DNA sequence characterisation and phylogeography of Lymnaea cousini and related species, vectors of fascioliasis in northern Andean countries, with description of L. meridensis n. sp. (Gastropoda: Lymnaeidae)

<p>Abstract</p> <p>Background</p> <p>Livestock fascioliasis is a problem throughout Ecuador, Colombia and Venezuela, mainly in Andean areas where the disease also appears to affect humans. Transmission patterns and epidemiological scenarios of liver fluke infection have shown to differ according to the lymnaeid vector snail species involved. These Andean countries present the vectors <it>Lymnaea cousini</it>, <it>L. bogotensis </it>and <it>L. ubaquensis</it>, unknown in the rest of Latin America. An exhaustive combined haplotype study of these species is performed by means of DNA sequencing of the nuclear ribosomal 18S RNA gene, ITS-2 and ITS-1, and mitochondrial DNA <it>cox</it>1 gene.</p> <p>Results</p> <p>The conserved 5.8S rDNA sequence corroborated that no pseudogenes are involved in the numerous non-microsatellite/minisatellite-related indels appearing between the ITS-2 and ITS-1 sequences when comparing different <it>L. cousini </it>- <it>L. bogotensis </it>populations. Sequence analyses and phylogenetic reconstruction methods including other lymnaeid vector species show that (i) <it>L. bogotensis </it>is a synonym of <it>L. cousini</it>, (ii) <it>L. ubaquensis </it>is a synonym of <it>Pseudosuccinea columella</it>, and (iii) populations of <it>L. cousini </it>hitherto known from Venezuelan highlands indeed belong to a new species for which the name <it>L. meridensis </it>n. sp. is proposed. This new species is described and a complete phenotypic differentiation provided.</p> <p>Conclusions</p> <p>ITS-2, ITS-1 and <it>cox</it>1 prove to be good markers for specimen classification and haplotype characterisation of these morphologically similar lymnaeids in endemic areas. Analysis of the 18S gene and phylogenetic reconstructions indicate that <it>L. cousini </it>and <it>L. meridensis </it>n. sp. cluster in an evolutionary line different from the one of <it>P. columella</it>, despite their external resemblance. This suggests an evolutionary phenotypic convergence related to similar environments and which has given rise to frequent specimen misclassification. Body size and phylogenetic relationships of <it>L. meridensis </it>n. sp. with well-known vectors as <it>Lymnaea cousini </it>and <it>P. columella</it>, as well as with <it>Galba</it>/<it>Fossaria </it>species, suggest that the new species may participate in disease transmission to both animals and humans in altitude areas during the yearly window in which temperatures are higher than the <it>F. hepatica </it>minimum development threshold. The involvement of <it>L. cousini </it>and <it>P. columella </it>in the transmission and geographical/altitudinal distribution of fascioliasis in these Andean countries is analysed.</p

DNA multigene characterization of Fasciola hepatica and Lymnaea neotropica and its fascioliasis transmission capacity in Uruguay, with historical correlation, human report review and infection risk analysis

Fascioliasis is a highly pathogenic zoonotic disease emerging in recent decades, in part due to the effects of climate and global changes. South America is the continent presenting more numerous human fascioliasis endemic areas and the highest Fasciola hepatica infection prevalences and intensities known in humans. These serious public health scenarios appear mainly linked to altitude areas in Andean countries, whereas lowland areas of non-Andean countries, such as Uruguay, only show sporadic human cases or outbreaks. To understand this difference, we characterized F. hepatica from cattle and horses and lymnaeids of Uruguay by sequencing of ribosomal DNA ITS-2 and ITS-1 spacers and mitochondrial DNA cox1, nad1 and 16S genes. Results indicate that vectors belong to Lymnaea neotropica instead of to Lymnaea viator, as always reported from Uruguay. Our correlation of fasciolid and lymnaeid haplotypes with historical data on the introduction and spread of livestock species into Uruguay allow to understand the molecular diversity detected. We study the life cycle and transmission features of F. hepatica by L. neotropica of Uruguay under standardized experimental conditions to enable a comparison with the transmission capacity of F. hepatica by Galba truncatula at very high altitude in Bolivia. Results demonstrate that although L. neotropica is a highly efficient vector in the lowlands, its transmission capacity is markedly lower than that of G. truncatula in the highlands. On this baseline, we review the human fascioliasis cases reported in Uruguay and analyze the present and future risk of human infection in front of future climate change estimations

Lymnaea schirazensis, an Overlooked Snail Distorting Fascioliasis Data: Genotype, Phenotype, Ecology, Worldwide Spread, Susceptibility, Applicability

BACKGROUND: Lymnaeid snails transmit medical and veterinary important trematodiases, mainly fascioliasis. Vector specificity of fasciolid parasites defines disease distribution and characteristics. Different lymnaeid species appear linked to different transmission and epidemiological patterns. Pronounced susceptibility differences to absolute resistance have been described among lymnaeid populations. When assessing disease characteristics in different endemic areas, unexpected results were obtained in studies on lymnaeid susceptibility to Fasciola. We undertook studies to understand this disease transmission heterogeneity. METHODOLOGY/PRINCIPAL FINDINGS: A ten-year study in Iran, Egypt, Spain, the Dominican Republic, Mexico, Venezuela, Ecuador and Peru, demonstrated that such heterogeneity is not due to susceptibility differences, but to a hitherto overlooked cryptic species, Lymnaea schirazensis, confused with the main vector Galba truncatula and/or other Galba/Fossaria vectors. Nuclear rDNA and mtDNA sequences and phylogenetic reconstruction highlighted an old evolutionary divergence from other Galba/Fossaria species, and a low intraspecific variability suggesting a recent spread from one geographical source. Morphometry, anatomy and egg cluster analyses allowed for phenotypic differentiation. Selfing, egg laying, and habitat characteristics indicated a migration capacity by passive transport. Studies showed that it is not a vector species (n = 8572 field collected, 20 populations): snail finding and penetration by F. hepatica miracidium occur but never lead to cercarial production (n = 338 experimentally infected). CONCLUSIONS/SIGNIFICANCE: This species has been distorting fasciolid specificity/susceptibility and fascioliasis geographical distribution data. Hence, a large body of literature on G. truncatula should be revised. Its existence has henceforth to be considered in research. Genetic data on livestock, archeology and history along the 10,000-year post-domestication period explain its wide spread from the Neolithic Fertile Crescent. It is an efficient biomarker for the follow-up of livestock movements, a crucial aspect in fascioliasis emergence. It offers an outstanding laboratory model for genetic studies on susceptibility/resistance in F. hepatica/lymnaeid interaction, a field of applied research with disease control perspectives

Wolbachia pipientis infections in populations of Aedes albopictus in the city of València (Spain): implications for mosquito control

[EN] Objective: The presence of Aedes albopictus, of high sanitary and social impact, was first reported in Valencia (Eastern Spain) in 2015. Innovative tools for its control include the use of the endosymbiotic bacterium Wolbachia pipientis. The release of mosquito males infected with the wPip strain, has proven very promising for large-scale Incompatible Insect Technique (IIT) applications. Before this strategy can be implemented in Valencia, it is important to know whether the natural local mosquito populations are Wolbachia-infected and, if so, identifying the infecting strains/supergroups, these being the objectives of the present work. Methods: Eggs were collected from the 19 districts of the València city between May and October 2019. A total of 50 lab-reared adult Ae. albopictus individuals were processed and analyzed for Wolbachia detection and molecular characterization. These actions took place within the framework of a collaboration established with the Department of Health and Consumer Affairs of the city council of Valencia. Fisher's exact test was used to detect the statistical significance of the differences between groups. Results: Our study revealed that 94% of the analyzed samples were naturally infected with Wolbachia. Both wAlbA and wAlbB supergroups were identified, with most samples (72% of the infected ones) carrying co-infections. Conclusions: These data provide the first characterization of the Wolbachia presence in natural populations of Ae. albopictus in the Mediterranean area of Spain. This information is relevant to evaluate the potential use of Wolbachia strains in order to achieve the suppression of the Asian tiger mosquito populations through massive release of artificially-infected males.[ES] Objetivo: La presencia de Aedes albopictus, de alto impacto sanitario y social, se informó por primera vez en Valencia en 2015. Las herramientas innovadoras para su control incluyen el uso de la bacteria endosimbiótica Wolbachia pipientis. La liberación de mosquitos machos infectados con la cepa wPip ha demostrado ser muy prometedora para aplicar la Técnica de Insectos Incompatibles (IIT) a gran escala. Antes de que esta estrategia pueda implementarse, es importante saber si las poblaciones locales de mosquitos silvestres están infectadas por Wolbachia y, de ser así, identificar las cepas/supergrupos infectantes, siendo estos los objetivos del presente trabajo. Metodos: Se recolectaron huevos de los diecinueve distritos de València entre mayo y octubre de 2019, y se mantuvieron en el laboratorio hasta llegar a adultos. Un total de cincuenta individuos adultos de Ae albopictus fueron procesados y analizados para detectar la presencia de Wolbachia y su caracterización molecular. Estas acciones se enmarcaron en la colaboración establecida con la Concejalía de Salud y Consumo del Ayuntamiento de València. La prueba exacta de Fisher fue utilizada para detectar la significación estadística de las diferencias entre grupos. Resultados: El 94% de las muestras analizadas estaban infectadas de forma natural con Wolbachia. Se identificaron los supergrupos wAlbA y wAlbB, y la mayoría de las muestras (72% de las infectadas) presentaban coinfecciones. Conclusiones: Los datos proporcionan la primera caracterización de la presencia de Wolbachia en poblaciones naturales de Ae. albopictus en el área mediterránea de España. Esta información es relevante para evaluar el potencial uso de cepas de Wolbachia de cara a la supresión de poblaciones de mosquito tigre asiático mediante la liberación masiva de machos infectados artificialmente.Este trabajo se realizó en el marco de la Acción AIM-COST CA17108 y ha sido parcialmente financiado mediante diferentes contratos específi cos por parte del Servicio de Sanidad y Consumo del Ajuntament de València.Peer reviewe

Fasciola hepatica reinfection potentiates a mixed Th1/Th2/Th17/Treg response and correlates with the clinical phenotypes of anemia

Background: Fascioliasis is a severe zoonotic disease of worldwide extension caused by liver flukes. In human fascioliasis hyperendemic areas, reinfection and chronicity are the norm and anemia is the main sign. Herein, the profile of the Th1/Th2/Th17/Treg expression levels is analyzed after reinfection, correlating them with their corresponding hematological biomarkers of morbidity. Methodology/Principal findings: The experimental design reproduces the usual reinfection/chronicity conditions in human fascioliasis endemic areas and included Fasciola hepatica primo-infected Wistar rats (PI) and rats reinfected at 8 weeks (R8), and at 12 weeks (R12), and negative control rats. In a cross-sectional study, the expression of the genes associated with Th1 (Ifng, Il12a, Il12b, Nos2), Th2 (Il4, Arg1), Treg (Foxp3, Il10, Tgfb, Ebi3), and Th17 (Il17) in the spleen and thymus was analyzed. After 20 weeks of primary infection, PI did not present significant changes in the expression of those genes when compared to non-infected rats (NI), but an increase of Il4, Arg1 and Ifng mRNA in the spleen was observed in R12, suggesting the existence of an active mixed Th1/Th2 systemic immune response in reinfection. Foxp3, Il10, Tgfb and Ebi3 levels increased in the spleen in R12 when compared to NI and PI, indicating that the Treg gene expression levels are potentiated in chronic phase reinfection. Il17 gene expression levels in R12 in the spleen increased when compared to NI, PI and R8. Gene expression levels of Il10 in the thymus increased when compared to NI and PI in R12. Ifng expression levels in the thymus increased in all reinfected rats, but not in PI. The clinical phenotype was determined by the fluke burden, the rat body weight and the hemogram. Multivariate mathematical models were built to describe the Th1/Th2/Th17/Treg expression levels and the clinical phenotype. In reinfection, two phenotypic patterns were detected: i) one which includes only increased splenic Ifng expression levels but no Treg expression, correlating with severe anemia; ii) another which includes increased splenic Ifng and Treg expression levels, correlating with a less severe anemia. Conclusions/Significance: In animals with established F. hepatica infection a huge increase in the immune response occurs, being a mixed Th2/Treg associated gene expression together with an expression of Ifng. Interestingly, a Th17 associated gene expression is also observed. Reinfection in the chronic phase is able to activate a mixed immune response (Th1/Th2/Th17/Treg) against F. hepatica but T and B proliferation to mitogens is strongly suppressed in all infected rats vs control in the advanced chronic phase independently of reinfection The systemic immune response is different in each group, suggesting that suppression is mediated by different mechanisms in each case. Immune suppression could be due to the parasite in PI and R8 rats and the induction of suppressive cells such as Treg in R12. This is the first study to provide fundamental insight into the immune profile in fascioliasis reinfection and its relation with the clinical phenotypes of anemia.Red de InvestigacioÂn Cooperativa en Enfermedades Tropicales (RICET, Instituto de Salud Carlos III RD16/0027/0023); Proyectos de InvestigacioÂn en Salud (Instituto de Salud Carlos III, MINECO, Madrid, Spain PI16/00520); No. SAF2006-09278 and No. SAF2010-20805 of the Ministry of Economy and Competitiveness, Madrid, Spain. MF is supported by the following funding sources: Ministerio Ciencia y Tecnologia (SAF2005-02220, SAF2007-61716 and SAF2010-18733)Peer Reviewe

Soft parts of <i>Lymnaea neotropica</i> infected with <i>Fasciola hepatica</i>, at the end of cercarial shedding.

<p><b>A.</b> Apical view showing extent of redial infection. <b>B.</b> Magnification showing massive presence of rediae.</p

Shell of <i>Lymnaea neotropica</i> used for the experimental study of the transmission of Uruguayan <i>Fasciola hepatica</i>.

<p><b>A.</b> Ventral view; <b>B.</b> Dorsal view.</p

Uruguayan fasciolid and lymnaeid materials used, hosts, localities and DNA haplotype information for each molecular marker.

<p>Uruguayan fasciolid and lymnaeid materials used, hosts, localities and DNA haplotype information for each molecular marker.</p

Livestock movement timeline in the early introduction and spread of <i>Fasciola hepatica</i> and lymnaeids in Uruguay.

<p>Maps of South America (<b>A</b>) and Uruguay (<b>B</b>) showing main livestock passageways during the early Spanish and Portuguese colonizations. The analysis concerns the time of the old Viceroyalty of Rio de la Plata, from Buenos Aires in the South and the "Banda Oriental" in the Southeast up to "Alto Peru" in the North. 1, 14, First introductions of pigs in 1541, horses in 1574, and goats in 1577; 2, 15, 16, First and second introduction of cattle derived from Corrientes population in 1611 and 1617; 3, 17, Introduction of cattle from Misiones by Jesuits at the beginning of 17th century; 4, 5, 18, 19, First introduction of sheep from Santa Fe in 1727 (4, 18) and subsequent large scale cattle introductions with "faeneros" from Asuncion, Corrientes and Santa Fe (4, 5, 18, 19); 6, Livestock route for silver transport from Potosi mines from mid 16th century; 7, Original route for introduced goats in 1611–1618; 8, 20, Introduction of sheep by the Portuguese in 1734–1735; 9, 21, Largest rustle of more than 400,000 cattle in 1705, from Vaqueria del Mar to Vaqueria de los Pinares, at the southern part of the Jesuit Misiones Orientales area (brownish area); 10, 22, Livestock spread at mid and end of 17th century; 11, Livestock route (Camino Real, Ruta del Viamont or Caminho do Viamão) for gold transport from Minas Geraes mines from 1690; 12, Interconnection livestock route (Ruta de las Misiones or Caminho das Missões); 13, Interconnection livestock route (Ruta de la Vaquería or Caminho da Vacaria); 23, Groups of Portuguese "bandeirantes" also using livestock; 24, Northward spread of livestock. Background for A from composed satellite map of South America orthographic projection by NASA (full resolution of 1,215 x 1,712 pixels; public domain) via Wikimedia Commons. Original S. Mas-Coma.</p