Lymnaea neotropica and Lymnaea viatrix, potential intermediate hosts for Fascioloides magna

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1 Journal of Helminthology, page 1 of 7 q Cambridge University Press 2012 doi: /s x Lymnaea neotropica and Lymnaea viatrix, potential intermediate hosts for Fascioloides magna R. Sanabria 1, R. Mouzet 2, J. Pankrác 3, F.F. Djuikwo Teukeng 2,4, B. Courtioux 2, A. Novobilský 5,J.Höglund 5, M. Kašný 3, P. Vignoles 2, G. Dreyfuss 2 *, D. Rondelaud 2 and J. Romero 1 1 CEDIVE, Fac. Cs. Veterinarias, Universidad Nacional de La Plata, Alvear 803, (7130) Chascomus, Buenos Aires, Argentina: 2 INSERM U 1094, Faculties of Medicine and Pharmacy, Limoges, France: 3 Laboratory of Helminthology, Faculty of Science, Charles University, Vinicna 7, Prague, Czech Republic: 4 Department of Animal Biology and Physiology, Faculty of Science, B.P. 812, Yaoundé, Cameroon: 5 Department of Biomedical Sciences and Veterinary Public Health, Section for Parasitology Swedish University of Agricultural Sciences (SLU), Uppsala, Sweden (Received 6 February 2012; Accepted 25 July 2012) Abstract Experimental infections of two South American lymnaeid populations with Fascioloides magna were carried out to determine whether these snails may sustain larval development of this digenean and, if so, to quantify their potential for cercarial production. The reference group was a French population of Galba truncatula infected and raised according to the same protocol. According to the internal transcribed sequence (ITS)-1 segment of their genomic rdna, these South American populations were identified as Lymnaea neotropica (origin, Argentina) and Lymnaea viatrix var. ventricosa (origin, Uruguay). In the snail groups followed for cercarial shedding, longer prepatent periods and lower numbers of shed cercariae were noted in South American lymnaeids. In other snails dissected at day 65 post-exposure, the redial and cercarial burdens of F. magna found in the bodies of L. neotropica and L. v. ventricosa were significantly lower than those noted in G. truncatula. Compared to the total cercarial production noted in the dissected snails, the percentage of cercariae that exited from snails was 51.3% for G. truncatula, 32.2% for L. neotropica and 46.8% for L. v. ventricosa. The two South American species of snails can thus be considered as potential intermediate hosts of F. magna. Introduction The giant liver fluke (Fascioloides magna) is a liver parasite which infects a variety of wild and domestic *Fax: gilles.dreyfuss@unilim.fr ruminants in North America and Europe (Swales, 1935; Erhardová-Kotrlá, 1971). On this last continent, fascioloidosis occurs currently in Austria, Croatia, the Czech Republic, Hungary, Italy, Serbia and Slovakia (Králová- Hromadová et al., 2011). During recent decades the range of F. magna distribution has increased, and other European countries are clearly at risk of its introduction. Several lymnaeid snails are known to be susceptible to

2 2 R. Sanabria et al. F. magna infection. In North America, five species: Fossaria bulimoides techella, F. modicella, F. parva, Stagnicola caperata and S. palustris nuttalliana have been reported as natural hosts, while another five lymnaeids have been successfully infected under laboratory conditions (Dunkel et al., 1996). In Europe, Galba truncatula is the most common intermediate host of F. magna, both in the field and in the laboratory (Erhardová-Kotrlá, 1971; Hirtová et al., 2003). Another three European species: Lymnaea palustris (Chroustová, 1979), Omphiscola glabra (Rondelaud et al., 2006) and Radix peregra (Faltýnková et al., 2006) can also sustain larval development of this parasite in the laboratory. Furthermore, Foreyt & Todd (1974) obtained full larval development of the parasite in an Australian species, Austropeplea (Lymnaea) tomentosa, when exposed to a North American isolate of F. magna. In view of these findings, one may wonder whether this digenean can infect additional lymnaeids in South American countries. As the importation of feral animals (mainly deer) for zoos or even for reproduction in hunting lodges exists in Argentina, the introduction of F. magnainfected animals and the infection of local snails are possible. For this reason, we have chosen two populations of South American lymnaeids, named Lymnaea viatrix prior to the present study, that are currently raised under laboratory conditions at CEDIVE (the Center for Veterinary Diagnostic and Investigations, Faculty of Veterinary Sciences, University of La Plata, Buenos Aires, Argentina) for production of F. hepatica metacercariae. These two populations were directly involved in the transmission of fasciolosis in their regions of origin (Argentina and Uruguay: Mera y Sierra et al., 2011) and their efficiency for F. hepatica transmission seemed to be two- or threefold greater than that of French G. truncatula (Sanabria et al., 2011). To verify the above hypothesis regarding F. magna, two experiments were carried out: (1) to determine the capacity of Lymnaea to sustain larval development of the parasite; and (2) to specify how many cercariae these snails produced when the method by Rondelaud et al. (2007) for the breeding of amphibious snails is used. The results were compared to those noted in French G. truncatula infected and raised according to the same protocol (reference group). Simultaneously, another experiment was conducted to identify precisely the two populations of South American snails, using molecular biology, because the systematics of these lymnaeids is controversial and identification cannot be made with the sole use of morphological criteria (Duffy et al., 2009; Mera y Sierra et al., 2009). Materials and methods Collection of snails and adult worms of Fascioloides magna The first South American population (strain U) of snails has been raised at CEDIVE since 1996 and originated from Paysandú, Uruguay ( S, W). The other population (strain SP) has been bred in the laboratory since 2008 and came from San Pedro, Buenos Aires, Argentina ( S, W). Two French populations of G. truncatula were also used. The first originated from a road ditch at Chézeau Chrétien, commune of Chitray, department of Indre ( N, E) and was used for molecular biology. The other population of G. truncatula was collected from a road ditch ( N, E) in the commune of Saint Michel de Veisse, department of Creuse, and was used for experimental infections. The habitat of this last population was located on siliceous soil so that the upper shell height of adults (8 9 mm) ranged in the same scale of values as that of South American adults. Snails of height 4 mm were collected from each population. Eggs of F. magna were collected from adult flukes recovered from the livers of naturally infected red deer (Cervus elaphus) hunted in the district of Krivoklátsko (Central Bohemia, Czech Republic). Prior to hatching, the eggs were washed several times with spring water and were incubated for 12 days at 248C in the dark (Ollerenshaw, 1971). Molecular identification of South American lymnaeids by polymerase chain reaction restriction fragment length polymorphism and sequencing Five snails of each population (SP, U and G. truncatula) were used for molecular taxonomical determination. The entire foot of each fresh specimen was sectioned with a scalpel blade. Foot samples were processed using the DNeasy Blood and Tissue Kit (QIAGEN, Hilden, Germany). Lysis using proteinase K was performed overnight. Once extracted, the internal transcribed spacer (ITS)-1 segment of their nuclear ribosomal DNA was amplified using the following primers: ETTS2 (5 0 -TAAC- AAGGTTTCCGTAGGTGAA-3 0 ) and ITS-1r (5 0 -CGAGC- GAGTGATCCACCGC-3 0 ), for forward and reverse senses, respectively (Carvalho et al., 2004). The polymerase chain reaction (PCR) mix was prepared to have a final volume of 25 ml, containing 12.5 ml of Qiagen Multiplex, 1 ml of each primer, 5 ml of genomic rdna and 5.5 ml of DNAse and RNAse-free water. The PCR reaction was performed using a Perkin-Elmer Thermo Cycler (Applied Biosystems, Warrington, Cheshire, UK) as follows: initial denaturation (958C, 3 min); 35 cycles, each comprising denaturation (958C, 45 s), annealing (558C, 1 min) and extension (728C, 2 min); final extension (728C, 5 min). Amplified products were digested with the endonuclease HpyF3 I(Dde I) (Fermentas GmbH, St. Leon-Rot, Germany) which restricts the sites C # TNAG This enzyme was chosen for the present study because it was previously reported as the most accurate for differentiating lymnaeid species (Carvalho et al., 2004). Incubation was performed at 378C for 3.5 h into a final volume of 15 ml, containing 5 ml of DNA, 8.5 ml of DNAse and RNAse-free water, 1 ml of Tango buffer (Fermentas GmbH) and 0.5 ml of enzyme. All products were separated on 2% agarose gels using a 100 bp ladder and stained with ethidium bromide. They were visualized using a UV transilluminator coupled with the software GELSMART 7.0 (Clara Vision, Verrières le Buisson, France). Band size was measured with the online available software Gel Analyzer 2010a ( gelanalyzer.com). For each South American population (SP or U), two amplicons were chosen randomly and purified using a rapid PCR purification kit (Marligen Biosciences,

3 Potential intermediate hosts for Fascioloides magna 3 Ijamsville, Maryland, USA) according to the manufacturer s instructions. Sequencing reactions were performed using the ABI Big Dye kits (Applied Biosystems) and the same forward and reverse primers as those employed in the PCR amplification of ITS-1. Both strands were sequenced utilizing the ABI Prism 3130xl genetic analyser (Applied Biosystems). The forward and reverse sequences obtained were aligned and analysed using the software FinchTV (Geospiza Inc., Seattle, Washington, USA) and Clustal X (EBI, Hinxton, Cambridgeshire, UK). Sequences were submitted to GenBank under the following accession numbers: JF and JF for population SP, and JF and JF for population U. A consensus sequence was then obtained for each population of South American snails and was compared to GenBank reported sequences using the online tool BLASTN ( Experimental infections of snails Two experiments were carried out. In experiment A, the propensity of each snail species for F. magna infection was studied. One hundred and fifty snails, each measuring 4 mm in shell height, were randomly chosen from each population. Each snail was then exposed to two miracidia for 4 h at 208C in 3.5 ml of spring water and then raised at the same temperature according to the method of Rondelaud et al. (2007). Snails were finally raised in groups of ten individuals in 14-cm Petri dishes (volume of spring water, 60 ml) for 30 days. Food consisted of dried leaves of lettuce and dead leaves of grass (Molinia caerulea), while several stems of live Fontinalis sp. ensured oxygenation of the water layer. The dissolved calcium in spring water was 35 mg/l. The Petri dishes were placed in an air-conditioned room under the following conditions: a constant temperature of 208C (^18C), natural photoperiod of 10 h light. At day 30 post-exposure (pe), each surviving snail was isolated in a 35-mm Petri dish with pieces of dead grass, lettuce and spring moss, and placed at 208C. A daily surveillance was made, (1) to change spring water and food if necessary, and (2) to count the metacercariae in the dishes. When the first cercarial shedding occurred, the surviving snails were subjected to a thermal shock every 3 days by placing their Petri dishes at C for 3 h (outdoors) to stimulate cercarial exit (this temperature was chosen according to our experience with cercarial shedding of snails infected with digeneans). At the death of each snail, the shell was measured using callipers. Experiment B was carried out to determine cercarial production of F. magna in snails dissected at day 65 pe at 208C. Each experimental group consisted of 100 snails. Snail exposure to miracidia and maintenance were similar to those of experiment A. At day 65, each surviving snail was dissected under a stereomicroscope to count free rediae, intraredial cercariae and free cercariae. The two parameters calculated were snail survival at day 30 pe and the prevalence of F. magna infection (calculated in relation to the number of snails surviving at day 30 pe). Prevalence took into account the number of cercariae-shedding snails (CS snails) and the number of individuals containing cercariae but without shedding (NCS snails) in experiment A, and all snails carrying larval forms of F. magna in experiment B. For each parameter, the difference between the values recorded for the three snail groups was analysed using a x 2 test. In experiment A, the other parameters calculated were the growth of CS snails during the experiment, the length of the prepatent period (time of cercarial differentiation inside rediae), the length of the patent period (time during which cercariae exit from the snail), the life period after the last exit of cercariae and the total number of metacercariae. In experiment B, the number of free rediae, the quantity of free cercariae and the quantity of intraredial cercariae were considered. Individual values recorded for these last eight parameters were averaged and their standard deviations were calculated considering snail groups. One-way analysis of variance was used to establish levels of statistical significance calculated using the Statview 5.0 software (Deltasoft Meylan, France). Results Identification of South American lymnaeids The approximate size of amplified bands for snail samples was 600 bp for population SP and G. truncatula, and 650 bp for population U. In South American lymnaeids, three bands (fig. 1) were noted in the patterns derived from the restriction fragments; 90, 130 and 380 bp for population SP and 120, 130 and 400 bp for population U. In contrast, four bands (80, 90, 130 and 300 bp) were noted for the French G. truncatula (fig. 1). The observed restriction patterns clearly allowed separation of population SP from population U. Sequence analysis of the ITS-1 amplicons showed differences among both populations of South American snails. Population SP (fig. 2) showed 99.6% identity with the previously reported sequence of Lymnaea neotropica (GenBank, accession number AM412228). On the other hand, population U (fig. 3) exhibited greater similarity to the sequences of L. viatrix var. ventricosa and L. viatrix (GenBank, accession numbers AM and HQ283254, respectively) and was thus classified as L. v. ventricosa in the present study. This population differed from the GenBank sequences by the addition of four bases at positions 272, 274, 275 and 322. Lymnaeid species and snail infection The results of experiment A are shown in table 1. Compared to the reference G. truncatula, the survival rate of snails at day 30 pe (table 1) was significantly greater for L. neotropica (x 2 ¼ 13.96, P, 0.001) and significantly lower for L. v. ventricosa (x 2 ¼ 7.34, P, 0.01). In both groups of South American snails, the prepatent period was significantly longer (F ¼ 3.58, P, 0.05) than that of the reference group, whereas the number of shed cercariae was significantly lower (F ¼ 6.70, P, 0.01). The prevalence of infection, the snail growth during the experiment, and the patent periods were not significantly different between the snail groups tested. In contrast, the South American snails survived on average days after the last exit of their cercariae and these periods

4 4 R. Sanabria et al. M K Strain SP Strain U Galba truncatula Fig. 1. Restriction fragments of rdna ITS-1 segment from each snail population treated with endonuclease HpyF3 I. Lanes: M, 100 bp marker; K, negative control; SP, Argentinean population of Lymnaea neotropica; U, Uruguayan population of Lymnaea viatrix; Galba truncatula, French G. truncatula. Band sizes are shown beside each band. were significantly longer (F ¼ 7.84, P, 0.01) than in the G. truncatula group. In experiment B, a significant difference (x 2 ¼ 10.97, P, 0.001) between survival rates of G. truncatula and L. neotropica was noted (table 2), whereas there was no difference between G. truncatula and L. v. ventricosa. Infection rates were not significantly different between the three snail groups. Compared to the reference group, the number of free rediae and that of free cercariae were significantly lower in the tested South American lymnaeids (free rediae: F ¼ 3.29, P, 0.05; free cercariae: F ¼ 16.58, P, 0.01). At day 65, the number of cercariae remaining in the body of rediae ranged within the same scale of values and so no significant difference was recorded. If the number of shed cercariae (table 1) is compared to the total cercarial production within the snail s body (table 2), the percentage of larvae that exited from the snail was 51.3% for G. truncatula and 46.8% for L. v. ventricosa, while it was only 32.2% for L. neotropica. In this respect, L. neotropica seemed to be a less suitable intermediate host of F. magna than L. v. ventricosa. Fig. 2. Comparison of ITS-1 sequences for population SP (GenBank, accession numbers JF and JF960166) and Lymnaea neotropica (GenBank, accession number AM ).

5 Potential intermediate hosts for Fascioloides magna 5 Fig. 3. Comparison of ITS-1 sequences for population U (GenBank, accession numbers JF and JF960168), L. viatrix var. ventricosa (GenBank, accession number AM412227) and L. viatrix (GenBank, accession number HQ283254). X indicates a lack of a base in the previously reported sequences. Discussion The presence of L. neotropica has been reported at Mendoza in western Argentina (Mera y Sierra et al., 2009) and is mentioned here for the first time in the eastern part of this country (Buenos Aires). In the same way, our results on L. v. ventricosa confirm the report by Carvalho et al. (2004) indicating the presence of this snail in Uruguay Table 1. Main characteristics of Fascioloides magna infection in three species of lymnaeids (experiment A). Parameters Galba truncatula Lymnaea neotropica Lymnaea viatrix ventricosa Number of pre-adult snails at exposure Number of surviving snails at day 30 pe (survival rate %) 119 (79.3) 141 (94.0) 98 (65.3) Number of CS snails Number of NCS snails Prevalence of infection (%) Growth of CS snails during the experiment (mm)* 2.8 (0.7) 2.5 (0.6) 2.7 (0.6) Length (days)*: prepatent period 65.2 (4.0) 74.9 (7.3) 70.5 (7.4) patent period 21.5 (7.2) 17.2 (5.4) 23.7 (4.7) life period after the last exit of cercariae 1.5 (1.7) 14.0 (5.1) 11.2 (5.0) Total number of metacercariae per snail* (47.2) 37.5 (21.4) 74.3 (37.5) pe, post-exposure; CS snails, cercariae-shedding snails; NCS, snails containing cercariae but without shedding. * Mean value (SD).

6 6 R. Sanabria et al. Table 2. The number and prevalence of larval stages of Fascioloides magna in three species of lymnaeids on day 65 post-exposure (pe) at 208C (experiment B). Parameters Galba truncatula Lymnaea neotropica Lymnaea viatrix ventricosa Number of pre-adult snails at exposure Number of surviving snails at day 30 pe (survival rate %) 73 (73.0) 91 (91.0) 61 (61.0) Number of snails containing cercariae (prevalence %) 40 (54.7) 51 (56.3) 30 (49.1) Number of larvae per snail*: free rediae 37.3 (5.1) 24.2 (8.1) 28.3 (6.5) free cercariae (32.4) 81.4 (17.3) (19.1) intraredial cercariae 58.2 (19.5) 34.7 (14.1) 35.1 (13.2) Total cercarial production per snail* (27.6) (16.5) (18.7) * Mean value (SD). (unspecified locality). Compared to sequences of L. viatrix (AM and HQ283254) the sequence of L. v. ventricosa showed four additional bases. As these differences were a constant in both forward and reverse strands for all sequences, it may be concluded that these characteristics represent a local genotype. Similar differences have already been reported, for example, in allopatric samples of Lymnaea stagnalis (Remigio & Blair, 1997). In view of this finding, it is necessary to take into account the fact that ITS-1 segments might show population variations, as demonstrated by Almeyda Artigas et al. (2000). Even if the use of ITS-1 sequence is sufficient for species discrimination of both South American lymnaeids, snail identification could be improved in further studies by additional molecular markers such as mitochondrial DNA. The present study demonstrates that Lymnaea neotropica and L. v. ventricosa can be included as new intermediate hosts for F. magna. The longer prepatent periods and lower numbers of cercariae noted for these two species (in the context of cercarial shedding, table 1) might represent the case of an incomplete adaptation process of the snail parasite system. This interpretation is supported by the larger values of standard deviations noted for these parameters (table 1). After the exit of the last cercariae, L. neotropica and L. v. ventricosa can survive on average for 14.0 and 11.2 days, respectively, before their death, whereas these snails still contained free cercariae in their bodies during this period. This finding is difficult to comment on because the post-shedding period was not recorded in our experimental infections of L. neotropica and L. v. ventricosa, nor in the French G. truncatula with a French isolate of F. hepatica. Two complementary hypotheses may be proposed. First, the two South American species could be more able to sustain larval development of F. magna than the French population of G. truncatula. Second, the life period after the last released cercariae might also be subjected to unknown characteristics of F. magna larval development. Under constant conditions in the laboratory, there was little cercarial shedding of F. magna from European populations of G. truncatula (Erhardová-Kotrlá, 1971; Vignoles et al., 2006; Rondelaud et al., 2007) and also from O. glabra (Rondelaud et al., 2006). Owing to the thermal shock used to stimulate cercarial shedding, the presence of metacercariae (table 1) was noted for a mean of 17.2 days (L. neotropica) and 23.7 days (L. v. ventricosa). The interruption of this process in the following days may be the consequence of too few free cercariae remaining in the snail body to cause another cercarial shedding. In conclusion, both South American lymnaeids, like A. tomentosa in Australia (Foreyt & Todd, 1974) and O. glabra in France (Rondelaud et al., 2006), can be potential intermediate hosts for larval development of F. magna, whereas fascioloidosis has not been recorded in these countries. These findings demonstrate the high potential of the parasite to spread in new areas by adapting to local snail species. The facility of adaptation of F. magna to new intermediate hosts enhances the risk of parasite transmission in these areas, mainly as a result of local deer migrations and/or importation of infected ruminants. Acknowledgements This study was partly supported by the Swedish Foundation for Agricultural Research (contract no. H ), the Czech Science Foundation (grant no. P502/ 10/P248), the Czech Ministry of Education (grant nos. MSM LC06009 and MSM ) and by the University Research Centre (UNCE) of Charles University, Prague (grant no ). The authors thank Dr David A. 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