This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. NEW DATA IN FRANCE ON THE TREMATODE ALARIA ALATA (GOEZE, 1792) OBTAINED DURING TRICHINELLA INSPECTIONS PORTIER J.*,**, JOUET D.*, FERTÉ H.*, GIBOUT O.***, HECKMANN A.**, BOIREAU P.** & VALLÉE I.** Summary: Résumé : NOUVELLES OBSERVATIONS D’ALARIA ALATA (GOEZE, 1792) LORS D’INSPECTIONS POUR TRICHINELLA EN FRANCE The trematode Alaria alata is a cosmopolite parasite found in red foxes (Vulpes vulpes), the main definitive host in Europe. In Alaria alata (Diplostomidae : Trematoda) est parasite à l’état adulte contrast only few data are reported in wild boars (Sus scrofa), a de l’intestin grêle du Renard roux (Vulpes vulpes), le principal paratenic host. The aim of this paper is to describe the importance hôte définitif en Europe. Les données existantes sur ce parasite and distribution of Alaria alata mesocercariae in wild boars, chez le Sanglier (Sus scrofa), le principal hôte paraténique, sont information is given by findings of these larvae during Trichinella fragmentaires. L’objectif de cet article est de préciser l’importance mandatory meat inspection on wild boars’ carcasses aimed for et la distribution d’Alaria alata chez les sangliers en France grâce human consumption. More than a hundred cases of mesocercariae aux déclarations de mésocercarioses obtenues lors des recherches positive animals are found every year in the East of France. First obligatoires de Trichine sur les carcasses ces dernières années. Le investigations on the parasite’s resistance to deep-freezing in meat recueil de ces déclarations montre que plus de 100 cas d’animaux are presented in this work. positifs sont signalés chaque année, principalement dans le nord- est de la France. Une première approche de la résistance à la KEY WORDS: trematode, Alaria alata, wild boar, Sus scrofa, Trichinella, congélation d’Alaria alata dans la viande est présentée dans ce resistance. travail. MOTS-CLÉS : trématode, Alaria alata, sanglier, Sus scrofa, Trichinella, résistance. INTRODUCTION After hatching of the eggs, the miracidia penetrate the first intermediate host (freshwater snails) to evolve after two generations of sporocysts who produce the he presence of Alaria alata has been described furcocercariae (Ruzskowski, 1922). The second inter- in wild boars and pigs (Sus scrofa) since the mediate host, an amphibian, is infected by this stage late 19th century (Duncker, 1896). In comparison T and gives in situ, the mesocercaria. Usually, the defi- with definitive hosts, few data on A. alata in Euro- nitive host is infected by predation. The mesocercariae pean Suidae are reported (JakšiĆ et al., 2002, Grösse & migrate to the lungs and evolve to the metacercarial Wüsste, 2006, MileševiĆ et al., 2004) and according to stage which migrates back to the intestines (the larvae the fluke’s life cycle, they are considered as paratenic migrate up the pulmonary tract and are swallowed) hosts (Dollfus & Chabaud, 1953). The life cycle of A. and finally develops to the mature adult stage. Some alata is complex and involves several hosts and tissue modifications are observed according to the species. locations according to the parasite stages. Indeed, the The transmammary infections by mesocercariae are adults of A. alata (Diplostomidae, Trematoda) are intes- possible with Alaria marcianae where adults serve tinal parasites of canids [Red Fox (Vulpes vulpes), Wolf as paratenic hosts and offspring might serve as defini- (Canis lupus), Raccoon Dog (Nyctereutes procyonides), tive hosts (Shoop et al., 1990). Alaria mustelae needs Dog (Canis lupus familiaris)], the definitive hosts. four hosts: the metacercarial stage does not develop to the adult stage in the same host (Bosma, 1931). * JE 2533 – USC Anses “VECPAR”, UFR de Pharmacie, Université de Reims Champagne-Ardenne, 51, rue Cognacq-Jay, 51096 Reims, Isolations of A. alata mesocercariae have occurred in France. France during meat inspections for Trichinella spp. ** Anses, Laboratory for Animal Health, Anses Maisons-Alfort, UMR in wild boars’ meat aimed for human consumption. BIPAR, ENVA, UPEC, 23, avenue du Général de Gaulle, 94706 Maisons-Alfort, France. The analysis for the zoonotic nematode Trichinella is *** Laboratoire départemental d’Analyses vétérinaires et alimentaires, indeed compulsory in pigs and wild boars and relies in Chemin des Champs de la Loge, BP 216, 10006 Troyes Cedex, the European Union on an EU regulation (Regulation France. EC 2075/2005). During these inspections, a new report Correspondence: Isabelle Vallée. Tel.: 33 (0)1 49 77 28 16. of Alaria sp. mesocercariae was made in December E-mail: [email protected] 2004 in the Aube department (North East of France). Parasite, 2011, 18, 271-275 Original contribution 271 PORTIER J., JOUET D., FERTÉ H. ET AL. These parasites were confirmed as mesocercariae of through a 180 μm mesh sized sieve and decanted in A. alata (Goeze, 1792). Since the description of Cha- a glass funnel for 30 min. One hundred ml are then baud et al. (1953), there was no report on A. alata in harvested in a graduated cylinder and decanted for France, but since its re-emergence in 2004, it is regu- 10 min. The top 90 ml are eliminated smoothly, and larly identified in some area from the East of France. the bottom 10 ml are poured in a Petri dish for Tri- Within the genus Alaria, the species of the new world chinella larvae search. Observations are made using are considered as zoonotic with seven published cases a stereomicroscope (× 40 magnification). of human alariosis, all from American or Canadian patients supposedly infested with these parasites (Möhl ISOLATION OF ALARIA MESOCERCARIA et al., 2009). One case was lethal: a young Canadian Parasitic elements corresponding morphological criteria died of a massive infestation, which caused multiple (Dollfus & Chabaud, 1953) for Alaria mesocercariae haemorrhages, mainly in the lungs (Fernandez et al., observed during Wild boars’ meat digestion are all sent 1976). This young man supposedly ate insufficiently from laboratories to the national reference laboratory cooked frogs legs. In other cases, larvae were found at the ANSES Maisons-Alfort for confirmation. under the skin (nodule), in the eye or in the respira- tory tract. The very little knowledge available on the MOLECULAR IDENTIFICATION OF ALARIA species A. alata must lead to an analysis of the risk for DEVELOPMENTAL STAGES human consumption. Only one experiment showed effects of massive experimental infestation on a pri- DNA was extracted from mesocercariae from wild mate (Odening, 1963). During this experimentation, boars and adult worms isolated from the intestine several hundreds of A. alata mesocercariae were given of red foxes (Vulpes vulpes) using the Qiamp DNA to a Rhesus monkey, which developed severe clinical mini kit (Qiagen, Germany) following manufacturer’s signs but did not die. Autopsy showed a generalized instructions. Sequencing of the D2 domain and the alariosis. No human case of alariosis has been reported second internal transcribed spacer (ITS-2) of the 28S in Europe and regarding more specifically A. alata, unit of the ribosomal DNA were used for the identi- there is no available data concerning its potential fication. PCR was performed in a 50 μl volume using danger given by human consumption. However, it is 5 μl of DNA, and 50 pmol of each of the primers. The evident that a zoonotic risk can be questioned and PCR mix contained (final concentrations) 10 mM Tris that the potential danger regarding food safety of wild HCl (pH 8.3), 1.5 mM MgCl2, 50 mM KCl, 0.01 % Triton boars’ meats has to be taken into consideration. In this X 100, 200 μM dNTP each base, and 1.25 units of Taq context, the aim of the present work was to analyse polymerase (Eppendorf, Germany). The D2 domain the distribution of A. alata in French wild boars and was amplified using the primers used by Mollaret et presents preliminary tests on its cryo-resistance. al. (1997): C2’B and D2. ITS-2 was amplified using primers ITS3Trem and ITS4Trem previously described by Dvořák et al. (2002). The initial denaturation at MATERIAL AND METHODS 94 °C for 3 min was followed by 40 cycles of dena- turation at 94 °C for 45 s, annealing at 50 °C for 45 s and extension at 68 °C for 2 min with a final elon- ARTIFICIAL DIGESTION OF MEAT gation time of 10 min at 68 °C. PCR products were ild boars meat was treated first for Trichi- directly sequenced in both directions with the primers nella inspection according to the method used for DNA amplification (QIAGEN, Germany). described in the EU regulation 2075/2005. The sequences were deposited in GenBank under W accession numbers JF340217 to JF340229. Sequence Briefly, the artificial digestion relies on a chlorhydro- pepsic digestion of muscle, which allows the release alignment was performed using the ClustalW routine of free larvae. In France, all routine laboratories per- included in the MEGA version 3.1 software (Kumar et forming Trichinella meat inspection use the method al., 2004) and checked by eye and comparison with with magnetic stirrer (Vallée et al., 2007), which is those deposited in Genbank. considered as the reference method for Trichinella meat inspection (EU Regulation 2075, 2005). Each CRYO-RESISTANCE TESTS carcass is analysed with at least 5 g of meat from Six positive carcasses were analysed for tests on tongue, diaphragm or foreleg in a pooled sample cryo-resistance. Samples were taken from diaphragm, weighting 100 g. coastal muscles and legs with a final weight of 100 g Samples are digested during 30 min in a beaker con- per condition. They were tested before freezing and taining 2 L water, 10 g powdered pepsin and 16 ml after 2, 3, 5, 10, 11, 18 and 19 days freezing (- 18 °C ± 25 % chlorhydric acid.