- Open Access
A new baseline for fascioliasis in Venezuela: lymnaeid vectors ascertained by DNA sequencing and analysis of their relationships with human and animal infection
© Bargues et al; licensee BioMed Central Ltd. 2011
Received: 15 March 2011
Accepted: 14 October 2011
Published: 14 October 2011
Human and animal fascioliasis poses serious public health problems in South America. In Venezuela, livestock infection represents an important veterinary problem whereas there appear to be few human cases reported, most of which are passively detected in health centres. However, results of recent surveys suggest that the situation may be underestimated in particular areas. To obtain a baseline for future fascioliasis assessment, studies were undertaken by means of rDNA ITS-2 and ITS-1 and mtDNA cox 1 sequencing to clarify the specific status of Venezuelan lymnaeids, their geographical distribution and fascioliasis transmission capacity, by comparison with other American countries and other continents.
Results obtained completely change the lymnaeid scenario known so far. The relatively rich lymnaeid fauna of Venezuela has been proven to include (i) Lymnaea meridensis and L. neotropica as the only native members, (ii) L. cubensis and Pseudosuccinea columella introduced from the Caribbean area, and (iii) Galba truncatula and L. schirazensis introduced from the Old World. The absence of representatives of the stagnicoline and Radix groups is remarkable. Four species are fascioliasis vectors: G. truncatula, L. cubensis and L. neotropica, which have the capacity to give rise to human endemic areas, and P. columella, which is a source of animal infection and is responsible for the spread of disease. Vector capacity in the apparently highland endemic L. meridensis is to be confimed, although may be expected given its phylogenetic relationships. Similarly as elsewhere, the non-transmitting L. schirazensis has been confused with L. cubensis, also with G. truncatula and possibly with L. neotropica.
The new scenario leads to the re-opening of many disease aspects. In Venezuela, altitude appears to be the main factor influencing fascioliasis distribution. Human infection shows an altitude pattern similar to other Andean countries, although a differing highland/lowland impact on animal infection does not appear evident. The overlap of G. truncatula, L. cubensis and probably also L. neotropica in temperate and cold zones suggests a higher risk for human infection in mid and high altitude areas. A lymnaeid species mapping by means of DNA markers becomes a priority to determine human and animal fascioliasis distribution in Venezuela, owing to the importance of lymnaeid vectors in defining transmission and epidemiological patterns.
Fascioliasis is a pathogenic liver parasitosis caused by fasciolid flukes which affects humans and livestock species almost everywhere [1, 2]. In the last two decades, this disease has emerged in many countries of Latin America, Europe, Africa and Asia [1, 3]. This emergence phenomenon has partly been related to climate change [4, 5], given the high dependence of both fasciolid larval stages and their freshwater lymnaeid snail vectors on climatic and environmental characteristics .
The infectivity of the metacercarial infective stage of isolates from different livestock species isolates have been shown to be similar [7, 8], whereas the lymnaeid vector species represent a crucial factor for the epidemiology of the disease [9, 10]. Geographical distribution, prevalences and intensities of both human and animal infection pronouncedly depend on the ecological characteristics (population dynamics, anthropophylic characteristics, type of water bodies, etc.) of the lymnaeid species involved in the transmission. Different lymnaeid species appear, therefore, linked to the different transmission patterns and epidemiological scenarios of this very heterogeneous disease in humans [2, 11]. Thus, similarly as in other vector-borne diseases, this relationship supports the use of lymnaeids as disease biomarkers and becomes useful for mathematical modelling and remote sensing - geographical information system (RS-GIS) tools for the control of fascioliasis [12, 13].
South America stands out due to the human endemic areas described in many Andean countries, including high prevalence and intensity in humans caused by Fasciola hepatica, such as in Chile , Bolivia [15–17], Peru [18, 19] and Ecuador . In Argentina the human fascioliasis situation seems to be underestimated  and in Colombia appropriate studies in risky rural areas are still pending .
Only two lymnaeid species have been traditionally reported to be present in Venezuela: Lymnaea cubensis and Pseudosuccinea columella[24–27]. The first is a member of the Galba/Fossaria group, a well-known vector of fascioliasis originally described from Cuba and distributed throughout southern North America and the Caribbean . Pseudosuccinea columella is a peculiar, widely dispersed species, believed to be originally from the region of southern North America, Central America and the Caribbean, but which has successfully colonised other continents (South America, Europe, Africa, Oceania) [29, 30], and is also an efficient vector leading to increased spread of the disease .
This reduced list of lymnaeid species reported in Venezuela changed as the consequence of the collection of specimens of different populations of L. cubensis in Mérida State in March 2000. A preliminary DNA sequencing process suggested that several of these populations belonged to the morphologically very similar species Galba truncatula, with which it may be easily confused, similarly as it happens with other small lymnaeid species [28, 32]. However, subsequent extensive DNA marker sequencing proved that indeed not only one but several different, yet similar species had been confused under the name of L. cubensis in Venezuela. Additionally, Lymnaea cousini, hithterto only known from Ecuador and Colombia , has recently been described and reported in Venezuela [33, 34].
However, lymnaeids pose serious specimen classification difficulties when only applying malacological methods such as anatomical studies [9, 10, 28, 35]. Moreover, intraspecific variation of shell shape is well marked in lymnaeids, although a genetic component in shell shape has been shown at least in some lymnaeid populations . In the Americas, specimen classification problems are mainly related to the so-called "fossarine" or Galba/Fossaria group of numerous, very similar, usually undifferentiable lymnaeid vector species [28, 32], as is the case of the aforementioned L. cubensis, G. truncatula and L. cousini.
The crucial implications of lymnaeid vectors for fascioliasis transmission, epidemiology and control urged the development of new tools to facilitate specimen classification, genetic characterisation of natural populations and laboratory strains, and to elucidate the systematics and taxonomy of the Lymnaeidae. This is the purpose of the worldwide lymnaeid molecular characterisation initiative . Nuclear ribosomal DNA (rDNA) and mitochondrial DNA (mtDNA) markers proved useful for this endeavour in invertebrates in general, although disadvantages and limitations depending on each marker should be taken into account . Their application also showed their usefulness in lymnaeids .
The internal transcribed spacers of the rDNA, mainly ITS-2 and secondarily ITS-1, are the most useful sequences for studies at both specific and supraspecific levels [2, 10, 38]. Similarly as in planorbid vectors of schistosomiasis , a fragment of the cytochrome c oxidase subunit I gene (cox 1) of the mtDNA has also been used in lymnaeids . However, its usefulness in lymnaeids has been recommended to be restricted to only population and close species analyses , due to saturation of nucleotide positions and problems posed by incomplete gene sequences .
The purpose of this present article is to report the results of more than ten years work by means of ITS-2, ITS-1 and cox 1 sequencing, which are required to clarify the number of lymnaeid species present in Venezuela and to ascertain their systematic status by comparison with lymnaeids not only of neighbouring or geographically near countries but also of different continents. The final analysis offers a completely new spectrum of six lymnaeid species whose composition considerably differs from what has been noted so far. This offers a new baseline on which to design and launch appropriate studies on human and animal fascioliasis in Venezuela henceforth. The implications of this new vector scenario on the disease are finally discussed.
Lymnaeid snail materials
Nuclear ribosomal and mitochondrial DNA haplotype code identification for lymnaeid species and populations studied from Venezuela
mtDNA cox 1
Combined H nomenclature
Coordinates and altitude
No. snails (month of collection)
Magdaleno, Lago Valencia-Maracay (Aragua)
10°07'36" N 67°35'42" W 428 m
water canal Calicanto flowing into the large Lake of Valencia-Maracay
L. cubensis ***
L.cub-4B, cox 1b
Mucura, Lago Valencia-Maracay (Aragua)
10°06'38" N; 67°28'47" W 410 m
pool of stagnant water close to the large Lake of Valencia-Maracay
L. cubensis ***
L.cub-4B, cox 1b
El Joque, Jají (Mérida)
8°35'10" N; 71°20'36" W 1995 m
irrigation canal in dairy cattle farm
Lymnaea sp. aff. cubensis ***
L.cub-2B, cox 1b
Estanques Lagunillas (Mérida)
°30'15" N; 71°23'41" W 1032 m
connecting canal from permanent pond inside urban area
L.cub-4B, cox 1b
La Linda, Güigüe (Carabobo)
10°04'53" N; 67°47'15" W 450 m
water canal at the southern part of the Lake of Valencia-Maracay
L. cubensis ***
L.neo-2A, cox 1c
Finca El Arenal, Tucacas (Falcón)
10°43'29" N; 68°23'25" W 30 m
irrigation canals from Aroa river in Parcelas Agrotécnicas of a farm, in the way to Las Lapas
L. cubensis ***
L.neo-2A, cox 1d
Iglesia Monterrey (Mérida)
8°38'39" N; 71°07'24" W 1900 m
irrigation canal along pasture grassland
G.tru-2D, cox 1b
San Rafael, Mucuchies A (Mérida)
8°44'42" N; 70°55'23" W 3150 m
grassland with slight slope flooded from small stream
G.tru-2D, cox 1b
San Rafael, Mucuchies B (Mérida)
8°44'46" N; 70°55'21" W 3200 m
small stream south from the village
G.tru-2D, cox 1b
San Isidro, Apartaderos (Mérida)
8°47'48" N; 70°51'33" W 3500 m
small stream behind the school of children San Isidro
G.tru-2D, cox 1b
Paso del Cóndor (Mérida)
8°50'41" N; 70°48'41" W 4080 m
road gutter with water descending from mountain fountain
G.tru-2D, cox 1b
Hotel Valle Grande (Mérida)
8°40'28" N; 71°06'03" W 2200 m
small gutter besides external wall of hotel building
Laguna Fe y Alegría (Mérida)
8°37'29" N; 70°49'33" W 1840 m
wild grass and stones besides small stream of fast running water
La Trampa (Mérida)
8°32'25" N; 71°27'23" W 2170 m
besides small water collection on hillside
G. truncatula ***
Laguna Mucubaji (Mérida)
8°47'52" N; 70°49'32" W 3550 m
surroundings and shore of large natural pool
La Linda, Güigüe (Carabobo)
10°04'53" N; 67°47'15" W 450 m
water canal at the southern part of the Lake of Valencia-Maracay
P. columella ***
El Valle (Mérida)
8°38'41" N; 71°07'24" W 1930 m
irrigation canal along pasture grassland close to Iglesia Monterrey
Lymnaeidae gen. sp.
Living specimens were fixed in 70% ethanol immediately after collection in the field and stored in the same fixative until analysis.
Preliminary classification of specimens (Table 1) was, whenever possible, based on shell shape and size and morpho-anatomical characteristics traditionally considered of systematic usefulness, mainly the sexual organs. Unfortunately, sometimes snail softparts were too contracted due to fixation by alcohol 96% in cold conditions or specimens available were too small to allow a clear anatomical classification and consequently specimens were classified on shell characteristics only. In given cases, specimens were collected and preliminarily classified before 2006, when publications including phenotypic re-assessments of species were not yet available. In other cases, the preliminary classification was only at genus level, because collectors (vets, i.e., non-malacology experts) already knew that there were many species under the term of "cubensis" together with the great difficulties or even sometimes impossibility to diferentiate between species of Galba/Fossaria. Even in a species a priori easily classifiable as P. columella, its unexpected finding at very high altitude led the collectors to leave its classification open. With regard to Galba/Fossaria species, several recent articles have furnished new complete phenotypic re-descriptions that may help henceforth [22, 28, 41–43].
DNA was extracted from more than one specimen of a given population when this was deemed necessary for sequence verification. DNA was only isolated from the foot of each snail [28, 44]. Snail feet fixed in 70% ethanol were used for DNA extraction procedures. After dissection under a microscope, half of the foot was suspended in 400 μl of lysis buffer (10 mM Tris-HCl, pH 8.0, 100 mM EDTA, 100 mM NaCl, 1% sodium dodecyl sulphate SDS) containing 500 μg/ml Proteinase K (Promega, Madison, WI, USA) and digested for 2 hr at 55°C with alternate shaking each 15 min. The extraction was then performed with chloroform and DNA was precipitated with isopropyl alcohol. The procedure steps were performed according to methods outlined previously [9, 28, 32, 45]. The pellet was dried and resuspended in 30 μl sterile TE buffer (pH 8.0). This suspension was stored at -20°C until use.
DNA sequence amplification
Each one of the three DNA markers was PCR amplified independently for each lymnaeid specimen and each PCR product was sequenced for a bona-fide haplotype characterisation. The rDNA spacers ITS-2 and ITS-1 were amplified using primers previously described [9, 28, 32, 46, 47]. A mitochondrial DNA cox 1 gene fragment was amplified using universal primers . Amplifications were generated in a Mastercycle epgradient (Eppendorf, Hamburg, Germany) using 4-6 μl of genomic DNA for each 50 μl PCR reaction. PCR conditions were 30 cycles of 30 sec at 94°C, 30 sec at 50°C and 1 min at 72°C, preceded by 30 sec at 94°C and followed by 7 min at 72°C for ITS-2 and ITS-1, and by 40 cycles of 30 sec at 90°C, 1 min at 48°C and 1 min at 72°C, preceded by 2.5 min at 94°C and followed by 10 min at 72°C for cox 1. Ten μl of each PCR product was checked by staining with ethidium bromide on 1% Nusieve® GTG agarose (FMC) gel electrophoresis, using the Molecular Weight Marker VI (Boehringer Mannheim) at 0.1 μg DNA/μl as control.
Purification and quantification of PCR products
Primers and nucleotides were removed from PCR products by purification on Wizard™ PCR Preps DNA Purification System (Promega, Madison, WI, USA) according to the manufacturer's protocol and resuspended in 50 μl of 10 mM TE buffer (pH 7.6). The final DNA concentration was determined by measuring the absorbance at 260 and 280 nm.
The sequencing of the complete rDNA ITS-2 and ITS-1 and the fragment of the mtDNA cox 1 gene was performed on both strands by the dideoxy chain-termination method . It was carried out with the Taq dye-terminator chemistry kit for ABI 3730 DNA Analyzer (Applied Biosystems, Foster City, CA, USA), using PCR primers.
Sequences were aligned using CLUSTAL-W version 1.8  and MEGA 4.0 , and assembly was made with the Staden Package . Subsequently, minor corrections were manually introduced for a better fit of nucleotide correspondences in microsatellite sequence regions. Homologies were performed using the BLASTN programme from the National Centre for Biotechnology information web site http://www.ncbi.nlm.nih.gov/BLAST.
DNA haplotype nomenclature
The codes for the sequences obtained follow the standard nomenclature proposed previously for lymnaeid snails [2, 10, 46]. It shall be noted that haplotype codes are only definitive in the case of complete sequences. When dealing with fragments or incomplete sequences, haplotype codes are provisional.
rDNA ITS-2: G. truncatula H1 [EMBL: AJ296271], H2 [EMBL: AJ243017] and H3 (= L. viatrix sensu Ueno et al., 1975; = L. cubensis sensu Ueno et al., 1975) [EMBL: AJ272051] [9, 28, 47]; L. cubensis H1 [EMBL: AM412223], H2 [EMBL: FN182200] and H3 [EMBL: FN182201] [28, 43], L. neotropica [EMBL: AM412225] , L. schirazensis H1 [GenBank: JF272601] and H2 [GenBank: JF272602] , L. cousini [EMBL: FN598153] and L. meridensis [EMBL: FN598154] ; P. columella H1 [EMBL: FN598155] and H2 [EMBL: FN598156]  and P. columella [GenBank: AY186751] .
rDNA ITS-1: G. truncatula HA [EMBL: AJ243018], HB [EMBL: AJ296270] and HC (= L. viatrix sensu Ueno et al., 1975; = L. cubensis sensu Ueno et al., 1975) [EMBL: AJ272052] [9, 28, 47]; L. cubensis HA [EMBL: AM412226], HB [EMBL: FN182202] and HC [EMBL: FN182203] [28, 43], L. neotropica [EMBL: AM412228] , L. schirazensis HA [GenBank: JF272603] and HB [GenBank: JF272604] , L. cousini [EMBL: FN598157] and L. meridensis [EMBL: FN598159] ; P. columella HA [EMBL: FN598160]  and P. columella [GenBank: AY186751] .
mtDNA cox 1 gene: G. truncatula from Spain [EMBL: AM494011]  and Germany [GenBank: EU818799] ; L. cubensis cox 1a [EMBL: AM494009] and cox 1b [EMBL: FN182205], L. neotropica cox 1a [EMBL: AM494008] and cox 1b [EMBL: FN356741] [28, 43, 55], L. schirazensis cox 1a [GenBank: JF272607], cox 1b [GenBank: JF272608], cox 1c [GenBank: JF272609], and cox 1d [GenBank: JF272610] , L. cousini [EMBL: FN598161] and L. meridensis [EMBL: FN598164] ; P. columella [EMBL: FN598165]  and P. columella [GenBank: AY227366] .
Phylogenetic analysis of ITS-2 and ITS-1 combined haplotypes was performed with a Maximum Likelihood (ML) approach using PAUP version 4.0b10. ML parameters and the evolutionary model best fitting our dataset were determined using Akaike and Bayesian information criteria (AIC and BIC) [57, 58], implemented in jModeltest vesion 0.1.1 . Starting branch lengths were obtained using the least-squares method with ML distances.
To provide an assessment of the reliability of the nodes in the ML tree, three methods were used. First, a bootstrap analysis using 1000 replicates was made with fast-heuristic search in PAUP. Second, a distance-based phylogeny using the neighbour-joining (NJ) algorithm  with the ML pairwise distances was obtained and statistical support for the nodes was evaluated with 1000 bootstrap replicates, with and without removal of gapped positions. Third, a Bayesian phylogeny reconstruction procedure was applied to obtain posterior probabilities (BPP) for the nodes in the ML tree, by using the same evolutionary model as above, implemented in MrBayes 3.1  with four chains during 1,000,000 generations, trees being sampled every 100 generations. The first 1000 trees sampled were discarded ("burn-in") and clade posterior probabilities (PP) were computed from the remaining trees.
Phylogenetic analyses were performed after adding the following reference sequences of rDNA ITS-2 and ITS-1 of lymnaeids stored in the databases: L. (Stagnicola) palustris palustris [EMBL: AJ319620, EMBL: AJ626849]; L. (S.) fuscus [EMBL: AJ319621, EMBL: AJ626856] ; Catascopia catascopium [GenBank: AF013143, GenBank: AF013143]; Hinkleyia caperata [GenBank: AF013139, GenBank: AF013139] ; Radix auricularia ITS-2 halplotype 1 [EMBL: AJ319628]; R. balthica (= R. peregra) ITS-2 haplotype 1 [EMBL: AJ319633] . The intergenic region sequence [GenBank: AY030361] including both ITSs of the planorbid species Biomphalaria pfeifferi was used as outgroup.
Nuclear rDNA ITS-2 and ITS-1 and mtDNA cox 1 nucleotide sequence data reported in this paper are available in the GenBank database under the accession numbers noted in Table 1.
Specimens from Magdaleno and Mucura (Aragua State), Estanques Lagunillas and Jají (Mérida State), preliminarily classified as L. cubensis or Lymnaea sp. aff. cubensis, proved to be L. cubensis by ribosomal and mitochondrial DNA markers (Table 1).
All the specimens from the four populations analysed present the same ITS-1 sequence, of 520 bp and a 56.35% GC content. When compared with the three ITS-1 haplotypes of L. cubensis available in EMBL (HA, HB, HC), this ITS-1 proved to be identical to the haplotype L.cub-HB also present in Sullivan Island, South Carolina, USA [EMBL: FN182202].
Only one haplotype was detected in all specimens and populations studied, with a length of 672 bp and an AT content of 68,60%. This sequence fits exactly with the previously described L.cub-cox 1b from South Carolina, USA [EMBL: FN182205], differing by only one mutation (T/C) with regard to L.cub-cox 1a in position 468 of the respective alignment. In the amino acidic sequence alignment (224 aa long), both cox 1a and cox 1b haplotypes are identical.
Specimens from La Linda (Carabobo State) and Finca el Arenal, Tucacas (Falcón State), preliminarily classified as L. cubensis, proved to be L. neotropica by ribosomal and mitochondrial DNA markers (Table 1).
All the specimens showed identical ITS-2 sequence, of 415 bp and a 56.87% GC content. When compared with the ITS-2 haplotype of L. neotropica available in EMBL (H1), the Venezuelan sequence proved to be different, showing a shorter length due to the lack of one microsatellite repeat (AT) in positions 402-403 of the sequence alignment of both ITS-2 haplotypes. This is the first time that this haplotype has been found and has consequently been deposited in GenBank under the new code L.neo-H2 (Table 1).
All specimens studied presented the same ITS-1 sequence of 533 bp and a 56.66% GC content. This sequence was compared with the ITS-1 haplotype of L. neotropica available in EMBL (L.neo-HA) and proved to be identical to this haplotype also present in Peru and Argentina [EMBL: AM412228].
Specimens from Iglesia Monterrey, two different populations in San Rafael de Mucuchíes, Apartaderos and Paso del Cóndor (Mérida State), preliminarily classified as Galba/Fossaria sp., L. viatrix or L. cubensis, proved to be G. truncatula by ribosomal and mitochondrial DNA markers (Table 1).
All the specimens showed an identical ITS-2 sequence, of 401 bp and a 59.10% GC content. When compared with the three ITS-2 haplotypes of G. truncatula available in EMBL (H1, H2, H3), this sequence proved to be identical to the previously described ITS-2 haplotype 2 (H2) for G. truncatula [EMBL: AJ243017].
All specimens studied showed identical ITS-1 sequence, of 504 bp and a 57.74% GC content. This sequence was compared with the three ITS-1 haplotypes of G. truncatula available in EMBL (HA, HB, HC) and proved to be different, with a specific mutation C instead of T in position 449 of the ITS-1 haplotype sequence alignment. This new haplotype has been deposited in GenBank under the code G.tru-HD (Table 1).
Snail specimens collected from Hotel Valle Grande, Laguna Fe y Alegría and La Trampa (Mérida State), preliminarily classified as Galba/Fossaria sp., L. cubensis or G. truncatula, proved to be L. schirazensis after ribosomal and mitochondrial DNA marker sequencing (Table 1).
All the specimens analysed presented the same ITS-2 sequence, of 444 bp and a 53.82% GC content. When compared to the two ITS-2 haplotypes of L. schirazensis available in GenBank, it proved to be identical to the previously described L.schi-H1 [GenBank: JF272601]. This haplotype differs from the other haplotype known L.schi-H2 [GenBank: JF272602] in only 8 polymorphic sites, corresponding to 8 indels caused by the microsatellite repeat (TGCT), being present twice in the haplotype 1 between positions 128 and 135 of the alignment and absent in the haplotype 2.
All the lymnaeid individuals showed identical ITS-1 sequences, of 533 bp long and a 59.91% GC content. This haplotype was compared with two ITS-1 haplotypes of L. schirazensis available in GenBank (HA, HB) and proved to be the same as the previously described L.schi-HA [GenBank: JF272605]. Differences between this haplotype A and L.schi-HB [GenBank: JF272604] are only 1 mutation and two indels.
The specimens from Laguna Mucubaji (Mérida State), previously identified as L. cousini, proved by ribosomal and mitochondrial DNA marker sequences to be another species to which the name L. meridensis has been recently given (Table 1).
Sequence length and its slightly GC biased average nucleotide composition are 457 bp and 58.85%, respectively. This sequence corresponds to the original haplotype L.mer-H1 of the recently described species L. meridensis [EMBL: FN598154]. In the ITS-2 sequence alignment with the haplotype H1 of L. cousini [EMBL: FN598157], a high number (63) of variable positions appear (12.45%), of which 14 are mutations and 49 indels.
ITS-1 length and its slightly GC biased average nucleotide composition are 570 bp and 58.41%, respectively. This sequence corresponds to the haplotype L.mer-HA of L. meridensis [EMBL: FN598159]. In the ITS-1 sequence alignment of L.mer-HA with haplotypes HA and HB of L. cousini [EMBL: FN598157, EMBL: FN598158], a total of 47 variable positions appear (7.75%), of which 29 were mutations and 18 indels.
The specimens from La Linda (Carabobo State) and El Valle (Mérida) previously identified as P. columella or ascribed to an undetermined lymnaeid (Lymnaeidae gen. sp.) respectively, proved to be P. columella also by ribosomal and mitochondrial DNA markers (Table 1).
Two haplotypes were found in the populations studied. All specimens from La Linda, showed identical ITS-2 sequence, of 404 bp and a biased GC content of 60.64%. This ITS-2 sequence is identical to P.col-H2 from Colombia [EMBL: FN598156]. Specimens from El Valle showed the same ITS-2 sequence of 470 bp and 57.02% GC content, which is identical to P.col-H1 from Puerto Rico [EMBL: FN598155]. Worth mentioning is the presence of T in the sequence position 6 in the La Linda population, whereas it presents C in El Valle population. When compared to the ITS-2 of P. columella from Cuba available in the database [GenBank: AY186751], a total of 2 mutations and 9 indels appear.
The ITS-1 sequence of both populations was the same, with a length of 536 bp and a slightly GC biased average nucleotide composition of 58.02%. This sequence was identical to that previously described for P.col-HA from Puerto Rico [EMBL: FN598160]. In a pairwise alignment comparison with P. columella ITS-1 from Cuba available in the database [GenBank: AY186751: 527 bp long and 58.44% GC], three indels appear in positions 262, 270 and 276. Worth noting is the presence of A in position 510, in which whether A or G were found in Cuba depending on the susceptibility or resistance characteristics of the population, respectively.
Only one haplotype was detected in all specimens of the populations studied. This fragment has a length of 672 bp and a highly AT-biased average nucleotide composition of 69.2%. This sequence was compared with the other two P. columella cox 1 sequences of similar length already published and available in databases. Nucleotide and amino acid compositions of P. columella from Venezuela are identical to those of the haplotype cox 1a from Puerto Rico [EMBL: FN598165] and Australia [GenBank: AY227366].
The ML model best fitting the ITS-1 an ITS-2 combined haplotype dataset was found to be GTR+G (-Ln likelihood = 8346.73598) with a shape parameter (alpha) of 0.6622, base frequencies for A, C, G, and T of 0.20677, 0.27935, 0.25969 and 0.25419, respectively, and a proportion of invariable sites = 0. The new ITS-1 sequences obtained from the two Radix species R. auricularia and R. balthica used were adequately deposited in the database [EMBL: AJ319628 and EMBL: AJ319633, respectively].
The monophyly of the ITSs haplotypes of the five lymnaeid species of the Galba/Fossaria group found in Venezuela was strongly supported (92/99/99 in NJ/ML/BBP). In this clade, G. truncatula does not appear clustering with other morphologically similar lymnaeids such as the New World L. cubensis and L. neotropica, nor to the phenotypically very close Old World L. schirazensis, but interestingly with L. meridensis, although supports are not high. Lymnaea schirazensis appears in a basal position, as a sister species, in this monophyletic group, although the clustering of the other four Galba/Fossaria species does not appear well supported.
Pseudosuccinea columella appears basal to the two groupings of the stagnicolines, including both Palaearctic and Nearctic species, and the Galba/Fossaria clade which comprises the F. hepatica main vector species. The branch of Pseudosuccinea, Galba/Fossaria and stagnicolines appears, moreover, well separated from the Radix branch, with very high supports (98/96/100 in NJ/ML/BBP).
Lymnaeid species reported in Venezuela
The first report on lymnaeid snail morphology and their habitat in Venezuela, as well as their possible involvement in F. hepatica transmission, appeared early in the 20th century . The presence and involvement of L. cubensis in the transmission of F. hepatica in different parts of the country was described from the mid-last century, mainly with regard to livestock infection [65–68], but also from places where human infection had been reported [69, 70]. The presence of P. columella was detected for the first time in Venezuela somewhat later in a water canal of Maracay city, Aragua State . Numerous multidisciplinary studies on L. cubensis, less numerous on P. columella, were performed mainly during the 1980s and first part of the 1990s, including research on distribution, biology, ecology, experimental F. hepatica infection, epidemiology and control in western areas [25, 27, 72–80].
Publications on lymnaeid snails in Venezuela have been more sporadic from the beginning of the 21st century. A high 23.3% F. hepatica infection prevalence in L. cubensis snails in endemic livestock farms of the Zulia State was noted . Surprisingly high prevalences of 39-43% were also reported from non-classified lymnaeids in different habitats in another farm of Mérida . After the addition of L. cousini and inclusion of G. truncatula, the four aforementioned species were noted as the only lymnaeids found in Venezuela within the very recent country wide malacological review .
A new lymnaeid species scenario obtained by DNA sequencing
Results obtained by DNA sequencing completely change the lymnaeid scenario in Venezuela. The new scenario of six lymnaeid species contributed in the present paper shows up to which level malacological methods may give rise to misclassifications when dealing with Lymnaeidae, mainly in problematic groups such as Galba/Fossaria[28, 43]. Except P. columella, a species with peculiar anatomo-morphological and shell shape characteristics, which facilitate its classification mainly in the largest specimens, the remaining L. cubensis, L. neotropica, G. truncatula, L. schirazensis and L. meridensis may be included in Galba/Fossaria (Figure 7). This may explain why the latter four species had been overlooked under the binomium L. cubensis during so much time. These five Galba/Fossaria species are very similar and almost indistinguishable when young and mid-sized.
This lymnaeid fauna thus appears to be markedly rich when compared to that known in countries of the regions of Central America, the Caribbean and South America. The lymnaeid richness of Venezuela results from the overlap of (i) species of Caribbean and/or Central American origin, such as L. cubensis and P. columella, (ii) elements which may be considered typical or perhaps endemic of the Neotropical region, such as L. neotropica and L. meridensis[22, 28], and (iii) lymnaeids indeed imported by human activities from other continents, such as G. truncatula of European origin and L. schirazensis of Asian origin [9, 28, 43]. This peculiar Venezuelan lymnaeid fauna is also characterised by two remarkable absences, such as the lack of representatives from the very large Radix and stagnicoline groups (Figure 7) [9, 35]. With regard to Radix, Venezuela agrees with its absence in the New World, although given Old World Radix species were imported to the USA time ago . Stagnicolines, widely spread throughout the Palaearctic region , are also represented by several species in the Nearctic region  and even by one endemic species in Mexico . The absence of stagnicolines in Venezuela supports their inability to colonise warmer latitudes, which agrees with their ecological preferences for cold-mild climates throughout the Holarctic.
Characterisation of Venezuelan lymnaeids
In L. cubensis, the detection of two different ITS-2 haplotypes highlights two different aspects. The fact that the combined haplotype L.cub-2B,cox 1b of the two Aragua populations is identical to that found in South Carolina, USA, suggests a derivation from the same geographical source. Hence, the presence of this combined haplotype in lowlands as a consequence of human importation not very long ago cannot be ruled out. On the contrary, the presence of another combined haplotype differing by only two A/T transversions and a different repeat number of an interrupted microsatellite at ITS-2 level (Figure 2) suggests potential adaptive mutations to the high altitude.
The present genetic confirmation of L. neotropica represents the first citation of this species in Venezuela, where it could have been confused with L. cubensis in lowland areas. The morphological similarity between these two species is at such a level that the synonymy of L. cubensis and L. viatrix was proposed  largely before the molecularly-based erection of the new species L. neotropica for the old, northern variety B elongata of L. viatrix. The finding of L. neotropica in Venezuela expands the geographical distribution of this species, hithterto only described from Peru  and Argentina , pronouncedly northward. Sequence differences, restricted to only the lack of a dinucleotide microsatellite repeat in the ITS-2 and to 6-7 mutations in cox 1 giving rise to only one different amino acid (Figure 3), may be interpreted as peripheral populations adapted to warmer Venezuelan lowlands of a lymnaeid species apparently very widely distributed throughout South America.
The present study reports the first published DNA sequence confirmation of the presence of G. truncatula in Venezuela, where it has been overlooked at high altitudes but perhaps also confused with L. cubensis in mid altitudes around 1900-2000 m. Galba truncatula, of Palaearctic origin and probable man-made importation from a European source , has already been described in highlands of other South American countries such as Bolivia [45, 47], Peru  and Argentina [85, 86]. Whereas the detection of only one mutation in the ITS-1 may easily be assumed, the high number of 27 mutations in a 672-bp-long cox 1 fragment when compared to other G. truncatula populations from elsewhere is surprising, even being silent (Figure 4).
Lymnaea schirazensis could have been confused in Venezuela with L. cubensis over a long time period and also with G. truncatula since the first detection of the latter in Mérida State in March 2000. Such confusion, however, does not differ from that which occurred in Asia, Africa, Europe, the Caribbean, Central America and South America. A recent large study has demonstrated that L. schirazensis and G. truncatula very pronouncedly differ at the level of rDNA and mtDNA, despite its marked anatomical and shell similarity: 130-139 nucleotide differences in ITS-2, 134-138 differences in ITS-1, and 57-67 mutations in cox 1 . This multidisciplinary study also allowed us to distinguish several phenotypic characteristics which help in the differentiation of both species . The combined haplotype L.schi-1A, cox1a found in Venezuela is the same also detected in other countries such as Iran, Egypt, Spain and the Dominican Republic (Figure 5). This suggests a human importation way from its probable original area in the Near East of Asia whether indirectly through the Dominican Republic in the first decades of the Spanish colonisation period or perhaps directly from the Iberian Peninsula by way of commercial ship activities in subsequent periods .
Lymnaea meridensis has recently been described as a new species after the DNA sequencing characterisation of the lymnaeid population of Laguna Mucubaji, Mérida State , initially classified as L. cousini by malacological techniques . Polymorphic sites are sufficiently numerous as to distinguish two species: 64 nucleotide differences (12.65% divergence) in ITS-2, 68-71 differences in ITS-1 (11.29-11.73%), and 37 differences in cox 1 (5.5%) (Figure 6). Moreover, a detailed morphometric comparison allowed the differentiation of both species by many anatomical characteristics . The finding of another lymnaeid population in the relatively near locality of Paso del Cóndor, at an altitude of 4040 m, recently ascribed to L. cousini, may most probably also concern L. meridensis. Both 18S gene sequence and the phylogenetic analysis based on ITS sequences supported a close evolutionary relationship between L. meridensis and L. cousini, suggesting an old common origin and a probable endemic divergence of L. meridensis by isolation in Venezuelan highlands .
Venezuelan P. columella show two ITS-2 haplotypes, H1 and H2, identical to those found in Puerto Rico and Colombia, respectively. The ITS-1 appears to be the same in these three countries. The cox 1 fragment sequence is identical to that in Puerto Rico and Colombia  and also Autralia . Pseudosuccinea columella is a rapidly colonising, more aquatic, more heat-tolerant species, considered to originate from Central America, the Caribbean and the southern part of North America. This lymnaeid is widely distributed throughout the world, including North, Central and South America and the Caribbean [26, 87], Europe , Africa , Australia, New Zealand and even Tahiti [29, 31]. Its typical presence in botanical gardens suggests its introduction with aquatic plants , a phenomenon especially increased in the last decades after the strong development of the trade of aquarium plants . Thus, the presence of P. columella in Venezuela should most probably be considered the result of a human introduction in recent times, similarly as in whole South America and in the other continents.
Summing up, DNA sequencing results suggest that (i) the original lymnaeid fauna of Venezuela was indeed only composed by L. meridensis and perhaps also L. neotropica, and that (ii) L. cubensis and P. columella were introduced from the Caribbean area, and (iii) G. truncatula and L. schirazensis from the Old World through human shipping activities in recent times (Figure 7).
Implications for human and animal fascioliasis epidemiology
Among the six lymnaeid species confirmed to be present in Venezuela, four of them are known to be good vectors of fascioliasis: L. cubensis, L. neotropica, G. truncatula and P. columella. This great diversity leads to the need of reopening several epidemiological and distributional aspects of the disease in both humans and animals.
Galba truncatula is considered the original and more efficient F. hepatica vector known . Moreover, it is the only transmitter in the highest human fascioliasis hyperendemic situations known, namely in high altitude Andean areas of Bolivia  and Peru . Such high fascioliasis transmission rates have been proved to be the consequence of life cycle modifications in both F. hepatica and G. truncatula as an adaptation response to the extreme conditions of the very high altitude . In high altitude areas, fascioliasis in children is usually detected in the advanced chronic stage, which has proved to have a great morbidity impact [90–92]. This poses a question mark of concern with regard to Andean highlands in Venezuela. The recent detection of liver fluke infection in children when performing random surveys in altitude areas of Mérida State  suggests that the human fascioliasis situation may be underestimated, mainly in high altitude Andean areas where G. truncatula is present. It is well known that children do not usually attend hospitals or health centres in such rural areas, as proved by the high fascioliasis prevalences and intensities detected in schoolchildren in other Andean countries [16–18]. Besides Mérida State, G. truncatula has also been recently reported from several high altitude areas ranging 2032-2511 m in Táchira State , where fascioliasis in cattle is known from lowlands and mid altitudes [69, 94] but human infection has never been reported.
Lymnaea cubensis and L. neotropica have also been found to be linked to human infection. Lymnaea cubensis is related to the Caribbean insular epidemiological pattern of human fascioliasis, typically represented by repeated outbreaks in a human hypoendemic area such as in Cuba . In Venezuela, the presence of L. cubensis has been described in several areas of Trujillo State [24, 73, 76, 78] and Portuguesa State , where or near to where human infection cases have been sporadically reported, as in highlands of Trujillo State such as Carache, at 1210 m [95, 96] and Jajó, at 1796 m , although subsequent surveys did not detect human infection despite the a priori adequate characteristics of the place , and lowlands of Portuguesa State such as Guanare, at 183 m . In the present study, L. cubensis has also been molecularly confirmed to be present in Mérida State up to almost 2000 m altitude, close to the 2050 m of the locality of Timotes where human infection was detected in a survey .
Lymnaea neotropica was originally described near Lima, Peru , where human infection has repeatedly been detected [100, 101]. Peruvian L. viatrix (= L. neotropica according to ) has been shown to transmit fascioliasis both experimentally and in nature . The transmission capacity of L. neotropica has also been molecularly confirmed in Argentina . In Venezuela, DNA sequencing results have demonstrated the presence of L. neotropica in lowlands of Carabobo and Falcon States. No human case has so far been described from Falcon, but sporadic human infection has been reported from Valle del Cabriales, at 479 m , where animal infection is endemic , and Valencia city, at 430 m .
Pseudosuccinea columella plays a prominent role in F. hepatica transmission to animals, as in the Caribbean [105, 106] and Brazil . However, its aquatic ecology and habitat preferences seem to explain why this vector species has never been particularly involved in human infection. Interestingly, one mutation at the level of the ITS-1 and another at ITS-2 have proved useful in distinguishing between susceptible and resistant populations of P. columella in Cuba , although nothing evidently suggests that these mutations are linked to resistance/susceptibility. The presence of A in position 510 of the pairwise ITS-1 alignment comparison with P. columella from Cuba suggests that this species presents F. hepatica-susceptible populations in Venezuela, whereas a T in position 6 of the ITS-2 indicates that a resistance-linked mutation is also there, at least in La Linda. In Venezuela, P. columella has only been found isolatedly in the States of Aragua (Maracay city), Carabobo (southern part of Valencia Lake), Guárico (Corozo Pando) and Mérida (El Valle) throughout an altitude range of 63-1929 m [23, 34]. This, together with the failure in refinding the populations of Maracay and Valencia in the 2006 survey , indicates that this exotic species has apparently not yet been able to further colonise and expand in Venezuela. This may be interpreted as the consequence of still insufficient time after a probable very recent man-made introduction. All in all, there is no evidence to support the idea that P. columella was the source of any of the human cases reported in Venezuelan lowlands. Lymnaea cubensis and the very similar L. neotropica were probably in the background of these sporadic infections, almost all concerning adult and old patients, passively detected in health centres in lowland areas. Indeed, L. cubensis has been cited in many lowlands of several States such as Aragua, Falcón, Lara, Portuguesa, Yaracuy, Zulia, Barinas, Trujillo, Sucre and Monagas [23, 34].
Experimental infection assays of L. schirazensis have proved that fasciolid larval stages are not able to fully develop within this lymnaeid, which does, therefore, not participate in disease transmission . In Venezuela, the presence of L. schirazensis poses a question mark on the geographical distribution of G. truncatula and all other Galba/Fossaria species with whose small and mid-sized specimens it may be very easily confused. One wonders whether unnoticed L. schirazensis specimens could be related to the different fascioliasis transmission capacities linked to different L. cubensis specimen size highlighted in mid-altitude localities of Trujillo State [76, 77]. Efforts are needed henceforth in Venezuela to clarify the geographical distribution of each Galba/Fossaria species, in order to furnish the baseline on which to correctly analyse the epidemiological characteristics and geographical distribution of both human and animal fascioliasis.
Nothing is known about the potential capacity of L. meridensis to transmit F. hepatica. However, both very close molecular and phylogenetic relationships with the species L. cousini suggest that it may most probably be involved in fascioliasis transmission . Studies, both in nature and in the laboratory, are needed to assess whether L. meridensis plays a role in fascioliasis transmission.
Distribution, both in space (latitudinal, longitudinal and altitudinal) and time (seasonal, yearly), of fascioliasis markedly depends on climate factors influencing (i) presence/absence and population dynamics of the freshwater vector species and (ii) fluke development of free larval stages in freshwater and of parasitic larval stages inside the snail. Air temperature, rainfall and evapotranspiration are the climate factors that more pronouncedly affect definitive host infection incidence . The only fasciolid present in Venezuela is F. hepatica, a fluke species well known due to its preference for temperate and cold climates . This broadly fits with animal fascioliasis reports in Venezuela, which show a clear geographical trend for north-western temperate and colder zones (Figure 1) .
Human infection has, however, only been reported from the Capital District, Miranda, Carabobo, Portuguesa, Trujillo, Mérida and Bolívar (Figure 1) . Although human reports are few when compared to other South American countries, such as Chile , Bolivia [15–17] and Peru [18, 19], a similar altitude pattern appears. In Venezuela, human infection shows more numerous patients, more child involvement and case concentration (same locality, same school) in altitude areas of the western Andean States. Cases appear to be isolated, sporadic and affecting adult and old subjects in the eastern lowland plains. Thus, the altitudinal distribution of this lower human infection situation in Venezuela appears to be similar to that in Ecuador  and Argentina .
Hence, in Venezuela all evidence indicates that altitude is the main factor influencing fascioliasis distribution, and therefore highlights importance of accurately assessing the altitudinal distribution range of each of the lymnaeid vector species. Altitudinal data for each species noted in Table 1 furnish a first approach. The new scenario provided here implies the need to completely re-assess the distribution of each one of the six lymnaeid species in space and time with the help of the DNA marker tools. Lymnaeid mapping becomes a priority to determine the distribution of human and animal fascioliasis inside Venezuela. The overlap of G. truncatula, L. cubensis and probably also L. neotropica in temperate and cold zones suggests that there is a higher risk for human infection in mid and high altitude rural areas. Appropriate human surveys, mainly focusing on children, in such areas are evidently needed to verify whether overlooked fascioliasis prevalence may exist.
The overlap, in the same endemic area, of more than one lymnaeid vector species with different ecological requirements and population dynamics will unfortunately make the application of mathematical forecast indexes  more complicated. Increased difficulties may similarly be expected for remote sensing and geographical information system (RS-GIS) methods  to obtain accurate results useful for fascioliasis risk assessment and monitoring.
Studies funded by Projects No. BOS2000-0570-C02-02, No. BOS2002-01978, No. SAF2006-09278 and SAF No. 2010-20805 of the Ministry of Education and Ministry of Science and Innovation, Madrid; and by the Red de Investigación de Centros de Enfermedades Tropicales - RICET (Projects No. C03/04, No. ISCIII2005-PI050574 and No. ISCIII-RETIC RD06/0021/0017 of the Programa de Redes Temáticas de Investigación Cooperativa RETICS/FEDER), FIS, Ministry of Health, Madrid, Spain. Research activities in the "El Joque" Experimental Farm Station funded by Project No. FA-464-09-03-C of the Consejo de Desarrollo Científico Humanístico, Tecnológico y de las Artes, Universidad de Los Andes (C.D.C.H.T.A.-ULA), Mérida, Venezuela.
Part of this work was made possible thanks to personal fellowships funded to P. Artigas and C. González by the Agencia Española de Cooperación Internacional (AECI), Ministry of Foreign Affairs, Madrid, Spain. Research stay of C. González in Parasitology Department of Valencia University additionally funded by the Universidad de los Andes-CONICIT, Venezuela. Thanks are given to Dr. A.M. Pérez (Maracay, Aragua State, Venezuela) for furnishing lymnaeids from Falcon State and and Dr. J.P. Pointier (Perpignan, France) for those from Aragua State, Carabobo State and Laguna Mucubaji of Mérida State.
Field work activities performed within the worldwide initiative of WHO (Headquarters Geneva, Switzerland) against human fascioliasis. Joint coordination activities carried out within Project No. RLA5049 of the International Atomic Energy Agency (Animal Production and Health Section, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture, Department of Nuclear Sciences and Applications, IAEA Headquaters Vienna, Austria). Technical support provided by the Servicio Central de Secuenciación para la Investigación Experimental (SCSIE) of the Universidad de Valencia (Dr. A. Martínez).
- Mas-Coma S, Bargues MD, Valero MA: Fascioliasis and other plant-borne trematode zoonoses. Int J Parasitol. 2005, 35: 1255-1278. 10.1016/j.ijpara.2005.07.010.PubMedView ArticleGoogle Scholar
- Mas-Coma S, Valero MA, Bargues MD: Fasciola, lymnaeids and human fascioliasis, with a global overview on disease transmission, epidemiology, evolutionary genetics, molecular epidemiology and control. Adv Parasitol. 2009, 69: 41-146.PubMedView ArticleGoogle Scholar
- World Health Organization: Control of foodborne trematode infections. WHO Techn Rep Ser. 1995, 849: 1-157.Google Scholar
- Mas-Coma S, Valero MA, Bargues MD: Effects of climate change on animal and zoonotic helminthiases. Rev Sci Techn Off Int Epiz. 2008, 27: 443-457.Google Scholar
- Mas-Coma S, Valero MA, Bargues MD: Climate change effects on trematodiases, with emphasis on zoonotic fascioliasis and schistosomiasis. Vet Parasitol. 2009, 163: 264-280. 10.1016/j.vetpar.2009.03.024.PubMedView ArticleGoogle Scholar
- Ollerenshaw CB: Forecasting liver-fluke disease. The Effects of Meteorological Factors upon Parasites, Symposium of the British Society for Parasitology. Edited by: Taylor AR, Muller R. 1974, Oxford: Blackwell Scientific Publications, 12: 33-52.Google Scholar
- Valero MA, Mas-Coma S: Comparative infectivity of Fasciola hepatica metacercariae from isolates of the main and secondary reservoir animal host species in the Bolivian Altiplano high human endemic region. Folia Parasitol. 2000, 47: 17-22.PubMedView ArticleGoogle Scholar
- Valero MA, Darce NA, Panova M, Mas-Coma S: Relationships between host species and morphometric patterns in Fasciola hepatica adults and eggs from the Northern Bolivian Altiplano hyperendemic region. Vet Parasitol. 2001, 102: 85-100. 10.1016/S0304-4017(01)00499-X.PubMedView ArticleGoogle Scholar
- Bargues MD, Vigo M, Horak P, Dvorak J, Patzner RA, Pointier JP, Jackiewicz M, Meier-Brook C, Mas-Coma S: European Lymnaeidae (Mollusca: Gastropoda), intermediate hosts of trematodiases, based on nuclear ribosomal DNA ITS-2 sequences. Inf Genet Evol. 2001, 1: 85-107. 10.1016/S1567-1348(01)00019-3.View ArticleGoogle Scholar
- Bargues MD, Mas-Coma S: Reviewing lymnaeid vectors of fascioliasis by ribosomal DNA sequence analyses. J Helminthol. 2005, 79: 257-267. 10.1079/JOH2005297.PubMedView ArticleGoogle Scholar
- Mas-Coma S: Epidemiology of fascioliasis in human endemic areas. J Helminthol. 2005, 79: 207-216. 10.1079/JOH2005296.PubMedView ArticleGoogle Scholar
- Fuentes MV, Valero MA, Bargues MD, Esteban JG, Angles R, Mas-Coma S: Analysis of climatic data and forecast indices for human fascioliasis at very high altitude. Ann Trop Med Parasitol. 1999, 93: 835-850. 10.1080/00034989957844.PubMedView ArticleGoogle Scholar
- Fuentes MV, Malone JB, Mas-Coma S: Validation of a mapping and predicting model for human fasciolosis transmission in Andean very high altitude endemic areas using remote sensing data. Acta Trop. 2001, 79: 87-95. 10.1016/S0001-706X(01)00106-1.PubMedView ArticleGoogle Scholar
- Apt W, Aguilera X, Vega F, Alcaino H, Zulantay I, Apt P, Gonzalez V, Retamal , Rodriguez J, Sandoval J: Prevalencia de fascoliasis en humanos, caballos, cerdos y conejos silvestres en tres provincias de Chile. Bol Of Sanit Panam. 1993, 115: 405-414.Google Scholar
- Hillyer GV, Soler de Galanes M, Rodriguez-Perez J, Bjorland J, Silva de Lagrava M, Guzman SR, Bryan RT: Use of the Falcon™ assay screening test-enzyme-linked immunosorbent assay (FAST-ELISA) and the enzyme-linked immunoelectrotransfer blot (EITB) to determine the prevalence of human fascioliasis in the Bolivian altiplano. Am J Trop Med Hyg. 1992, 46: 603-609.PubMedGoogle Scholar
- Esteban JG, Flores A, Angles R, Strauss W, Aguirre C, Mas-Coma S: A population-based coprological study of human fascioliasis in a hyperendemic area of the Bolivian Altiplano. Trop Med Int Health. 1997, 2: 695-699. 10.1046/j.1365-3156.1997.d01-356.x.PubMedView ArticleGoogle Scholar
- Esteban JG, Flores A, Angles R, Mas-Coma S: High endemicity of human fascioliasis between Lake Titicaca and La Paz valley, Bolivia. Trans Roy Soc Trop Med Hyg. 1999, 93: 151-156. 10.1016/S0035-9203(99)90289-4.PubMedView ArticleGoogle Scholar
- Esteban JG, Gonzalez C, Bargues MD, Angles R, Sanchez C, Naquira C, Mas-Coma S: High fascioliasis infection in children linked to a man-made irrigation zone in Peru. Trop Med Int Health. 2002, 7: 339-348. 10.1046/j.1365-3156.2002.00870.x.PubMedView ArticleGoogle Scholar
- González LC, Esteban JG, Bargues MD, Valero MA, Ortiz P, Naquira C, Mas-Coma S: Hyperendemic human fascioliasis in Andean valleys: An altitudinal transect analysis in children of Cajamarca province, Peru. Acta Trop. 2011, 120: 119-129. 10.1016/j.actatropica.2011.07.002.PubMedView ArticleGoogle Scholar
- Trueba G, Guerrero T, Fornasini M, Casariego I, Zapata S, Ontaneda S, Vasco L: Detection of Fasciola hepatica infection in a community located in the Ecuadorian Andes. Am J Trop Med Hyg. 2000, 62: 518-PubMedGoogle Scholar
- Mera y Sierra R, Agramunt V, Cuervo P, Mas-Coma S: Human fascioliasis in Argentina: retrospective overview, critical analysis and baseline for future research. Parasit Vector. 2011, 4: 104-10.1186/1756-3305-4-104.View ArticleGoogle Scholar
- Bargues MD, Artigas P, Khoubbane M, Mas-Coma S: DNA sequence characterisation and phylogeography of Lymnaea cousini and related species, vectors of fascioliasis in northern Andean countries, with description of Lymnaea meridensis n. sp. (Gastropoda: Lymnaeidae). Parasit Vector. 2011, 4: 132-10.1186/1756-3305-4-132.View ArticleGoogle Scholar
- González LC, Blanco MA, Gil F, Castro T, Mas-Coma S: Fascioliasis in Venezuela: past and present knowledge on human and animal infection. Zoonotic Fascioliasis in Latin America. Edited by: Mas-Coma S, Bargues MD, Viljoen GJ. 2011, Vienna-Roma: IAEA-FAOGoogle Scholar
- Morales G, Pino LA: Infection de Lymnaea cubensis par Fasciola hepatica dans une région d'altitude au Venezuela. Ann Parasitol Hum Comp. 1983, 58: 27-30.PubMedGoogle Scholar
- Morales G, Pino LA, Rodriguez E: Diseño de estrategias de control para poblaciones de Lymnaea cubensis Pfeiffer, 1839 y Lymnaea columella Say, 1817. Bol Dir Malariol Saneam Ambient. 1983, 23: 11-17.Google Scholar
- Malek EA: Snail hosts of schistosomiasis and other snail-transmitted diseases in tropical America: a manual. 1985, Washington: Pan American Health Organization, Scientific Publication, 478: 325-Google Scholar
- Chrosciechowski P: Problema de la fascioliasis en Venezuela: su aspecto malacológico. Bol Dir Malariol Saneam Amb. 1987, 27: 53-58.Google Scholar
- Bargues MD, Artigas P, Mera y Sierra R, Pointier JP, Mas-Coma S: Characterisation of Lymnaea cubensis, L. viatrix and L. neotropica n. sp., the main vectors of Fasciola hepatica in Latin America, by analysis of their ribosomal and mitochondrial DNA. Ann Trop Med Parasitol. 2007, 101: 621-641.PubMedView ArticleGoogle Scholar
- Hubendick B: Recent Lymnaeidae. Their variation, morphology, taxonomy, nomenclature, and distribution. 1951, 3 (1): 1-223. Kungliga Svenska Vetenskapsakademiens Handlingar, Fjärde Serien; + 5 plGoogle Scholar
- Pointier JP, Cousteau C, Rondelaud D, Theron A: Pseudosuccinea columella (Say, 1817) (Gastropoda, Lymnaeidae), snail host of Fasciola hepatica: first record for France in the wild. Parasitol Res. 2007, 101: 1389-1392. 10.1007/s00436-007-0656-y.PubMedView ArticleGoogle Scholar
- Baldock FC, Arthur RJ: A survey of fascioliasis in beef cattle killed at abattoirs in southern Queensland. Austr Vet J. 1985, 62: 324-326. 10.1111/j.1751-0813.1985.tb07650.x.View ArticleGoogle Scholar
- Artigas P, Bargues MD, Mera y Sierra R, Agramunt VH, Mas-Coma S: Characterisation of fascioliasis lymnaeid intermediate hosts from Chile by DNA sequencing, with emphasis on Lymnaea viator and Galba truncatula. Acta Trop. 2011, 120: 245-257.PubMedView ArticleGoogle Scholar
- Pointier JP, Noya O, Amarista M, Theron A: Lymnaea cousini Jousseaume, 1887 (Gastropoda: Lymnaeidae): first record for Venezuela. Mem Inst Oswaldo Cruz. 2004, 99: 567-569.PubMedView ArticleGoogle Scholar
- Pointier JP, Noya O, Alarcon de Noya B, Theron A: Distribution of Lymnaeidae (Mollusca: Pulmonata), intermediate snail hosts of Fasciola hepatica in Venezuela. Mem Inst Oswaldo Cruz. 2009, 104: 790-796. 10.1590/S0074-02762009000500022.PubMedView ArticleGoogle Scholar
- Bargues MD, Horak P, Patzner RA, Pointier JP, Jackiewicz M, Meier-Brook C, Mas-Coma S: Insights into the relationships of Palaearctic and Nearctic lymnaeids (Mollusca: Gastropoda) by rDNA ITS-2 sequencing and phylogeny of stagnicoline intermediate host species of Fasciola hepatica. Parasite. 2003, 10: 243-255.PubMedView ArticleGoogle Scholar
- Samadi S, Roumegoux A, Bargues MD, Mas-Coma S, Yong M, Pointier JP: Morphological studies of lymnaeid snails from the human fascioliasis endemic zone of Bolivia. J Moll Stud. 2000, 66: 31-44. 10.1093/mollus/66.1.31.View ArticleGoogle Scholar
- Mas-Coma S, Bargues MD: Populations, hybrids and the systematic concepts of species and subspecies in Chagas disease triatomine vectors inferred from nuclear ribosomal and mitochondrial DNA. Acta Trop. 2009, 110: 112-136. 10.1016/j.actatropica.2008.10.013.PubMedView ArticleGoogle Scholar
- Puslednik L, Ponder WF, Dowton M, Davis AR: Examining the phylogeny of the Australasian Lymnaeidae (Heterobranchia: pulmonata: Gastropoda) using mitochondrial, nuclear and morphological markers. Mol Phylogenet Evol. 2009, 52: 643-659. 10.1016/j.ympev.2009.03.033.PubMedView ArticleGoogle Scholar
- Kane RA, Stothard JR, Emery AM, Rollinson D: Molecular characterization of freshwater snails in the genus Bulinus: a role for barcodes?. Parasit Vector. 2008, 1: 15-10.1186/1756-3305-1-15.View ArticleGoogle Scholar
- Paraense WL: Lymnaea columella in northern Brazil. Mem Inst Osw Cruz. 1983, 78: 477-482. 10.1590/S0074-02761983000400011.View ArticleGoogle Scholar
- Pointier JP, Cazzaniga NJ, González-Salas C, Gutiérrez A, Arenas JA, Bargues MD, Mas-Coma S: Anatomical studies of sibling species within Neotropical lymnaeids snail intermediate hosts of fascioliasis. Mem Inst Osw Cruz. 2006, 101: 431-435. 10.1590/S0074-02762006000400015.View ArticleGoogle Scholar
- Rondelaud D, Vignoles P, Dreyfuss G: La limnée tronquée, un mollusque d'intérêt médical et vétérinaire. 2009, Limoges: Presses Universitaires de Limoges, 283-Google Scholar
- Bargues MD, Artigas P, Khoubbane M, Flores R, Glöer P, Rojas-Garcia R, Ashrafi K, Falkner G, Mas-Coma S: Lymnaea schirazensis, an overlooked snail distorting fascioliasis data: Genotype, phenotype, ecology, worldwide spread, susceptibility, applicability. PLoS ONE. 2011, 6 (9): e24567-10.1371/journal.pone.0024567. (33 pp. + 3 Suppl. Tables + 5 Suppl. Figures)PubMed CentralPubMedView ArticleGoogle Scholar
- Bargues MD, Mangold A, Muñoz-Antoli C, Pointier JP, Mas-Coma S: SSU rDNA characterization of lymnaeid snails transmitting human fascioliasis in South and Central America. J Parasitol. 1997, 83: 1086-1092. 10.2307/3284367.PubMedView ArticleGoogle Scholar
- Bargues MD, Mas-Coma S: Phylogenetic analysis of lymnaeid snails based on 18S rDNA sequences. Mol Biol Evol. 1997, 14: 569-577.PubMedView ArticleGoogle Scholar
- Bargues MD, Artigas P, Jackiewicz M, Pointier JP, Mas-Coma S: Ribosomal DNA ITS-1 sequence analysis of European stagnicoline Lymnaeidae (Gastropoda). Heldia (Münchner Malakologische Mitteilungen), München. 2006, 6 (1/2): 29-40.Google Scholar
- Mas-Coma S, Funatsu IR, Bargues MD: Fasciola hepatica and lymnaeid snails occurring at very high altitude in South America. Parasitology. 2001, 123: S115-S127.PubMedView ArticleGoogle Scholar
- Folmer O, Black M, Hoch W, Lutz R, Vrijenhoek R: DNA primers for amplification of mitochondrial cytochrome c oxidase subunit I from diverse metazoan invertebrates. Mol Marine Biol Biotech. 1994, 3: 294-299.Google Scholar
- Sanger F, Nicklen S, Coulson AR: DNA sequencing with chain-terminating inhibitors. Proc Natn Acad Sc USA. 1977, 74: 5463-5467. 10.1073/pnas.74.12.5463.View ArticleGoogle Scholar
- Thompson JD, Higgins DG, Gibson TJ: CLUSTAL W: improving the sensitivity and progressive multiple sequence alignment through sequence weighting, positions-specific gap penalties and weight matrix choice. Nucl Acids Res. 1994, 22: 4673-4680. 10.1093/nar/22.22.4673.PubMed CentralPubMedView ArticleGoogle Scholar
- Tamura K, Dudley J, Nei MB, Kumar S: MEGA4: Molecular Evolutionary Genetic Analysis (MEGA) sofware version 4.0. Mol Biol Evol. 2007, 24: 1596-1599. 10.1093/molbev/msm092.PubMedView ArticleGoogle Scholar
- Staden R, Judge DP, Bonfield JK: Sequence assembly and finishing methods. Meth Bioch Anal. 2001, 43: 302-322.Google Scholar
- Gutierrez A, Pointier JP, Fraga J, Jobet E, Modat S, Perez RT, Yong M, Sanchez J, Loker ES, Theron A: Fasciola hepatica: identification of molecular markers for resistant and susceptible Pseudosuccinea columella snail hosts. Exp Parasitol. 2003, 105: 211-218. 10.1016/j.exppara.2003.12.006.PubMedView ArticleGoogle Scholar
- Albrecht C, Wolf C, Glöer P, Wilke T: Concurrent evolution of ancient sister lakes and sister species: the freshwater gastropod genus Radix in lakes Ohrid and Prespa. Hydrobiologia. 2008, 615: 157-167. 10.1007/s10750-008-9555-1.View ArticleGoogle Scholar
- Mera y Sierra R, Artigas P, Cuervo P, Deis E, Sidoti L, Mas-Coma S, Bargues MD: Fascioliasis transmission by Lymnaea neotropica confirmed by nuclear rDNA and mtDNA sequencing in Argentina. Vet Parasitol. 2009, 166: 73-79. 10.1016/j.vetpar.2009.08.001.PubMedView ArticleGoogle Scholar
- Remigio EA, Hebert PD: Testing the utility of partial COI sequences for phylogenetic estimates of gastropod relationships. Mol Phylogenet Evol. 2003, 29: 641-647. 10.1016/S1055-7903(03)00140-4.PubMedView ArticleGoogle Scholar
- Akaike H: A new look at the statistical model identification. Ieee Trans Automat Control. 1974, 19: 716-723. 10.1109/TAC.1974.1100705.View ArticleGoogle Scholar
- Posada D, Buckley TR: Model selection and model averaging in phylogenetics: advantages of the AIC and Bayesian approaches over likelihood ratio tests. Syst Biol. 2004, 53: 793-808. 10.1080/10635150490522304.PubMedView ArticleGoogle Scholar
- Posada D: jModelTest: phylogenetic model averaging. Mol Biol Evol. 2008, 25: 1253-1256. 10.1093/molbev/msn083.PubMedView ArticleGoogle Scholar
- Saitou N, Nei M: The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol. 1987, 4: 406-425.PubMedGoogle Scholar
- Ronquist F, Huelsenbeck JP: MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics. 2003, 19: 1572-1574. 10.1093/bioinformatics/btg180.PubMedView ArticleGoogle Scholar
- Remigio EA, Blair D: Relationships among problematic North American stagnicoline snails (Pulmonata: Lymnaeidae) reinvestigated using nuclear ribosomal DNA internal transcribed spacer sequences. Can J Zool. 1997, 75: 1540-1545. 10.1139/z97-779.View ArticleGoogle Scholar
- De Jong RJ, Morgan JAT, Paraense WL, Pointier JP, Amarista M, Ayeh-Kumi PFK, Babiker A, Barbosa CS, Bremond Ph, Canese AP, Pereira de Souza C, Dominguez C, File S, Gutierrez A, Incani RN, Kawano T, Kazibwe F, Kpikpi J, Lwambo NJS, Mimpfoundi R, Njiokou F, Poda JN, Sene M, Velasquez LE, Yong M, Adema CM, Hofkin BV, Mkoji GM, Loker ES: Evolutionary relationships and biogeography of Biomphalaria (Gastropoda: Planorbidae) with implications regarding its role as host of the human bloodfluke, Schistosoma mansoni. Mol Biol Evol. 2001, 18: 2225-2239.View ArticleGoogle Scholar
- Bello H: Sobre la distomatosis hepática en Venezuela. Vargas. 1916, 7: 7-Google Scholar
- Lutz A: Moluscos de agua dulce recogidos y observados en Venezuela. Estudios de Zoología y Parasitología Venezolanas. Edited by: Rio de Janeiro. 1928, 75-84.Google Scholar
- Lutz A: Estudios de Zoología y Parasitología Venezolanas. 1955, Reimpresión de la edición de 1928 ordenada por la Universidad Central de Venezuela en Homenaje al Dr. Lutz en el centenario de su muerte. Caracas: Universidad Central de Venezuela, 137-Google Scholar
- Vergani F: Datos biológicos experimentales sobre el caracol Lymnaea (Galba) cubensis P., 1911. Rev Vet Venezuela. 1957, 52: 193-210.Google Scholar
- Martinez R, Miranda R: Aspectos de la reproducción en moluscos pulmonados del área Metropolitana de Caracas. Estudio de Caracas, Univ Cent Venezuela. 1968, 1: 123-165.Google Scholar
- Ramirez-Villamediana JJ, Vergani F: Contribución al estudio del ciclo evolutivo de la Fasciola hepática en Venezuela. Rev Ganad Col. 1949, 3: 817-826.Google Scholar
- Briceño Rossi AL: Trabajo experimental sobre Fasciola hepatica. Rev San Asis Soc. 1950, 14: 380-388.Google Scholar
- Malek EA, Chrosciechowski P: Lymnaea (Pseudosuccinea) columella from Venezuela, and notes on distribution of Pseudosuccinea. Nautilus. 1964, 78: 54-56.Google Scholar
- Chavez K, Surumay Q, Olivares R, Montiel N: Prevalencia de distomatosis hepática en fincas del Distrito Mara del estado Zulia. Vet Trop. 1979, 4: 52-63.Google Scholar
- Morales G, Pino LA: Lymnaea cubensis Pfeiffer, 1839, Hospedador Intermediario de Fasciola hepatica en la zona Alta de Los Andes Trujillanos, Venezuela. Bol Dir Malariol Saneam Ambient. 1981, 21: 1-9.Google Scholar
- Morales G, Pino LA: Fasciola hepatica: Aspectos ecoepidemiológicos de interés para el desarrollo de estrategias de control. Ganadería Mestiza de Doble Propósito. Edited by: González Stognaro C. 1992, Astro Data, 301-329.Google Scholar
- Morales G, Rodriguez E, Pino LA, Perdomo L: Estadísticas vitales de Lymnaea cubensis Pfeiffer, 1839 en condiciones de laboratorio. Bol Dir Malariol Saneam Ambient. 1985, 25: 89-99.Google Scholar
- Morales G, Pino LA, Morales J: Distribución de las redias y cercarias de Fasciola hepatica en una población silvestre de Lymnaea cubensis del Occidente de Venezuela. Acta Cient Venez. 1986, 37: 535-541.Google Scholar
- Morales G, Pino LA, Perdomo L: Utilidad del conocimiento del tamaño del molusco Lymnaea cubensis en la implementación de programas de control de la distomatosis hepática. Rev Fac Cien Vet UCV. 1986, 33: 28-37.Google Scholar
- Pino LA, Morales G: Habitats de Lymnaea cubensis Pfeiffer, 1839, hospedador intermediario de Fasciola hepatica, detectados en el estado Trujillo, Venezuela. Acta Cient Venez. 1982, 33: 61-65.Google Scholar
- Pino LA, Morales G, Marquez A: Presencia de Lymnaea cubensis hospedador intermediario de Fasciola hepatica en el Municipio La Cañada de Urdaneta, Estado Zulia. Vet Trop. 1995, 20: 85-93.Google Scholar
- Melendez R, Coronado A, Diaz J, Crespo G: Aspectos epidemiológicos de la fascioliasis bovina en el centro-occidente venezolano; con énfasis en la prevalencia del trematode y de su hospedador intermediario. Acta Cien Venez. 1983, 34: 65-71.Google Scholar
- Fuenmayor A, Simoes D, Gonzalez R, Chirinos A: La Distomatosis hepática y su asociación con los factores de riesgo en los Municipios Mara y Páez del estado Zulia, Venezuela. Rev Cient FCV-LUZ. 2000, 103: 183-190.Google Scholar
- Nieves E, Rondón M, Zamora E, Salazar M: Fasciola hepatica en la zona alta de Mérida, Venezuela. Rev Elec Vet REDVET. 2005, 6 (12): [http://www.veterinaria.org/revistas/redvet/n121205.html]Google Scholar
- Gomez Agudelo T, Perez Reyes R, Zeron Braco F: Fasciolosis en México. Estado actual y huéspedes intermediarios. Rev Latinoamer Microbiol. 1978, 20: 121-127.Google Scholar
- Paraense WL: Lymnaea viatrix: a study of topotypic specimens (Mollusca: Lymnaeidae). Rev Bras Biol. 1976, 36: 419-428.Google Scholar
- Bargues MD, Mera y Sierra R, Gomez HG, Artigas P, Mas-Coma S: Ribosomal DNA ITS-1 sequencing of Galba truncatula (Gastropoda: Lymnaeidae) and its potential impact on fascioliasis transmission in Mendoza, Argentina. Animal Biodiv Conserv. 2006, 29: 191-194.Google Scholar
- Bargues MD, Mera y Sierra R, Gomez HG, Artigas P, Mas-Coma S: Caracterización molecular de Galba truncatula, vector principal de la Fascioliasis, en Argentina. Implicaciones en salud pública. Enf Emerg, Barcelona. 2007, 9: 77-82.Google Scholar
- Paraense WL: Lymnaea viatrix and Lymnaea columella in the Neotropical region: a distributional outline. Mem Inst Oswaldo Cruz. 1982, 77: 181-188.Google Scholar
- Brown D: Freshwater Snails of Africa and Their Medical Importance. 1994, London: Taylor & Francis Ltd, 608-2Google Scholar
- Mas-Coma S, Angles R, Esteban JG, Bargues MD, Buchon P, Franken M, Strauss W: The Northern Bolivian Altiplano: a region highly endemic for human fascioliasis. Trop Med Int Health. 1999, 4: 454-467. 10.1046/j.1365-3156.1999.00418.x.PubMedView ArticleGoogle Scholar
- Valero MA, Santana M, Morales M, Hernandez JL, Mas-Coma S: Risk of gallstone disease in advanced chronic phase of fascioliasis: an experimental study in a rat model. J Inf Dis. 2003, 188: 787-793. 10.1086/377281.View ArticleGoogle Scholar
- Valero MA, Navarro M, Garcia-Bodelon MA, Marcilla A, Morales M, Garcia JE, Hernandez JL, Mas-Coma S: High risk of bacterobilia in advanced experimental chronic fasciolosis. Acta Trop. 2006, 100: 17-23. 10.1016/j.actatropica.2006.09.002.PubMedView ArticleGoogle Scholar
- Valero MA, Girones N, Garcia-Bodelon MA, Periago MV, Chico-Calero I, Khoubbane M, Fresno M, Mas-Coma S: Anemia in advanced chronic fasciolosis. Acta Trop. 2008, 108: 35-43. 10.1016/j.actatropica.2008.08.007.PubMedView ArticleGoogle Scholar
- Alarcon de Noya B, Rojas E, Colmenares C, Morales C, Contreras R, Valero SK, Hernandez D, Briceño S, Scorza JV, Noya O: Brote familiar de fascioliasis en Venezuela. Bol Malariol Salud Amb. 2007, 47: 47-54.Google Scholar
- Perruolo G, Perdomo E, Silva Coronado J: Estudio de la Fasciola hepatica en el Estado Táchira, Venezuela. Incidencia y prevalencia en la zona sur del Estado Táchira. Rev Cient UNET. 1987, 1: 14-19.Google Scholar
- Tombazzi C, Abdul-Hadi S, Lecuna V, Contreras R, Marquez D: Endoscopic retrograde cholangiopancreatography in hepatic fascioliasis. GEN. 1994, 48: 278-80.Google Scholar
- Abdul-Hadi S, Contreras R, Tombazzi C, Alvarez M, Melendez M: Hepatic fascioliasis: case report and review. Rev Inst Med Trop Sao Paulo. 1966, 14: 69-73.Google Scholar
- Scorza JV, Villegas E, Morales C: Fascioliasis hepática en el Estado Trujillo, Venezuela. Un segundo caso clínico. Arch Venez Med Trop. 1999, 3: 9-13.Google Scholar
- Abdul-Hadi S, Figueira I, Madera C, Olaizola C, Contreras R, Sanchez MA, Colmenares C, Safar ML: Estudio de la fascioliasis hepática humana y parasitosis intestinales en el caserío Mesa Arriba del municipio Carache, estado Trujillo, Venezuela. Rev Soc Venez Microbiol. 2009, 29: 128-132.Google Scholar
- Rodriguez A, Gonzalez H: Sobre un nuevo caso humano de fascioliasis hepática en Venezuela. Rev Inst Nac Hig. 1975, 8: 111-114.Google Scholar
- Blancas G, Terashima A, Maguina C, Vera L, Alvarez H, Tello R: Fasciolosis humana y compromiso gastrointestinal: estudio de 277 pacientes en el Hospital Nacional Cayetano Heredia 1970-2002. Rev Gastroenterol Perú. 2004, 24: 143-157.Google Scholar
- Marcos LA, Romani L, Florencio L, Terashima A, Canales M, Nestares J, Huayanay L, Gotuzzo E: Zonas hiperendémicas y mesoendémicas de la infección por Fasciola hepatica aledañas a la ciudad de Lima: Una enfermedad emergente?. Rev Gastroenterol Perú. 2007, 27: 21-26.Google Scholar
- Tantalean M, Huiza A, Capuñay R: Los hospederos intermediarios de Fasciola hepatica en el Perú: estudio de la infección natural y experimental de Lymnaea viator, L. diaphana y Physa venustula. Biota. 1974, 10: 243-250.Google Scholar
- Mayaudon TH, Gallo P: Algunas consideraciones sobre las helminto-zoonosis en Venezuela. Rev Fac Cien Vet, Univ Cent Venezuela. 1963, 20: 5-21.Google Scholar
- Incani RN, Vieira JM, Pacheco M, Planchart S, Amarista M, Lazdins J: Human infection by Fasciola hepatica: report of a geriatric case. Invest Clin. 2003, 44: 255-260.PubMedGoogle Scholar
- De Leon-Dancel D: Life history of Lymnaea columella (Say) and its experimental infection with Fasciola hepatica (L.). J Agric Univ Puerto Rico. 1970, 54: 297-305.Google Scholar
- Ferrer J, Perera G, Yong M, Amador O: Life tables of Fossaria cubensis and Pseudosuccinea columella, intermediate hosts of Fasciola hepatica in Cuba. J Med Appl Malacol. 1989, 1: 189-194.Google Scholar
- Coelho LHL, Lima WS: Population dynamics of Lymnaea columella and its natural infection by Fasciola hepatica in the State of Minas Gerais, Brazil. J Helminthol. 2003, 77: 7-10. 10.1079/JOH2002138.PubMedView ArticleGoogle Scholar
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