Variability of Calodium hepaticum eggs from sigmodontine host species through geometric morphometric analysis.

Calodium hepaticum is a zoonotic nematode with a worldwide distribution. Although the host range of C. hepaticum includes a wide spectrum of mammals (including humans), this parasite is predominantly associated with the families Muridae and Cricetidae. Several Sigmodontinae species from Argentina were found to be infected by C. hepaticum, with a high prevalence in Akodon azarae. The present study focuses on C. hepaticum eggs from natural infection of three species of sigmodontine rodents from Argentina. Eggs were genetically characterized (intergenic 18S rRNA region). The objectives of this work are: (i) to propose a new analytical methodology; and (ii) to morphologically characterize C. hepaticum eggs, from three Sigmodontinae species (A. azarae, Calomys callidus and Oligoryzomys flavescens). Analyses were made by the Computer Image Analysis System based on the new standardized measurements and geometric morphometric tools. The resulting factor maps clearly illustrate global size differences in the parasite eggs from the three Sigmodontinae species analysed. The degree of similarity between egg populations was assessed through pairwise Mahalanobis distances, showing that the largest distances were detected between parasite eggs from C. callidus and O. flavescens. Herein, the phenotypical plasticity of C. hepaticum eggs is shown. Significant positive correlations were obtained between each egg parasite principal component 1 and rodent corporal characteristics: weight; liver weight; rodent length; and rodent body condition. The usefulness of the geometric morphometric analysis in studies of the relationship between C. hepaticum and its host must be highlighted. The high prevalence observed in A. azarae, associated with the wide size range of the parasite eggs evidenced by principal component analysis, suggests A. azarae to be the Sigmodontinae host species that plays the most important role as reservoir host for C. hepaticum in the New World.

Equines as reservoirs of human fascioliasis: transmission capacity, epidemiology and pathogenicity in Fasciola hepatica-infected mules.

Fascioliasis is a zoonotic disease caused by liver flukes transmitted by freshwater lymnaeid snails. Donkey and horse reservoir roles have been highlighted in human endemic areas. Liver fluke infection in mules has received very limited research. Their role in disease transmission, epidemiological importance and Fasciola hepatica pathogenicity are studied for the first time. Prevalence was 39.5% in 81 mules from Aconcagua, and 24.4% in 127 from Uspallata, in high-altitude areas of Mendoza province, Argentina. A mean amount of 101,242 eggs/mule/day is estimated. Lymnaeids from Uspallata proved to belong to ribosomal DNA internal transcribed spacer (ITS) markers ITS-1 and ITS-2 combined haplotype 3C of Galba truncatula. These lymnaeids were experimentally susceptible to infection by egg miracidia from mules. Infectivity, number of cercariae/snail and shedding period fit the enhanced F. hepatica/G. truncatula transmission pattern at very high altitude. This indicates that the mule is able to maintain the F. hepatica cycle independently. Individual burdens of 20 and 97 flukes were found. Mule infection susceptibility is intermediate between donkey and horse, although closer to the latter. Anatomo-pathology and histopathology indicate that massive infection may cause mule death. Haematological value decreases of red blood cells, haemoglobin, leucocytes and lymphocytes indicate anaemia and strong immunosuppression. Strongly increased biochemical marker values indicate liver function alterations. The mule probably played a role in the past exchanges with Chile and Bolivia through Mendoza province. Evidence suggests that mules could contribute to the spread of both F. hepatica and G. truncatula to human fascioliasis-endemic areas in these countries.

Numerous Fasciola plasminogen-binding proteins may underlie blood-brain barrier leakage and explain neurological disorder complexity and heterogeneity in the acute and chronic phases of human fascioliasis.

Human fascioliasis is a worldwide, pathogenic food-borne trematodiasis. Impressive clinical pictures comprising puzzling polymorphisms, manifestation multifocality, disease evolution changes, sequelae and mortality, have been reported in patients presenting with neurological, meningeal, neuropsychic and ocular disorders caused at distance by flukes infecting the liver. Proteomic and mass spectrometry analyses of the Fasciola hepatica excretome/secretome identified numerous, several new, plasminogen-binding proteins enhancing plasmin generation. This may underlie blood-brain barrier leakage whether by many simultaneously migrating, small-sized juvenile flukes in the acute phase, or by breakage of encapsulating formations triggered by single worm tracks in the chronic phase. Blood-brain barrier leakages may subsequently occur due to a fibrinolytic system-dependent mechanism involving plasmin-dependent generation of the proinflammatory peptide bradykinin and activation of bradykinin B2 receptors, after different plasminogen-binding protein agglomeration waves. Interactions between diverse parasitic situations and non-imbalancing fibrinolysis system alterations are for the first time proposed that explain the complexity, heterogeneity and timely variations of neurological disorders. Additionally, inflammation and dilation of blood vessels may be due to contact system-dependent generation bradykinin. This baseline allows for search of indicators to detect neurological risk in fascioliasis patients and experimental work on antifibrinolytic treatments or B2 receptor antagonists for preventing blood-brain barrier leakage.

Human fascioliasis infection sources, their diversity, incidence factors, analytical methods and prevention measures.

Human fascioliasis infection sources are analysed for the first time in front of the new worldwide scenario of this disease. These infection sources include foods, water and combinations of both. Ingestion of freshwater wild plants is the main source, with watercress and secondarily other vegetables involved. The problem of vegetables sold in uncontrolled urban markets is discussed. Distinction between infection sources by freshwater cultivated plants, terrestrial wild plants, and terrestrial cultivated plants is made. The risks by traditional local dishes made from sylvatic plants and raw liver ingestion are considered. Drinking of contaminated water, beverages and juices, ingestion of dishes and soups and washing of vegetables, fruits, tubercles and kitchen utensils with contaminated water are increasingly involved. Three methods to assess infection sources are noted: detection of metacercariae attached to plants or floating in freshwater, anamnesis in individual patients, and questionnaire surveys in endemic areas. The infectivity of metacercariae is reviewed both under field conditions and experimentally under the effects of physicochemical agents. Individual and general preventive measures appear to be more complicated than those considered in the past. The high diversity of infection sources and their heterogeneity in different countries underlie the large epidemiological heterogeneity of human fascioliasis throughout.

Diagnosis of human fascioliasis by stool and blood techniques: update for the present global scenario.

Before the 1990s, human fascioliasis diagnosis focused on individual patients in hospitals or health centres. Case reports were mainly from developed countries and usually concerned isolated human infection in animal endemic areas. From the mid-1990s onwards, due to the progressive description of human endemic areas and human infection reports in developing countries, but also new knowledge on clinical manifestations and pathology, new situations, hitherto neglected, entered in the global scenario. Human fascioliasis has proved to be pronouncedly more heterogeneous than previously thought, including different transmission patterns and epidemiological situations. Stool and blood techniques, the main tools for diagnosis in humans, have been improved for both patient and survey diagnosis. Present availabilities for human diagnosis are reviewed focusing on advantages and weaknesses, sample management, egg differentiation, qualitative and quantitative diagnosis, antibody and antigen detection, post-treatment monitoring and post-control surveillance. Main conclusions refer to the pronounced difficulties of diagnosing fascioliasis in humans given the different infection phases and parasite migration capacities, clinical heterogeneity, immunological complexity, different epidemiological situations and transmission patterns, the lack of a diagnostic technique covering all needs and situations, and the advisability for a combined use of different techniques, at least including a stool technique and a blood technique.

Spread of the fascioliasis endemic area assessed by seasonal follow-up of rDNA ITS-2 sequenced lymnaeid populations in Cajamarca, Peru.

Fascioliasis is a worldwide emerging snail-borne zoonotic trematodiasis with a great spreading capacity linked to animal and human movements, climate change, and anthropogenic modifications of freshwater environments. South America is the continent with more human endemic areas caused by Fasciola hepatica, mainly in high altitude areas of Andean regions. The Peruvian Cajamarca area presents the highest human prevalences reported, only lower than those in the Bolivian Altiplano. Sequencing of the complete rDNA ITS-2 allowed for the specific and haplotype classification of lymnaeid snails collected in seasonal field surveys along a transect including 2007-3473 m altitudes. The species Galba truncatula (one haplotype preferentially in higher altitudes) and Pseudosuccinea columella (one haplotype in an isolated population), and the non-transmitting species Lymnaea schirazensis (two haplotypes mainly in lower altitudes) were found. Climatic seasonality proved to influence G. truncatula populations in temporarily dried habitats, whereas L. schirazensis appeared to be more climatologically independent due to its extreme amphibious ecology. Along the southeastern transect from Cajamarca city, G. truncatula and L. schirazensis shared the same site in 7 localities (46.7% of the water collections studied). The detection of G. truncatula in 11 new foci (73.3%), predominantly in northern localities closer to the city, demonstrate that the Cajamarca transmission risk area is markedly wider than previously considered. Lymnaea schirazensis progressively increases its presence when moving away from the city. Results highlight the usefulness of lymnaeid surveys to assess borders of the endemic area and inner distribution of transmission foci. Similar lymnaeid surveys are still in need to be performed in the wide northern and western zones of the Cajamarca city. The coexistence of more than one lymnaeid transmitting species, together with a morphologically indistinguishable non-transmitting species and livestock movements inside the area, conform a complex scenario which poses difficulties for the needed One Health control intervention.

Effects of climate change on animal and zoonotic helminthiases.

Current knowledge of animal and zoonotic helminthiases in which effects of climate change have been detected is reviewed. Climate variables are able to affect the prevalence, intensity and geographical distribution of helminths, directly influencing free-living larval stages and indirectly influencing mainly invertebrate, but also vertebrate, hosts. The impact of climate change appears to be more pronounced in trematodes, and is mainly shown by increased cercarial production and emergence associated with global warming. Fascioliasis, schistosomiasis (S. japonicum) and cercarial dermatitis caused by avian schistosomes have been the focus of study. Alveolar echinococcosis is currently the only cestode disease that climate change has been found to influence. Nematodiases, including heterakiasis, different trichostrongyliases and protostrongyliases, ancylostomiases and dirofilariases, are the helminth diseases most intensively analysed with regard to climate change. It may be concluded that helminth diseases should be listed among the infectious diseases with which special care should be taken because of climate change in the future, especially in temperate and colder northern latitudes and in areas of high altitude.

Geographical variation in populations of Phlebotomus (Paraphlebotomus) caucasicus (Diptera: Psychodidae) in Iran.

A comparative morphological and molecular study was carried out on 11 different populations of Phlebotomus (Paraphlebotomus) caucasicus Marzinovsky 1917 caught in 7 provinces in Iran (2004-2005). Differences in the implantation level of the two distal spines of the style, the number of setae of the basal lobe of coxite, and the length of the third antennal segment, revealed the existence of two morphotypes within P. (Pa) caucasicus, a species having a confused history if we take into account an unclear synonymisation with Phlebotomus (Paraphlebotomus) grimmi Porchinsky 1874. Sequencing of mtDNA (a fragment of cytchromeB gene, tRNA for serine gene and a fragment of NADH1 gene) and Neighbour-Joining analysis showed a partial correlation between morphotypes and haplotypes. We also found a correlation between the latter and the geographical origin of the specimens. These results need further studies in order to appreciate the role of each morphotype/haplotype in the transmission of Leishmania major.

Origin and phylogeography of the Chagas disease main vector Triatoma infestans based on nuclear rDNA sequences and genome size.

For about half of all Chagas disease cases T. infestans has been the responsible vector. Contributing to its genetic knowledge will increase our understanding of the capacity of geographic expansion and domiciliation of triatomines. Populations of all infestans subcomplex species, T. infestans, T. delpontei, T. platensis and T. melanosoma and the so-called T. infestans "dark morph", from many South American countries were studied. A total of 10 and 7 different ITS-2 and ITS-1 haplotypes, respectively, were found. The total intraspecific ITS-2 nucleotide variability detected in T. infestans is the highest hitherto known in triatomines. ITS-1 minisatellites, detected for the first time in triatomines, proved to be homologous and thus become useful markers. Calculations show that ITS-1 evolves 1.12-2.60 times faster than ITS-2. Despite all species analyzed presenting the same n=22 chromosome number, a large variation of the haploid DNA content was found, including a strikingly high DNA content difference between Andean and non-Andean specimens of T. infestans (mean reduction of 30%, with a maximum of up to 40%) and a correlation between presence/absence of minisatellites and larger/smaller genome size. Population genetics analysis of the eight composite haplotypes of T. infestans and net differences corroborate that there are clear differences between western and eastern populations (60%), and little genetic variation among populations (1.3%) and within populations (40%) within these two groups with migration rates larger than one individual per generation corresponding only to pairs of populations one from each of these groups. These values are indicative either of a large enough gene flow to prevent population differentiation by drift within each geographic area or a very recent spread, the latter hypothesis fitting available data better. Phylogenetic trees support a common ancestor for T. infestans and T. platensis, an origin of T. infestans in Bolivian highlands and two different dispersal lines, one throughout Andean regions of Bolivia and Peru and another in non-Andean lowlands of Chile, Paraguay, Argentina, Uruguay and Brazil.

Fascioliasis and other plant-borne trematode zoonoses.

Fascioliasis and other food-borne trematodiases are included in the list of important helminthiases with a great impact on human development. Six plant-borne trematode species have been found to affect humans: Fasciola hepatica, Fasciola gigantica and Fasciolopsis buski (Fasciolidae), Gastrodiscoides hominis (Gastrodiscidae), Watsonius watsoni and Fischoederius elongatus (Paramphistomidae). Whereas F. hepatica and F. gigantica are hepatic, the other four species are intestinal parasites. The fasciolids and the gastrodiscid cause important zoonoses distributed throughout many countries, while W. watsoni and F. elongatus have been only accidentally detected in humans. Present climate and global changes appear to increasingly affect snail-borne helminthiases, which are strongly dependent on environmental factors. Fascioliasis is a good example of an emerging/re-emerging parasitic disease in many countries as a consequence of many phenomena related to environmental changes as well as man-made modifications. The ability of F. hepatica to spread is related to its capacity to colonise and adapt to new hosts and environments, even at the extreme inhospitality of very high altitude. Moreover, the spread of F. hepatica from its original European range to other continents is related to the geographic expansion of its original European lymnaeid intermediate host species Galba truncatula, the American species Pseudosuccinea columella, and its adaptation to other lymnaeid species authochthonous in the newly colonised areas. Although fasciolopsiasis and gastrodiscoidiasis can be controlled along with other food-borne parasitoses, fasciolopsiasis still remains a public health problem in many endemic areas despite sustained WHO control programmes. Fasciolopsiasis has become a re-emerging infection in recent years and gastrodiscoidiasis, initially supposed to be restricted to Asian countries, is now being reported in African countries.

European Lymnaeidae (Mollusca: Gastropoda), intermediate hosts of trematodiases, based on nuclear ribosomal DNA ITS-2 sequences.

Freshwater snails of the family Lymnaeidae are of a great parasitological importance because of the very numerous helminth species they transmit, mainly trematodiases of large medical and veterinary impact. The present knowledge on the genetics of lymnaeids and on their parasite-host inter-relationships is far from being sufficient. The family is immersed in a systematic-taxonomic confusion. The necessity for a tool which enables species distinction and population characterization is evident. This paper aims to review the European Lymnaeidae basing on the second internal transcribed spacer ITS-2 of the nuclear ribosomal DNA. The ITS-2 sequences of 66 populations of 13 European and 1 North American lymnaeid species, including the five generic (or subgeneric) taxa Lymnaea sensu stricto, Stagnicola, Omphiscola, Radix and Galba, have been obtained. The ITS-2 proves to be a useful marker for resolving supraspecific, specific and population relationships in Lymnaeidae. Three different groupings according to their ITS-2 length could be distinguished: Radix and Galba may be considered the oldest taxa (370-406 bp lengths), and Lymnaea s. str., European Stagnicola and Omphiscola (468-491 bp lengths) the most recent, American Stagnicola and Hinkleyia being intermediate (434-450 bp lengths). This hypothesis agrees with the phylogeny of lymnaeids based on palaeontological data, chromosome numbers and radular dentition. ITS-2 sequences present a conserved central region flanked by two variable lateral regions corresponding to the 5' and 3' ends. The number of repeats of two microsatellites found in this conserved central region allows to differentiate Radix from all other lymnaeids. Phylogenetic trees showed four clades: (A) Lymnaea s. str., European Stagnicola and Omphiscola; (B) Radix species; (C) Galba truncatula; and (D) North American stagnicolines. ITS-2 results suggest that retaining Stagnicola as a subgenus of Lymnaea may be the most appropriate and that genus status for Omphiscola is justified. Radix shows a complexity suggesting different evolutionary lines, whereas G. truncatula appears to be very homogeneous. North American and European stagnicolines do not belong to the same supraspecific taxon; the genus Hinkleyia may be used for the American stagnicolines. Genetic distances and sequence differences allowed us to distinguish the upper limit to be expected within a single species and to how different sister species may be. S. palustris, S. fuscus and S. corvus proved to be valid species, but S. turricula may not be considered a species independent from S. palustris. Marked nucleotide divergences and genetic distances detected between different S. fuscus populations may be interpreted as a process of geographic differentiation developping in the present. Among Radix, six valid species could be distinguished: R. auricularia, R. ampla, R. peregra (=R. ovata;=R. balthica), R. labiata, R. lagotis and Radix sp. The information which the ITS-2 marker furnishes is of applied interest concerning the molluscan host specificity of the different trematode species. The phylogenetic trees inferred from the ITS-2 sequences are able to differentiate between lymnaeids transmitting and those non-transmitting fasciolids, as well as between those transmitting F. hepatica and those transmitting F. gigantica. The Fasciola specificity is linked to the two oldest genera which moreover cluster together in the phylogenetic trees, suggesting an origin of the Fasciola ancestors related to the origin of this branch. European Trichobilharzia species causing human dermatitis are transmitted only by lymnaeids of the Radix and Lymnaea s. str.-Stagnicola groups. Results suggest the convenience of reinvestigating compatibility differences after accurate lymnaeid species classification by ITS-2 sequencing. Similarly, ITS-2 sequencing would allow a step forward in the appropriate rearrangement of the actual systematic confusion among echinostomatids.

Nuclear rDNA ITS-2 sequences reveal polyphyly of Panstrongylus species (Hemiptera: Reduviidae: Triatominae), vectors of Trypanosoma cruzi.

Panstrongylus species are widely distributed throughout the Americas, where they act as vectors of Trypanosoma cruzi, agent of Chagas disease. Their intraspecific relationships, taxonomic position and phylogeny in relation to other Triatomini were explored using ribosomal DNA (rDNA) internal transcribed spacer 2 (ITS-2) sequence polymorphisms and maximum parsimony, distance and maximum likelihood analyses of 10 populations representing six species of the genus (P. megistus, P. geniculatus, P. rufotuberculatus, P. lignarius, P. herreri and P. chinai). At the subspecific level, P. megistus appeared more homogeneous than P. rufotuberculatus and P. geniculatus (both with broader distribution). Several dinucleotide microsatellites were detected in the sequences of given species. Many of these microsatellites (GC, TA, GT and AT) showed different number of repeats in different populations and thus, may be very useful for population differentiation and dynamics analyses in future studies. The sequences of P. lignarius (considered sylvatic) and P. herreri (a major disease vector in Peru) were identical, suggesting that these species should be synonymised. Intrageneric analysis showed a clear separation of P. rufotuberculatus, with closest relationships between P. geniculatus and P. chinai, and P. megistus occupying a separate branch. Genetic distances between Panstrongylus species (0.11585-0.22131) were higher than those between Panstrongylus and other Triatomini (16 species from central and North America and South America) (0.08617-0.11039). The distance between P. megistus and P. lignarius/herreri (0.22131) was the largest so far recorded in the tribe. The pronounced differences in length and nucleotide composition suggest a relatively old divergence of Panstrongylus species. P. rufotuberculatus was closer to Mesoamerican Triatoma, Meccus and Dipetalogaster species than to other Panstrongylus. All Panstrongylus clustered with the Mesoamerican clade; P. rufotuberculatus clustered with the phyllosoma complex and T. dimidiata, with D. maxima and T. barberi in a basal position. The rest of Panstrongylus appeared paraphyletically in the tree. This is evidence suggesting polyphyly within the genus Panstrongylus, whose species may be related to the ancestors giving rise to central and North American Triatomini.

Diplogonoporiasis presumably introduced into Spain: first confirmed case of human infection acquired outside the Far East.

A 58-year-old man who was very fond of eating raw fish and had not left Zaragoza Province in Spain in the last 20 years excreted a short chain of gravid proglottids. Treatment with 50 mg/kg of paromomycin sulfate was divided into three doses, all given within the same day, followed by administration of a laxative at night. On days 1 and 2 post-treatment, several chains of degenerated proglottids were evacuated. Only eggs were expelled on days 3 and 4. A long complete strobila including the scolex, and immature, mature, and gravid segments was spontaneously discharged on day 25 after treatment. It was identified as Diplogonoporus balaenopterae, and was the first confirmed case of diplogonoporiasis outside the Far East (all previous cases were reported from Japan, except for one recent case reported from Korea). The origin of this case, outside of its normal geographic location, may be associated with the importation of fish into Spain. The viability of the infective larval stage is evidence of its resistance to export/import conditions. Treatment with paromomycin sulfate did not result in the whole worm being discharged but several short strobilar chains showed drug-induced degeneration.

Occurrence of a sibling species complex within neotropical lymnaeids, snail intermediate hosts of fascioliasis.

The delimitation of cryptic species within the genus Lymnaea, which are the main vectors of fascioliasis, remains a topic of controversy. An analysis of genetic variability based on 12 enzyme loci revealed different fixed alleles at 9 loci between two sympatric samples of Lymnaea viatrix at the type locality in Lima, Peru. The absence of heterozygotes within this locality indicates the presence of isolated populations or cryptic species within L. viatrix. Significant genetic differences were also found between these two L. viatrix samples from Lima and other populations of L. viatrix in South America and in addition to species such as L. truncatula, L. cubensis and L. columella. Moreover, the lack of variability within each Lymnaea samples studied indicates the existence of a high selfing rate in each species.

Phylogeography and genetic divergence of some lymnaeid snails, intermediate hosts of human and animal fascioliasis with special reference to lymnaeids from the Bolivian Altiplano.

A population genetic study using starch gel electrophoresis was performed on populations of several species of lymnaeid snails acting as intermediate hosts for Fasciola hepatica (Trematoda, Plathyhelminth). Lymnaea viatrix was collected in 16 sites from the Bolivian Northern Altiplano. L. cubensis were obtained in one site from Venezuela, one site from Guadeloupe, three sites from Cuba and one site from the Dominican Republic. L. truncatula were collected in one site from France, one from Portugal and one from Morocco. Multilocus enzyme electrophoresis (MEE) were determined for 282 snails at 18 loci. A complete monomorphism was encountered at each geographic site. However, among these 18 loci, 13 are polymorphic and low and high levels of genetic divergence were observed between samples. Two genotypic groups can be differentiated by their multilocus genotypes. The western genotypic group associates together samples from Venezuela, Guadeloupe, Cuba and Dominican Republic (L. cubensis) while samples from France, Portugal and Morocco (L. truncatula) belong to the eastern genotypic group. Surprisingly, the Northern Bolivian Altiplano populations (L. viatrix) do not present any genetic divergence with the Portuguese sample. Therefore, the Bolivian snails belong entirely to the eastern genetic group. Within each group slight genetic divergences were observed. These results strongly support the European origin of the lymnaeid snails from the Northern Bolivian Altiplano.

ITS-2 rDNA sequencing of Gnathostoma species (Nematoda) and elucidation of the species causing human gnathostomiasis in the Americas.

From several gnathostome species the complete internal transcribed spacer ITS-2 ribosomal DNA (rDNA) repeat sequence and a fragment of the 5.8S rDNA were obtained by direct polymerase chain reaction cycle-sequencing and silver-staining methods. The size of the complete ITS-1 sequence in agarose gel electrophoresis was also obtained. The ITS-2 enabled the differentiation of Gnathostoma spinigerum from Thailand and Gnathostoma binucleatum from Mexico and Ecuador and confirmed the validity of the latter. Gnathostoma turgidum, Gnathostoma sp. I (=Gnathostoma procyonis sensu Almeyda-Artigas et al., 1994), and Gnathostoma sp. II (=G. turgidum sensu Foster, 1939 pro parte), all from Mexico, proved to be independent species, but Gnathostoma sp. III, also from Mexico, could not be differentiated from G. turgidum. In Mexico and Ecuador, gnathostomes involved in human infection and that had been classified as G. spinigerum belong to G. binucleatum. The 5.8S rDNA sequences of the 6 Gnathostoma species studied were identical. The results of the ITS-1 agreed with those results of ITS-2.

SSU rDNA characterization of lymnaeid snails transmitting human fascioliasis in South and Central America.

The small subunit (18S) rRNA gene sequences of the lymnaeid morphs I and II (Mollusca: Gastropoda: Basommatophora: Lymnaeidae) transmitting human fascioliasis in the high endemic zone of the northern Bolivian Altiplano and of Lymnaea cubensis from Mexico and Guadeloupe island (Caribbean) have been obtained by direct polymerase chain reaction PCR cycle sequencing and silver staining methods and compared to that of the 6 most common European Lymnaeidae species. Results allow us to establish definitively the distinction between the lymnaeids from the northern Bolivian Altiplano and L. cubensis. Lymnaea cubensis is a valid species distributed in North and Central America but absent in the northern Bolivian Altiplano. Lymnaeid morphs I and II from the northern Bolivian Altiplano both present the same 18S rDNA sequence, which is moreover identical to that of the European species Lymnaea truncatula. Significant nucleotide substitutions in helix E10-1 of the variable region V2 of the secondary structure suggest the need for distinguishing L. cubensis in the subgenus Lymnaea (Bakerilymnaea) with L. (B.) cubensis as type species. The subgenus Lymnaea (Fossaria) is retained, with L. (F.) truncatula as type species. The larger number of nucleotides in the 18S rDNA sequence of L. cubensis (1,860 bp) with regard to the other Lymnaea species (1,843-1,852 bp) is tentatively related to the more ancient paleogeographic origin of L. cubensis. The grouping of L. cubensis with L. truncatula and the relationship of Lymnaea auricularia with Lymnaea peregra in the phylogenetic trees obtained show an evolutionary parallelism with the digenean parasite species they transmit, namely Fasciola hepatica and Fasciola gigantica, respectively.

Insights into the relationships of Palearctic and Nearctic lymnaeids (Mollusca: Gastropoda) by rDNA ITS-2 sequencing and phylogeny of stagnicoline intermediate host species of Fasciola hepatica.

Fascioliasis by Fasciola hepatica is the vector-borne disease presenting the widest latitudinal, longitudinal and altitudinal distribution known. F. hepatica shows a great adaptation power to new environmental conditions which is the consequence of its own capacities together with the adaptation and colonization abilities of its specific vector hosts, freshwater snails of the family Lymnaeidae. Several lymnaeid species only considered as secondary contributors to the liver fluke transmission have, however, played a very important role in the geographic expansion of this disease. Many of them belong to the so-called "stagnicoline" type group. Stagnicolines have, therefore, a very important applied interest in the Holarctic region, to which they are geographically restricted. The present knowledge on the genetics of stagnicolines and on their parasite-host interrelationships is, however, far from being sufficient. The present paper analyses the relationships between Palaearctic and Nearctic stagnicoline species on the base of the new light furnished by the results obtained in nuclear rDNA ITS-2 sequencing and corresponding phylogenetic studies of the lymnaeid taxa Lymnaea (Stagnicola) occulta, L. (S.) palustris palustris (topotype specimens) and L. (S.) p. turricula from Europe. Natural infections with F. hepatica have been reported in all of them. Surprisingly, ITS-2 length and GC content of L. occulta were similar and perfectly fitted within the respective ranges known in North American stagnicolines. Nucleotide differences and genetic distances were higher between L. occulta and the other European stagnicolines than between L. occulta and the North American ones. The ITS-2 sequence of L. p. turricula from Poland differed from the other genotypes known from turricula in Europe. The phylogenetic trees using the maximum-parsimony, distance and maximum-likelihood methods confirmed (i) the inclusion of L. occulta in the branch of North American stagnicolines, (ii) the link between the North American stagnicolines-L. occulta group with Galba truncatula, and (iii) the location of the L. p. turricula genotype from Poland closer to L. p. palustris than to other European L. p. turricula genotypes. The Palaearctic species occulta is included in the genus Catascopia, together with the Nearctic species catascopium, emarginata and elodes. The results suggest a potential of transmission capacity for C. occulta higher than that of other European stagnicolines or Omphiscola glabra. The relatively low genetic distances between C. occulta and G. truncatula and the clustering of both species in the same clade suggest that C. occulta may be potentially considered as the second lymnaeid intermediate host species of F. hepatica in importance in eastern and northern Europe, and probably also western and central Asia, after G. truncatula. L. p. turricula may be considered as a potential secondary vector of F. hepatica, at a level similar to that of L. p. palustris.