New insight in lymnaeids snails as intermediate hosts of Fasciola

hepatica in Ecuador

Caron Yannick – ycaron@ulg.ac.be

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 !