITS-2 (24 random snails)
• All the sequence were identical• BLASTn : ITS2 : 100% G. schirazensis (JF272602)
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
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
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
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
Generation Lymnaea fuscus Radix balthica
F1 - 11.52
F2 4.1 22.2
F3 18.5 27.2 →
F4 34.4 → 48.3 →
→ Cercarial shedding
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
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
Thank you for your attention !