New insight in the Lymnaeids intermediate host of Fasciola hepatica in Belgium. Caron Yannick – June 2011 Meeting of the Belgian Society for Parasitology - Liège – June 10th, 2011

Introduction Overview - Trematoda / Digenea - 2 hosts - Economically importance : - Daily weight gain - Fertility - Milk - Liver Some generality first, The liver fluke is a trematoda and a digenea parasite The life cycle of this parasite needs two host, one intermediate that is the lymnaeid snail and one definitive generaly a cattle but the man can also act as an definitive host. This pathology is economically important because of the decreasing of the daily weight gain, the decreasing of the fertility and the milk production and of course the liver condemnation. In a recent study, the financial loss due to bovine fasciolosis in switzerland reached approximately 52 million euros per year.

Fasciola hepatica - Life cycle Definitive host : - Ovine, Caprine - Cattle - Man - Rodent, … Metacercaria Egg Miracidium Cercaria Quickly, here is the definitive host, with eggs in the stools, a miracidia will develop in the eggs, then hatch in the environement; this miracidium will infest an intermediate host where asexual multiplication takes place with sporocyst, redia until cercariae shedding in the environement, then cercariae will loose their tail and become metacercariae, the infective stage for the cattle. The prepatent period is about ten weeks. Intermediate host : - Galba truncatula, …

Introduction Fasciola hepatica intermediate host (Europe) Galba truncatula Omphiscola glabra Lymnaea stagnalis Pseudosuccinea columella Radix peregra (=R. balthica = R. ovata) Radix labiata Radix auricularia Galba trucatula is the main intermediate host of Fasciola hepatica in Europe. But Fasciola hepatica was found in several other intermediate host during previous studies. Since the fifetees Fh was found in O.glabra, L. stagnalis, R. peregra, R. ovata, R. auricularia naturally or experimentally infested.

Aims of the study Implementation of an optimized protocol suitable in the field - From DNA extraction … - … to Multiplex PCR To assess the prevalence of Fasciola hepatica in Belgium regarding to lymnaeid species and environmental factors. The first aim of this study was the implementation of an optimized protocol suitable in the fields, from DNA extraction to the multiplex PCR. And the second aim was to assess the prevalence of F. hepatica in front of in lymnaeids snails and environemental factors.

Context Pondscape project (SD/BD/02A) - Financed by EU and BELSPO « Towards a sustainable management of pond diversity at the landscape level » - Financed by EU and BELSPO - Multi- (http://www.pondscape.be) - disciplinary (ecology, socio-economic, toxicology, …) - Universities/Institute (RBINSC, UCL, UGENT, FUNDP, LUX) The context is the pondscape project (or this study was done in the frame of the pondscape project) A short description of this project could be « towards … This project is financed by Europe and Beslspo. This is a multi-disciplinary project because there are several workpackage dealing about ecology, social economic concern, toxicology, and so on Several university or institution are involved (…)

Experimental protocol Sampling (July-August 2008) Snails 1 - Polders 2 - Sandy region 3 - Loamy region 4 - Chalky region 5 - Gutland We can begin the first part, this is the experimental protocol. A big sampling campaign were realized in July and August 2008. 5 biogeographic area were selected in Belgium (Polders, Sand Region, Loam region, Chalk region and Gultand). In each of this 5 biogeographic area, 5 cluster were defined, and in each of this cluster 5 ponds were randomly selected. Thus we sampled 125 ponds and 16 other interesting biotopes. We sampled snails but also environemental factors. 142 biotopes

Experimental protocol Sampling (July-August 2008) Biotopes 52.8% (75/142) Snails (7103) G. truncatula: 34.8% Radix sp.: 65.2% Lymnaeid snails were found in 52.8% of the visited biotopes. Seven thousand one hundred and three snails were collected: 34.8% of G. truncatula and 65.2% of Radix sp. We can see on the graph that the number of snails are very variable. G. truncatula were mainly found in the four first biogeographic area. Radix sp. was found everywhere in Belgium but especially in polders.

Experimental protocol Sampling (July-August 2008) Factors Natural Pond Extensive Pond Intensive pond Pond Code BKLE1 Cluster : 1- Polders Sub-cluster : Klemskerke GPS coordinate: N51° 13,438’ E03° 01,970’ Province : Occidental Flander Sampling date  : 30/7/8 Pond dimension  : 30 m diameter pH: 8.4 Water temperature: 23.4°C Ground type: Mud No fence WBB Trampling (+++) Stool (+++) Pond type: intensive Total collected snail number : 34 Species number : 1 (G. truncatula) We sampled snails but we sampled also environmental factors for exemple here in this pond in polders, we collect, Ph, ground type, if there is fence or not, if there is cattle, if there is trampling, stools , … and we collect also the type of the ponds, we classified pond in three types: Natural for pond in natural reserve, intensive for pond in high agriculture or breeding area (activity) and extensive pond for intermediate area

Experimental protocol DNA extraction Chelex® based Pooling (max 10 snails) DNA was extracted using Chelex. The chelex is a molecular biology grade resin. To decrease the number of PCR the samples were pooled in batch, we put a maximum of 10 snails in one pool. Ajouter tableau ?

Experimental protocol Multiplex PCR (2 bands) Fasciola sp. specific sequence (124 bp) Snail rDNA ITS-2 sequence (+/- 500 bp) Positive pools: (8) and (9) Limit of detection: 10 pg No cross reaction with Dicrocoelium dendriticum nor Fascioloides magna. A band was visible (400 bp) for Paramphistomum daubneyi. After the DNA extraction, the multiplex PCR was done. In this multiplex PCR 2 bands were amplified. The fasciola sp sequence and the rDNA ITS-2 sequence. This gel illustrate a PCR of 10 pools. We can see that the pools 8 and 9 were positives for Fasciola sp. The Limit of detection for Fasciola sp. sequence is 10 pg of Fasciola hepatica DNA. (+100 ng snail DNA) and 100 pg for a positive pool. No cross reaction with Dicrocoelium dendriticum nor Fascioloides magna but a band was visible (400 bp) for Paramphistomum daubneyi. Anyway this band doesn’t invalidate the interpretation of the infestation status. ITS-2 Fasciola sp.

Fasciola sp. in Belgium Multiplex PCR G. truncatula: 28 positive pools 30 positive snails Prevalence: 1.31% Radix sp.: 6 positive pools 7 positive snails Prevalence: 0.16% Here is the multiplex results 1.31% of the G. truncatula were positives and 0.16 % of The Radix sp. were positives. We can see on the map the localisation of the different sites where positive snails were found. Galba in red and Radix in blue. 7.89% G. truncatula eliminated of the study because of PCR inhibition 8.77% Radix sp. eliminated of the study because of PCR inhibition 1 - Polders 2 - Sandy region 3 - Loamy region 4 - Chalky region 5 - Gutland

Fasciola sp. in Belgium Radix sp. ? ITS-2 Sequencing 7 positive Radix sp. 93 random negative Radix sp. Radix peregra Radix labiata It si very difficult to identify correctly Radix sp. with morphological features. We talk about species complex. To overcome this difficulty we sequence the ITS-2 of the 7 positive Radix sp. and we randomly select 93 others Radix sp. For this one hundred ITS-2 sequence 98 sequences were 100% identical, we call this sequence PRT1 and for the 2 other sequences they were 100% sequences, we call this sequence PRT2. Then We construct a phylogenetic tree based on Maximum likelihood and 500 bootstrap and we can see that PRT1 is in the R.peregra taxon and PRT2 is in the R. labiata taxon. We can add that the 7 positive Radix sp. were all identified as Radix peregra. the ITS2 sequence of rDNA of Radix snails using Maximum Likekihood method in MEGA 5.05 (500 bootstraps and General Time Reversible model as method for model substitution and Gamma Distributed as rates among sites). The scale shows the number of nucleotide substitutions per site between DNA sequences. L.stagnalis was set as outgroup. Values on node represent bootstrap percentages. All the positive Radix sp. belong to Radix peregra. 100 ITS-2 sequences: - 98 sequences 100% identical :PRT1 2 sequences 100 % identical: PRT2

Fasciola sp. in Belgium CART analysis Presence/absence G. truncatula R. peregra Commune 100% Commune 100% pH 73% Ground type 59.25% Water temperature 46.81% Pond type 23.04% Ground type 45.32% Water temperature 23.04% Pond type 13.85% pH 14.25% Tree sensibility 84.44% Tree sensibility 81.25% Tree specificity 81.25% Tree specificity 60.22% The locality explains 100% of presence/absence G.truncatula seems to have specific needs in comparison with Radix peregra Then we’ve made CART analysis. CART is for Classification and Regression tree analysis. We’ve chose several environemental factor to assess the presence/absence of G. truncatula and Radix sp. in Belgium. For exemple Commune, ph, Water temperature … The analysis successively splits the dataset into increasingly homogenous subsets until it is stratified and meet specific criteria. We can see that the commune explains 100% of presence/absence of snails. And G.truncatula seems to have exacting needs in front of Radix sp. (stenoecious vs euryoecious). For example, the ph explains 73% of the presence of Galba in the ponds, and it is not the case for Radix sp.

Fasciola sp. in Belgium CART analysis Infestation status The number of collected snails explained 100 % of infestation status. The pond type is not very informative - No link with cattle G. truncatula R. peregra Number 100% Pond type 22.14% Pond type 29.84% Tree sensibility 83.33% Tree sensibility 100% Tree specificity 97.78% Tree specificity 82.35% We’ve made another CART analysis to assess the infestation status of the snail. We chose the number of snails, the pond type and we make a aconcatenation of cattle link factors with trampling, breeding, and stools presence. number of collected snails explains 100% of the infestation status of snails. The number of snails explains 100% of the infestation status but the pond type is not very informative. Surprisingly, there is no link between infestation status and cattle. That means that the probablilty for one Gt to be infested is not higher when stools or trampling are present in the pond.

Discussion/Conclusion Reliable protocol DNA extraction Internal control Pooling Low prevalence in G. truncatula Sampling in permanent water habitats This is the last part of the presentation. The protocol used is very reliable because the DNA extraction is very fast, cheap and without toxic solution as phenol. The internal control is interesting because it permits the detection of PCR inhibition. The pooling is also interesting because it dramatically drecrease the number of PCR. The prevalence of G. truncatula is very low. Probably because the sampling was done in permanent water habitats only and not also in temprorary water habitats. (Perhaps alos because of the geographical difference with other study (colder in Belgium) Perhaps also that the cattle are over treated …)

Discussion/Conclusion R. peregra as intermediate host of F. hepatica ? = R. balthica = R. ovata ? R. peregra = « incidental » intermediate host Prevalence very low = epidemiological weight ? High density and wide distribution R. labiata ? Experimental infestation in our lab successful up to metacercariae … Seems to be a rare species Is R. peregra an intermediate host of F.hepatica ? Is R. peregra the same species as R. balthica as R.ovata : this point is very controversial in recent literature. Someone says yes, someone says no … If we admit R. peregra, R. balthica and R. ovata are the same species, This snail is able to harbour asexual multiplication of F.hepatica in Europe and act as incidental intermediate host. It was proved with natural and experimental infestation in old and recent studies. Prevalence is very low so What about the epidemiological weight ? We can say that the prevalence of Gt is low too … This species has a high density and a very wide distribution in Europe, This species is called the travelling Radix this contributing to its importance in Fasciola hepatica cycle. Concerning R. labiata some experimental infestations were made in our lab and it was a success till metacercariae. This species seems to be in the minority in contrast with R. peregra. (Experimental infestation failure in our lab? Variability between population ? (described for Gt))

Discussion/Conclusion CART analysis Presence/absence R. peregra is euryoecious and present a good adaptability Adaptibility for parasitism in biotopes without G. truncatula ? Infestation status No link with cattle at the pond level During the first CART analysis we saw that R. peregra is probably more tolrant than G. truncatula. Radis peregra ahs a better adaptability. What about this aadaptability in biotope without G. truncatula ? For the infestation status we saw that there is no link with cattle and it is an intersting results because in general farmers think that the presence of a pond in their meadow has a negative impact in the health of their cattle. It seems that this negative impact is for Fasciola hepatica wrong. (Indeed, Radix peregra is known as « travelling lymnaeid » and thus present a very good adaptability. G. truncatula is more stenoecious. What about its adaptability in front of Fasciola hepatica in a biotope without G. truncatula ? In site were Radix sp. is infected, four times on five Radix peregra is alone.)

Acknowledgment Pondscape staff Sampling teams Koen martens Els de Roeck Robert Mandiki Sampling teams Lionel, Richard, Dirk, Tom, Michel, Verl, Philippe, Jos, Olivier, Luc, Rik, An, Wim, Bert, Kris, Jan … Thank you very much to all the pondscpe staff and particularly to Koen, Els and Robert. And of course, thank you also to the sampling teams

Thank you for your attention !