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One of the most interesting biological models is that of snail-trematode interactions, many of which ultimately result in the transmission of several important diseases, particularly in the tropics. Herein, we review the scientific advances on a trematode-snail system in which certain populations of Pseudosuccinea columella (a common host species for trematodes) have been demonstrated naturally-resistant to Fasciola hepatica, in association with an effective encapsulation of the parasite by innate immune cells of the host, the hemocytes. Emphasis is made on the molecular and immunological features characterizing each P. columella phenotype in relation to their anti-parasitic competence, their distinctive ecological patterns and the existence of a significant cost of resistance. An integrative overview of the resistance to F. hepatica through comparative immunobiology, genetics and ecology is presented to hypothesize on the possible origins and evolution of this phenomenon and to postulate significant roles for parasite mediated-selection and environmental factors in shaping and maintaining the resistant phenotype in the field. Lastly, clues into future experimental perspectives to deeply characterize the interplay between P. columella and F. hepatica and the immunobiology of the resistance are also included. The advances revised in the present paper are only beginning to unravel mechanisms of anti-parasite innate defense responses and their evolutionary bases, and can facilitate the development of prospective approaches towards practical applications of P. columella resistance.

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Fasciola hepatica is a worldwide emerging and re-emerging parasite heavily affecting several regions in South America. Some lymnaeid snail species of American origin are among the major hosts of F. hepatica worldwide. Recent paleoparasitological findings detected its DNA in a 2300-year-old sample in Patagonia, countering the common hypothesis of the recent arrival of F. hepatica in the Americas during European colonization. Thus, the theory of an initial introduction in the 1500s can no longer be sustained. This article discusses how it was possible for F. hepatica to reach and spread in the Americas in relation to the availability and compatibility of hosts through natural and incidental introductions. Our study will serve to better understand the ongoing Neotropical scenario of fasciolosis.

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The increasing distribution and prevalence of fasciolosis in both human and livestock are concerning. Here, we examine the various types of factors influencing fasciolosis transmission and burden and the interrelations that may exist between them. We present the arsenal of molecules, 'adjusting' capabilities and parasitic strategies of Fasciola to infect. Such features define the high adaptability of Fasciola species for parasitism that facilitate their transmission. We discuss current environmental perturbations (increase of livestock and land use, climate change, introduction of alien species and biodiversity loss) in relation to fasciolosis dynamics. As Fasciola infection is directly and ultimately linked to livestock management, living conditions and cultural habits, which are also changing under the pressure of globalization and climate change, the social component of transmission is also discussed. Lastly, we examine the implication of increasing scientific and political awareness in highlighting the current circulation of fasciolosis and boosting epidemiological surveys and novel diagnostic techniques. From a joint perspective, it becomes clear that factors weight differently at each place and moment, depending on the biological, environmental, social and political interrelating contexts. Therefore, the analyses of a disease as complex as fasciolosis should be as integrative as possible to dissect the realities featuring each epidemiological scenario. Such a comprehensive appraisal is presented in this review and constitutes its main asset to serve as a fresh integrative understanding of fasciolosis.

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Antimicrobial peptides (AMPs) are biomolecules with antimicrobial activity against a broad group of pathogens. In the past few decades, AMPs have represented an important alternative for the treatment of infectious diseases. Their isolation from natural sources has been widely investigated. In this sense, mollusks are promising organisms for the identification of AMPs given that their immune system mainly relies on innate response. In this report, we characterized the peptide fraction of the Cuban freshwater snail Pomacea poeyana (Pilsbry, 1927) and identified 37 different peptides by nanoLC-ESI-MS-MS technology. From these peptide sequences, using bioinformatic prediction tools, we discovered two potential antimicrobial peptides named Pom-1 (KCAGSIAWAIGSGLFGGAKLIKIKKYIAELGGLQ) and Pom-2 (KEIERAGQRIRDAIISAAPAVETLAQAQKIIKGG). Database search revealed that Pom-1 is a fragment of Closticin 574 previously isolated from the bacteria Clostridium tyrobutyrium, and Pom-2 is a fragment of cecropin D-like peptide first isolated from Galleria mellonella hemolymph. These sequences were chemically synthesized and evaluated against different human pathogens. Interestingly, structural predictions of both peptides in the presence of micelles showed models that comprise two alpha helices joined by a short loop. The CD spectra analysis of Pom-1 and Pom-2 in water showed for both structures a high random coil content, a certain content of α-helix and a low ß-sheet content. Like other described AMPs displaying a disordered structure in water, the peptides may adopt a helical conformation in presence of bacterial membranes. In antimicrobial assays, Pom-1 demonstrated high activity against the Gram-negative bacteria Pseudomonas aeruginosa and moderate activity against Klebsiella pneumoniae and Listeria monocytogenes. Neither of the two peptides showed antifungal action. Pom-1 moderately inhibits Zika Virus infection but slightly enhances HIV-1 infectivion in vitro. The evaluation of cell toxicity on primary human macrophages did not show toxicity on THP-1 cells, although slight overall toxicity was observed in high concentrations of Pom-1. We assume that both peptides may play a key role in innate defense of P. poeyana and represent promising antimicrobial candidates for humans.

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The Fasciola hepatica/Pseudosuccinea columella interaction in Cuba involves a unique pattern of phenotypes; while most snails are susceptible, some field populations are naturally resistant to infection and parasites are encapsulated by snail hemocytes. Thus, we investigated the hemocytes of resistant (R) and susceptible (S) P. columella, in particular morphology, abundance, proliferation and in vitro encapsulation activity following exposure to F. hepatica. Compared to susceptible P. columella, hemocytes from exposed resistant snails showed increased levels of spreading and aggregation (large adherent cells), proliferation of circulating blast-like cells and encapsulation activity of the hemocytes, along with a higher expression of the cytokine granulin. By contrast, there was evidence of a putative F. hepatica-driven inhibition of host immunity, only in susceptible snails. Additionally, (pre-)incubation of naïve hemocytes from P. columella (R and S) with different monosaccharides was associated with lower encapsulation activity of F. hepatica larvae. This suggests the involvement in this host-parasite interaction of lectins and lectins receptors (particularly related to mannose and fucose sensing) in association with hemocyte activation and/or binding to F. hepatica.

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Fasciolosis is an important food-borne parasitic disease affecting over two million people worldwide with economic losses related to cattle production of up to US$ 3 billion annually. Despite the long known presence of Fasciola hepatica in the Caribbean islands its transmission is not well known. This study reviews historical and recent data on fasciolosis in the West Indies, revealing for the first time the outcomes of sympatric and allopatric fluke/snail interactions in the area by exploring the susceptibility of four lymnaeid species after exposure to F. hepatica isolates from Cuba, the Dominican Republic and France. Overall, Galba cubensis showed a mean prevalence of 71.8% and appears to be the most suitable intermediate host species irrespective of the isolate used. Sympatric combinations (snail and parasite from the same country) were generally more compatible (higher susceptibility, parasite intensity and snail survival post-exposure) and only the allopatric interaction of French G. truncatula/Cuban F. hepatica attained 100% prevalence and mean intensity over 33 rediae/snail. However, certain Dominican populations of Pseudosuccinea columella showed high parasite intensities (>30 rediae/snail) when infected with Cuban flukes, highlighting the potential risks of biological introductions. Overall, high compatibility in most sympatric combinations compared to low or moderate compatibility in allopatric ones, suggests the existence of local adaptation from a long sustained interaction that has led to high rates of transmission. Interestingly, attempts to infect G. schirazensis with sympatric and allopatric flukes failed and coupled with the lowest survival rates which supposes a low risk of fasciolosis transmission in areas where this is the only snail species. Although there are significant gaps in the actual status of fasciolosis transmission from several islands in the West Indies these results show a permanent risk. We conclude that fasciolosis transmission is high in areas where the local snail, G. cubensis, occurs, and will be even higher in the presence of the invasive P. columella.

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Pseudosuccinea columella snails transmit the trematode Fasciola hepatica, but in Cuba, six naturally occurring populations successfully resist parasite infection. Here, we present an updated distribution of P. columella in Cuba; 68 positive sites with the earliest records more abundant in west-central Cuba and with east-central populations generally corresponding to the newest samples. No records were found farther east. The IPA site reported 10.5% prevalence of F. hepatica-infected snails. Population genetics, studied through microsatellites, showed low allelic and multilocus genotypic richness (MLGT), mainly in susceptible populations, strong deviations from panmixia and high self-fertilization rates. Susceptible individuals were grouped in one major cluster containing the majority of MLGT, and two independent clusters grouped the MLGT of resistant individuals from western and central populations, respectively. From these, we propose that several introductions of P. columella occurred in Cuba, primarily in the west, with the early arrivals deriving on the resistant populations. A more recent introduction of susceptible P. columella carrying MLGT T and Y may have occurred, where the latter spread quickly through the island and possibly increase the risk of parasite transmission in Cuba since all snails naturally infected with F. hepatica were carriers of the MLGT Y. Interestingly, even though resistant populations are highly diverse and are likely the oldest within Cuba, they are only found in six localities characterized by soft (total hardness, TH = 6.3 ± 1.03°d) and slightly acidic (pH = 6.2 ± 0.12) waters with low richness in snail species (3.2 ± 1.02). This tendency was also observed in a two-year follow-up ecological study that was conducted on a farm where both phenotypes occurred in sympatry; colonization events by resistant over susceptible snails coincided with a reduction in the pH and TH of the water. A comparison of life traits in susceptible and resistant isolates reared at two different pH/TH conditions (5.9/4°d or 7.8/14°d) showed that low pH/TH negatively affects P. columella, irrespective of the phenotype. However, evidence of higher tolerance (higher survival, life expectancy, egg viability) to such conditions was observed in resistant isolates. Finally, we speculate that the limited distribution of resistant populations might be related to a better exploitation of sites that are less suitable to snails (thus, with lower competition), rather than to a differential ecological restriction to specific environmental conditions from susceptible P. columella.

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Introducción: en el paisaje epidemiológico de la fascioliasis humana, dos aspectos son de fundamental importancia: a) presencia de hospederos definitivos (especialmente ganado bovino y humanos) parasitados con adultos del tremátodo y; b) presencia de hospederos intermediarios invertebrados, moluscos de la familia Lymnaeidae, en los que se desarrollan los estadios larvarios, además de plantas acuáticas de consumo humano. Material y métodos: con el objetivo de prevenir brotes de fascioliasis humana mediante la correcta identificación de aspectos de interés epidemiológico, un humedal fue seleccionado en el sector de Palo Blanco, municipio de Jarabacoa, provincia de La Vega, en el cual se realizó un estudio malacológico en octubre de 2016, en busca de especies de moluscos limneidos e hidrófitos asociados. En el laboratorio se obtuvieron los índices de infección natural y experimental a Fasciola hepatica. Resultados: se identificó el limneido Pseudosuccinea columella, en proporciones de 250 especímenes por persona/hora. El vegetal predominante fue el berro (Nasturtiun officinale), cultivado comercialmente en tres cuadrantes del humedal y en el otro cuadrante se observaron bovinos pastando. El índice de infección experimental de los moluscos fue de 94 % a la cepa simpátrica de F. hepatica de Dajabón; no se encontraron moluscos parasitados naturalmente. Conclusiones: Pseudosuccinea columella debe considerarse como un molusco de relevancia epidemiológica en la transmisión de la fascioliasis en República Dominicana, a tenor de su dispersión y elevado índice de infección experimental obtenido en el presente estudio

Introduction: in the epidemiological landscape of human fascioliasis, two aspects are of fundamental importance: a) presence of definite hosts (especially cattle and humans) parasitized with trematode adults and b) presence of invertebrate intermediate hosts, mollusks of the family Lymnaeidae, in which larval stages develop, in addition to aquatic plants for human consumption. Material and methods: in order to prevent outbreaks of human fascioliasis by correctly identifying aspects of epidemiological interest, a wetland was selected in the sector of Palo Blanco, municipality of Jarabacoa, province of La Vega, in which a malacological study was carried out in October 2016, in search of limneid mollusk species and associated hydrophytes. Natural and experimental infection rates to Fasciola hepaticawere obtained in the laboratory. Results: the lymnaeid Pseudosuccinea columella was identified, in proportions of 250 specimens collected per person/hour. The predominant vegetable was the watercress (Nasturtiun officinale), commercially cultivated in three quadrants of the wetland and in the other quadrant were observed cattle grazing. The rate of experimental infection of mollusks was 94 % to the sympatric strain of F. hepatica of Dajabón; no naturally parasitized mollusks were found. Conclusions: Pseudosuccinea columella should be considered as a mollusk of epidemiological relevance in the transmission of fascioliasis in the Dominican Republic, due to its dispersion and high rate of experimental infection obtained in the present study

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Resumen La fasciolosis es una trematodosis que afecta fuertemente al ganado de Cuba. Se estudiaron 10 vaquerías del municipio más occidental de Cuba para determinar la prevalencia de esta parasitosis en el ganado y en los hospederos intermediarios. Se recolectaron y estudiaron muestras fecales de 455 vacas lecheras seleccionadas al azar. Las muestras fueron conservadas a -20 oC hasta su estudio. La detección de la infección por Fasciola hepatica fue realizada a partir de un método de Elisa denominado FasciDIG®. El análisis e interpretación de los resultados demostró que 146 muestras fueran positivas a F. hepatica (32,09 %). Todas las vaquerías presentaron animales infectados con rangos de prevalencia entre las vaquerías que oscilaron de 9,5 % al 84 %. También fue posible encontrar las dos especies de hospederos intermediarios de este parásito en Cuba, Galba cubensis y Pseudosuccinea columella. Debe prestarse atención a la prevalencia en bovinos encontrada en este estudio para prevenir la aparición de brotes humanos de esta enfermedad.

Abstract Fasciolosis is a foodborne trematodosis affecting many cattle farms in Cuba. Ten dairy farms in western Cuba were studied to assess the prevalence in both intermediate and definitive hosts. A single stool sample from 455 dairy cows was taken and studied. The animals were randomly selected and the samples were kept at -20oC until infection was determined. Detection of the Fasciola hepatica infection was carried out using a non-commercial sandwich-ELISA called FasciDIG®. Results showed that 146 samples were positive for F. hepatica (32.09%) with every farm testing positive for F. hepatica infection, while prevalence ranged from 9.5% to 84% among farms. It was also possible to detect the presence of the two species of intermediate hosts of this trematode in Cuba, Galba cubensis and Pseudosuccinea columella. Main attention must be given to bovine prevalence found in this study in order to prevent eventual human outbreaks of this disease.

Resumo A fasciolose é uma trematoda que afeta fortemente ao gado de Cuba. Foram estudadas 10 leiterias do município mais ocidental de Cuba para determinar a prevalência desta parasitose no gado e nos hospedeiros intermediários. Se coletaram e estudiaram amostras fecais de 455 vacas leiteiras selecionadas aleatoriamente. As amostras foram conservadas a −20 oC até seu estudo. A detecção da infecção por Fascíola hepática foi realizada a partir de um método de Elisa denominado FasciDIG®. A análise e interpretação dos resultados demonstrou que 146 amostras foram positivas à F. hepática (32,09 %). Todas as leiterias apresentaram animais infectados com rangos de prevalência entre as leiterias que oscilaram de 9,5 % a 84 %. Também foi possível encontrar a duas espécies de hospedeiros intermediários deste parasita em Cuba, Galba cubensis e Pseudosuccinea columella. Deve prestar-se atenção à prevalência em bovinos encontrada neste estudo para prevenir a aparição de surtos humanos desta enfermidade.

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BACKGROUND: Pseudosuccinea columella is one of the most widespread vectors of Fasciola hepatica, a globally distributed trematode that affects humans, livestock and wildlife. The exclusive occurrence in Cuba of susceptible and naturally-resistant populations to F. hepatica within this snail species, offers a fascinating model for evolutionary biology, health sciences and vector control strategies. In particular, resistance in P. columella is characterized by the encapsulation of the parasite by host's immune cells and has been experimentally tested using different Cuban F. hepatica isolates with no records of successful infection. Here, we aimed to explore for the first time, the effect of different parasite doses, successive exposures and different parasite origins on the infection outcomes of the two phenotypes of P. columella occurring in Cuba. METHODS: To increase the chances for F. hepatica to establish, we challenged Cuban P. columella with increasing single parasite doses of 5, 15 or 30 miracidia and serial exposures (three-times) of 5 miracidia using a sympatric F. hepatica isolate from Cuba, previously characterized by microsatellite markers. Additionally, we exposed the snails to F. hepatica from different geographical origins (i.e. Dominican Republic and France). Parasite prevalence, redial burden and survival of snails were recorded at 25 days post-exposure. RESULTS: No parasite development was noted in snails from the resistant populations independent of the experimental approach. Contrastingly, an overall increase in prevalence and redial burden was observed in susceptible snails when infected with high miracidia doses and after serial exposures. Significant differences in redial burden between single 15 miracidia and serial 3 × 5 miracidia infected snails suggest that immune priming potentially occurs in susceptible P. columella. Compatibility differences of allopatric (Caribbean vs European) F. hepatica with susceptible snails were related to the geographical scale of the combinations. CONCLUSIONS: Here, the effectiveness of P. columella resistance to F. hepatica does not decline with increasing parasite doses, successive infection or different geographical origins of parasite isolates, while presenting new evidence for specificity for infection in susceptible P. columella snails. Understanding the peculiarities of the P. columella-F. hepatica interaction and the extent of the resistant phenotype is crucial for an effective parasite control and for developing alternatives to tackle fasciolosis transmission.

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OBJECTIVE: To study the distribution of vertical transmission of dengue viruses in field-collected Aedes aegypti larvae in the municipality of Arroyo Naranjo in Havana, Cuba. METHODS: Aedes aegypti larvae and pupae were collected monthly between September 2013 and July 2014 in the seven Municipal Health Areas of Arroyo Naranjo. Pools formed of 30-55 larvae were examined through PCR and sequencing to detect the presence of each serotype. RESULTS: We analysed 111 pools of larvae and pupae (4102 individuals) of which 37 tested positive for at least one DENV. More than one DENV type was observed in 10 of the 37 positive pools. Infected pools were detected every month, except in January, suggesting a sustained circulation of DENV in the vector populations. DENV-1 and DENV-3 were the most frequent and dispersed, though all four DENV types were detected. Nucleotide sequencing from positive pools confirmed RT-PCR results for DENV-1 (genotype V), DENV-3 (genotype III) and DENV-4 (genotype II). DENV-2 was detected by RT-PCR but could not be confirmed by nucleotide sequencing. CONCLUSION: Our study of the distribution of natural vertical transmission of dengue virus types highlights extrinsic virus activity patterns in the area and could be used as a new surveillance tool.

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The giant African snail Lissachatina fulica has been reported invading Cuba since 2014 and is now well established in areas of Havana and several nearby regions. This invasive species is of major concern to health authorities given its role as an important vector of parasites such as Angiostrongylus cantonensis, the causative agent of eosinophilic meningoencephalitis in humans. Here, we update the distribution within Cuba. We also report on our studies of experimental life-history traits to assess the population dynamics and potential for spread of this species in Cuba. The experimental population had a very low probability of dying at first age intervals (Type I survival curve) with a life expectancy of 71 weeks. During our experiment, sexual maturity was reached after 22 weeks (individuals of 70 mm shell length) and eggs were laid regularly (mean batch size: 188 ±â€¯111.79 SD). We failed to detect any deviations from the mortality curve and individuals reached an average of 77.3 mm (shell length) and weighted 57.7 g after one year. Predicted curve models indicate that snails reaching their average lifespan of five years should attain 10-12 cm (shell length) and weight 160 g. The spreading of this invasive and vector snail has been tracked for four years in Cuba showing a steady increase of invaded localities. How fast and how far this species develops in Cuba is unknown but the life history parameters indicated in this paper show that it has a large potential to invade all areas of Cuba quickly unless a systematic abatement strategy is developed.

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The Blue-winged Teal (Spatula discors) is known for engaging in long-distance North-South migrations and back in the Americas with Cuba being an important wintering ground. Teals serve as hosts for a wide range of parasite species that can be "discharged" at each end of the migration route. Here, we explore for the first time the diversity of trematodes that the Blue-winged Teal may be introducing to -or exporting from- Cuba. We found 15 digenean parasites in 65 sampled teals of which 13 represent first reports for Cuba with one species (Echinostoma revolutum) of zoonotic importance. Overall prevalence was very high (96.92%) whereas Trichobilharzia spp. resulted the most ubiquitous parasite. Highest intensities were recorded for Australapatemon sp. (arriving teals) and Levinseniella amnicolae (departing teals). Altogether, departing teals harboured the highest number of trematode species and abundance. The short development and lifespan, inside migratory birds, of adult stages of trematodes like Trichobilharzia sp., Cotylurus flabelliformis and Microphallus pygmaeus strongly suggest that the occurrence of these trematodes in the departing teals could be related to their long establishment in Cuban ecosystems. In a global scenario where parasitology, particularly wildlife parasitology, is mostly overlooked, there should be an increasing need of gathering information and increasing surveillance of wildlife diseases that might eventually become important for the health of ecosystems and of domestic animals and humans. The present study constitutes the first major attempt to explore trematode infection from S. discors in Cuba under the view of parasite flow via bird migration.

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While horizontal transmission (human-mosquito-human) of dengue viruses largely determines the epidemiology of the disease, vertical transmission (infected female mosquito- infected offspring) has been suggested as a mechanism that ensures maintenance of the virus during adverse conditions for horizontal transmission to occur. The purpose of this study was to analyze the natural infection of larval stages of Aedes aegypti (Diptera: Culicidae) with the dengue virus (DENV) in Cuba. Here, we report vertical transmission of DENV-3 genotype III in natural populations of Ae. aegypti through RT-PCR detection and serotyping plus sequencing. Our report constitutes the first record of vertical transmission of DENV in Ae. aegypti from Cuba with details of its serotype and genotype.

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BACKGROUND: Fasciolosis is one of the food-borne neglected trematodioses that has reemerged as a human disease while its effects on domestic animal health remains of significant economic consideration. Being snail-borne disease, the accurate and time-saving epidemiological surveillance of the transmission foci where infected lymnaeid snails occur could be essential to effectively focus or redirect control strategies. For this purpose, the first monoclonal antibody-based immunoenzymatic assay to detect Fasciola hepatica-infected snails (FasciMol-ELISA) was recently developed and showed a high sensitivity and specificity when tested in an experimental F. hepatica - Galba cubensis system. METHODS: Here, we surveyed populations of G. cubensis occurring in western Cuba for the assessment of the FasciMol-ELISA in determining natural F. hepatica infection in this intermediate host. A multiplex PCR, previously developed to detect F. hepatica in G. cubensis, was used for sample classification. Snail dissection method was also employed as screening technique. A Χ(2) test and a Kappa index were calculated to evaluate the positivity and the level of agreement between the FasciMol-ELISA and the snail dissection methods with the multiplex PCR, respectively. RESULTS: Galba cubensis was found in nine out of 12 sampled localities of which four were positive for F. hepatica infection as detected by both immunoenzymatic and PCR-based assays. The overall prevalence was higher than the natural infection rates previously reported for Cuban G. cubensis (range from 4.1 to 7.42% depending on the screening method). No significant differences were found between FasciMol-ELISA and multiplex PCR when determining parasite positivity (Χ(2) = 6.283; P = 0.0981) whereas an excellent agreement was also noted (Kappa = 0.8224). CONCLUSIONS: Our results demonstrate the importance of malacological surveys in assessing parasite transmission risk and constitute an alert on the need of accurate measures to control fasciolosis in western Cuba. The sensitivity and specificity of the FasciMol-ELISA as well as its time-saving capacity and the easy of performing the determination of a large number of samples, point at this assay as a novel tool suitable for large-scale monitoring of natural snails populations. To our knowledge, this is the first study that explores natural infection by F. hepatica in field-occurring lymnaeid snails using an immunoenzymatic assay.

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Natural infections of lymnaeid snails by Fasciola hepatica are of primary importance to study transmission. Also, infected snails in the field can be used to explore the existing compatibility in host-parasite interactions. This paper aimed to describe the infection rate of Galba cubensis populations in fasciolosis transmission areas. Eight sites were sampled in western Cuba and 24 infected snails at six sites were found. The mean prevalence was 2.94% and the maximum value was 11.4%. The intensity of parasite infection was assessed as the number of rediae inside a single snail. High variation within the sites examined was observed, but a maximum of 76 rediae was recovered from one individual. Although the presence of two other trematode families (Schistosomatidae and Paramphistomatidae) was discovered in dissected individuals, no co-infection with F. hepatica was observed. This is the first time a study of natural prevalence of F. hepatica infection is carried out in Cuba, considered a hyper endemic country for bovine fasciolosis. Our results suggest that fasciolosis transmission may occur even when the number of infected snails remains relatively low.

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Fasciolosis is a snail-borne trematode infection that has re-emerged as a human disease, and is considered a significant problem for veterinary medicine worldwide. The evaluation of the transmission risk of fasciolosis as well as the efficacy of the strategies for its control could be carried out through epidemiological surveillance of the snails that act as intermediate hosts of the parasites. The present study aimed to develop the first multiplex PCR to detect Fasciola hepatica in Galba cubensis, an important intermediate host of the parasite in the Americas and especially in the Caribbean basin. The multiplex PCR was optimized for the amplification of a 340 bp fragment of the second internal transcribed spacer (ITS-2) of F. hepatica rDNA, while another set of primers was designed and used to amplify a conserved segment of the nuclear 18S rDNA of the snail (451 bp), as an internal control of the reaction. The assay was able to detect up to 100 pg of the parasite even at high concentrations of snail DNA, an analytical sensitivity that allows the detection of less than a single miracidium, which is the minimal biological infestation unit. A controlled laboratory-reared G. cubensis - F. hepatica system was used for the evaluation of the developed multiplex PCR, and 100% sensitivity and specificity was achieved. This assay constitutes a novel, useful and suitable technique for the survey of fasciolosis transmission through one of the main intermediate hosts in the Western hemisphere.

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