New data in France on the trematode Alaria alata (Goeze, 1792) obtained during Trichinella inspections

The trematode Alaria alata is a cosmopolite parasite found in red foxes (Vulpes vulpes), the main definitive host in Europe. In contrast only few data are reported in wild boars (Sus scrofa), a paratenic host. The aim of this paper is to describe the importance and distribution of Alaria alata mesocercariae in wild boars, information is given by findings of these larvae during Trichinella mandatory meat inspection on wild boars’ carcasses aimed for human consumption. More than a hundred cases of mesocercariae positive animals are found every year in the East of France. First investigations on the parasite’s resistance to deep-freezing in meat are presented in this work.


INTRODUCTION
T he presence of Alaria alata has been described in wild boars and pigs (Sus scrofa) since the late 19 th century (Duncker, 1896). In comparison with definitive hosts, few data on A. alata in European Suidae are reported (JakšiĆ et al., 2002, Grösse & Wüsste, 2006, MileševiĆ et al., 2004 and according to the fluke's life cycle, they are considered as paratenic hosts (Dollfus & Chabaud, 1953 After hatching of the eggs, the miracidia penetrate the first intermediate host (freshwater snails) to evolve after two generations of sporocysts who produce the furcocercariae (Ruzskowski, 1922). The second intermediate host, an amphibian, is infected by this stage and gives in situ, the mesocercaria. Usually, the definitive host is infected by predation. The mesocercariae migrate to the lungs and evolve to the metacercarial stage which migrates back to the intestines (the larvae migrate up the pulmonary tract and are swallowed) and finally develops to the mature adult stage. Some modifications are observed according to the species. The transmammary infections by mesocercariae are possible with Alaria marcianae where adults serve as paratenic hosts and offspring might serve as definitive hosts (Shoop et al., 1990). Alaria mustelae needs four hosts: the metacercarial stage does not develop to the adult stage in the same host (Bosma, 1931 These parasites were confirmed as mesocercariae of A. alata (Goeze, 1792). Since the description of Chabaud et al. (1953), there was no report on A. alata in France, but since its re-emergence in 2004, it is regularly identified in some area from the East of France. Within the genus Alaria, the species of the new world are considered as zoonotic with seven published cases of human alariosis, all from American or Canadian patients supposedly infested with these parasites (Möhl et al., 2009). One case was lethal: a young Canadian died of a massive infestation, which caused multiple haemorrhages, mainly in the lungs (Fernandez et al., 1976). This young man supposedly ate insufficiently cooked frogs legs. In other cases, larvae were found under the skin (nodule), in the eye or in the respiratory tract. The very little knowledge available on the species A. alata must lead to an analysis of the risk for human consumption. Only one experiment showed effects of massive experimental infestation on a primate (Odening, 1963). During this experimentation, several hundreds of A. alata mesocercariae were given to a Rhesus monkey, which developed severe clinical signs but did not die. Autopsy showed a generalized alariosis. No human case of alariosis has been reported in Europe and regarding more specifically A. alata, there is no available data concerning its potential danger given by human consumption. However, it is evident that a zoonotic risk can be questioned and that the potential danger regarding food safety of wild boars' meats has to be taken into consideration. In this context, the aim of the present work was to analyse the distribution of A. alata in French wild boars and presents preliminary tests on its cryo-resistance.

ARTIFICIAL DIGESTION OF MEAT
W ild boars meat was treated first for Trichinella inspection according to the method described in the EU regulation 2075/2005. Briefly, the artificial digestion relies on a chlorhydropepsic digestion of muscle, which allows the release of free larvae. In France, all routine laboratories performing Trichinella meat inspection use the method with magnetic stirrer (Vallée et al., 2007), which is considered as the reference method for Trichinella meat inspection (EU Regulation 2075, 2005. Each carcass is analysed with at least 5 g of meat from tongue, diaphragm or foreleg in a pooled sample weighting 100 g. Samples are digested during 30 min in a beaker containing 2 L water, 10 g powdered pepsin and 16 ml 25 % chlorhydric acid. The digestion fluid is passed through a 180 μm mesh sized sieve and decanted in a glass funnel for 30 min. One hundred ml are then harvested in a graduated cylinder and decanted for 10 min. The top 90 ml are eliminated smoothly, and the bottom 10 ml are poured in a Petri dish for Trichinella larvae search. Observations are made using a stereomicroscope (× 40 magnification).

ISOLATION OF ALARIA MESOCERCARIA
Parasitic elements corresponding morphological criteria (Dollfus & Chabaud, 1953) for Alaria mesocercariae observed during Wild boars' meat digestion are all sent from laboratories to the national reference laboratory at the ANSES Maisons-Alfort for confirmation.

DEVELOPMENTAL STAGES
DNA was extracted from mesocercariae from wild boars and adult worms isolated from the intestine of red foxes (Vulpes vulpes) using the Qiamp DNA mini kit (Qiagen, Germany) following manufacturer's instructions. Sequencing of the D2 domain and the second internal transcribed spacer (ITS-2) of the 28S unit of the ribosomal DNA were used for the identification. PCR was performed in a 50 μl volume using 5 μl of DNA, and 50 pmol of each of the primers. The PCR mix contained (final concentrations) 10 mM Tris HCl (pH 8.3), 1.5 mM MgCl 2 , 50 mM KCl, 0.01 % Triton X 100, 200 μM dNTP each base, and 1.25 units of Taq polymerase (Eppendorf, Germany). The D2 domain was amplified using the primers used by Mollaret et 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 94 °C for 3 min was followed by 40 cycles of denaturation at 94 °C for 45 s, annealing at 50 °C for 45 s and extension at 68 °C for 2 min with a final elongation time of 10 min at 68 °C. PCR products were directly sequenced in both directions with the primers used for DNA amplification (QIAGEN, Germany). The sequences were deposited in GenBank under accession numbers JF340217 to JF340229. Sequence alignment was performed using the ClustalW routine included in the MEGA version 3.1 software (Kumar et al., 2004) and checked by eye and comparison with those deposited in Genbank.

CRYO-RESISTANCE TESTS
Six positive carcasses were analysed for tests on cryo-resistance. Samples were taken from diaphragm, coastal muscles and legs with a final weight of 100 g per condition. They were tested before freezing and after 2, 3, 5, 10, 11, 18 and 19 days freezing (-18 °C ± 2 °C). Temperature was monitored with an automatic Original contribution Parasite, 2011, 18, 271-275 recording system during all the freezing process with a control every 2 min. After freezing, meat was defrosted at room temperature and treated by artificial digestion for larvae releasing as previously described. Mesocercariae were then analysed under a stereomicroscope (× 40 magnification) to evaluate their mobility.

IDENTIFICATION OF MESOCERCARIAE
T he morphology of the trematode larvae showed high similarity with A. alata mesocercariae described by Chabaud & Dollfus (1953). Regarding molecular identification, all sequences of the D2 domain of the mesocercariae (n = 1) the adult (n = 2) and the egg (n = 2,) tested were similar and 100 % homologous with the sequences of A. alata deposited in Genbank (accession number AF184263), from an adult fluke isolated from Raccoon Dog (Nyctereutes procyonoides) (Tkach et al., 2001). No variations were observed for the ITS-2 domain between the nine samples sequenced: mesocercariae (n = 6), and eggs (n = 2).

POSITIVES WILD BOARS' CARCASSES
The presence of A. alata was detected for the first time in 2004 from a pool of wild boars in Aube department.

CRYO-RESISTANCE TESTS
A. alata mesocercariae were found alive in fresh meat and in meat frozen at -18 °C ± 2 °C for two and five days. Most larvae identified were mobile at least five days post-freezing, however it was not possible to evaluate survival after five days since no mesocercariae were found in the meat at days 10 to 19 (Table I).

DISCUSSION
T his work provides an updating on the presence of A. alata in French wild boars' populations since the last report published by Dollfus & Chabaud (1953). The identification of A. alata joins in the context of the compulsory diagnosis for trichinellosis and have indeed benefits of the Quality Assurance plan developed in France since 2004 to increase the sensitivity of Trichinella detection in meat (Vallée et al., 2007). Since this first description in 2004, the fluke has been found in 11 French departments mainly in East of France. During these three last years, A. alata did not expand widely throughout the French territory. However, Trichinella inspection has also enabled detection of Alaria larvae in wild boars' meat in other countries like Croatia (MileševiĆ, 2004) or Germany (Grosse and Wüste, 2006). Although A. alata larvae have been identified during Trichinella meat inspection, the chlorhydro-pepsic digestion is meant to detect Trichinella sp. larvae and not Alaria mesocercariae. A. alata seems to behave as a larva migrans in wild boars and does not appear as encysted (direct observations at the laboratory) although other descriptions show cysts (Leuckart, 1901). The digestion method used for Trichinella inspection may dissolve mesocercariae, leading to false negative results (Riehn et al., 2010). Sieves used for this method have 180 μm mesh size, whereas fresh larvae are 460 to 680 μm length and 118 to 338 μm width (Brumpt, 1945;Dollfus & Chabaud, 1953). In these conditions, Alaria larvae may not all pass through the sieve. Chlorhydro-pepsic digestion as defined by European regulation CE 2075/2005 may only permit detection of A. alata mesocercariae in highly infested meat samples. Recent works have shown that other methods may be more efficient than the artificial digestion method designed for Trichinella (Riehn et  al., 2010). Moreover, wild boars meat samples used for Trichinella inspections are the muscles from the tongue, the masseter or the diaphragm. Digestion must be performed only on muscle tissues and not on fat tissues. Very little information is available on the distribution of Alaria mesocercariae in the wild boars' carcasses and mesocercariae distribution may not match Trichinella distribution. More studies are thus needed to analyse the distribution of mesocercariae in paratenic hosts but this would involve experimental infections of wild animals, which are difficult to envisage. However such work would allow to target some tissues to increase the sensitivity of the detection. The parasite A. alata is thus present in a high number of wild boars carcasses but very little is known about the risk for human consumption. The cryo-resistance tests described in this work have shown that larvae can resist at least five days in frozen meat. Unfortunately, the absence of larvae after five days' freezing did not allow to assess the survival of larvae after this duration. Cryo-resistance tests have not shown a limit of mesocercariae resistance to freezing. The best method of killing these larvae is still cooking: 71 °C remains the recommended temperature to inactivate Trichinella larvae (Gamble et al., 2000). This is the reason why the same treatment should be used for A. alata inactivation. The compulsory inspection for Trichinella in wild boars has allowed to show the importance of the distribution of A. alata in France. This parasite has been present for a long time and its distribution is probably larger than showed in this work. Although no human case has been described in Europe, A. alata may be able to cause illness as the American species of Alaria did. This potential risk for food safety must be taken into account and the resistance of this parasite to freezing treatments as well as its distribution in wild boar carcasses remain to be evaluated. More sensitive detection methods should also be investigated, which include the choice of the tissues to be targeted for analysis as well as the method of detection.