Evaluation of Loop-Mediated Isothermal Amplification (LAMP) in Urine Samples for the Diagnosis of Imported Schistosomiasis.

Migratory flows and international travel are triggering an increase in imported cases of schistosomiasis in non-endemic countries. The present study aims to evaluate the effectiveness of the LAMP technique on patients' urine samples for the diagnosis of imported schistosomiasis in a non-endemic area in comparison to a commercial immunochromatographic test and microscopic examination of feces and urine. A prospective observational study was conducted in sub-Saharan migrants attending the Tropical Medicine Unit, Almería, Spain. For schistosomiasis diagnosis, serum samples were tested using an immunochromatographic test (Schistosoma ICT IgG-IgM). Stool and urine samples were examined by microcopy. Urine samples were evaluated by combining three LAMP assays for the specific detection of Schistosoma mansoni, S. haematobium, and for the genus Schistosoma. To evaluate the diagnostic accuracy, a latent class analysis (LCA) was performed. In total, 115 patients were included (92.2% male; median age: 28.3 years). Of these, 21 patients (18.3%) were diagnosed with schistosomiasis confirmed by microscopy, with S. haematobium being the most frequent species identified (18/115; 15.7%). The Schistosoma ICT IgG-IgM test result was 100% positive and Schistosoma-LAMP was 61.9% positive, reaching as high as 72.2% for S. haematobium. The sensitivity and specificity estimated by LCA, respectively, were: 92% and 76% for Schistosoma ICT IgG-IgM, 68% and 44% for Schistosoma-LAMP, and 46% and 97% for microscopy. In conclusion, the Schistosoma-LAMP technique presented a higher sensitivity than microscopy for the diagnosis of imported urinary schistosomiasis, which could improve the diagnosis of active infection, both in referral centers and in centers with limited experience or scarce resources and infrastructure.

Fasciola Infection Unexpectedly Found During Cholecystectomy: Review on How to Avoid Increasing Surgery Interventions in Non-human Endemic Areas.

PURPOSE: Fascioliasis is caused by Fasciola hepatica of almost worldwide distribution and F. gigantica in wide regions of Asia and Africa. Their adult stage develops in the biliary canals and gallbladder. Infection follows an initial, 3-4 month long invasive, migratory or acute phase, and a several year-long biliary, chronic or obstructive phase. METHODS: The unexpected finding of a fasciolid inside the gallbladder during a cholecystectomy for obstructive lithiasis suspicion in a patient is reported from an area of Iran where human infection had been never reported before and studies on fascioliasis in livestock are absent. RESULTS: The fluke obtained was phenotypically classified as F. hepatica by morphometry and genotypically as F. gigantica by mtDNA cox1 fragment sequencing, although with F. hepatica scattered mutations in species-differing nucleotide positions. The clinical, radiological, and biological signs observed at the acute and chronic phases often lead to some misdiagnosis. Serological methods may be useful in cases of negative coprology. Diagnostic techniques with insufficient resolution leading to unnecessary invasive interventions are analyzed. The way to avoid unnecessary surgery is described, including analyses to be made, diagnostic tools to be used, and aspects to be considered. CONCLUSION: Reaching a correct diagnosis in the confusing presentations avoids procedure delays and unnecessary surgery. A correct drug treatment may be sufficient. Except in extreme pathological presentations, lesions decrease in number and size and finally disappear or calcify after a successful treatment. Finally, the need to increase awareness of physicians about fascioliasis is highlighted, mainly in non-human endemic areas.

Schistosoma haematobium infection and morbidity risk factors for pre-school age children in western Angola: A knowledge, attitudes and practices survey.

BACKGROUND: Urogenital schistosomiasis is one of the most prevalent parasitic diseases in sub-Saharan Africa. It is a poverty-related disease conditioned by behavioural practices. METHODS: Our objective is to evaluate the awareness, mindset and habits about urogenital schistosomiasis in the community of Cubal (Angola), as well as its association with infection and urinary tract morbidity in pre-school age children. A cross-sectional study of knowledge, attitudes and practices at home was conducted between February and May 2022 with 250 participants. RESULTS: Overall, 93.6% of those surveyed had some prior knowledge about schistosomiasis and, among all the symptoms associated with this disease, blood in the urine was the best known (54.4%). Nevertheless, 57.6% obtained a medium knowledge score. Regarding attitude, the majority of respondents had a high attitude score (79.2%) with 96.0% willing to participate in mass drug administration campaigns. Laundry in the river was the most common risk practice (61.2%) and 55.2% out of the total were classified with a low practice score. CONCLUSION: Low knowledge about symptoms and transmission by caregivers was the outstanding risk factor for infection in pre-school age children (OR = 16.93, 95%CI: 3.93-72.82), and lack of knowledge that avoiding entering the river prevents schistosomiasis was the main risk factor for morbidity in PSAC (OR = 8.14, 95%CI: 1.14-58.25).

Potential intermediate hosts of Angiostrongylus cantonensis in the European Mediterranean region (Mallorca, Spain).

Angiostrongylus cantonensis is the main etiological agent of eosinophilic meningitis, a disease that often leads to severe neurological manifestations in mammals and birds. In recent years, the prevalence of this zoonotic nematode has dramatically increased as it expands into new territories beyond its native range in Southeast Asia and the Pacific Basin.Its arrival to Europe, the last continent to be invaded, has raised many questions concerning the parasite's life cycle, particularly in the Mediterranean region, where it is now endemic. This study aims to collect evidence about intermediate hosts (snail and slug species) involved in the transmission of the A. cantonensis in Mallorca. We have conducted a systematic surveillance of gastropods within 2 km radius areas, surrounding the specific locations where infected hedgehogs were found. We employed a sequence-based detection approach that included a species-specific PCR amplification followed by DNA sequencing of the internal transcribed spacer I (ITS-1). This conservative approach was essential to avoid cross reactions with the numerous metastrongylid species known to be circulating in Mallorca. Overall, we collected and identified 398 gastropods belonging to 17 species, of which 11% tested positive for A. cantonensis. These gastropods were collected from touristic settlements to agricultural lands. Five gastropod species: Theba pisana, Cornu aspersum, Deroceras reticulatum, Milax gagates and Otala lactea, had been previously incriminated as A. cantonensis intermediate hosts, while 6 species: Papillifera papillaris, Cochlicella acuta, Eobania vermiculata, Ganula lanuginosa, Milax nigricans and Rumina decollata, are newly recorded hosts. The findings of this study have important epidemiological implications, and further measures are discussed to prevent neuroangiostrongylosis cases.

Molecular phylogeny of Psychodopygina (Diptera, Psychodidae) supporting morphological systematics of this group of vectors of New World tegumentary leishmaniasis.

New World sandflies are vectors of leishmaniasis, bartonellosis, and some arboviruses. A classification based on 88 morphological characters was proposed 27 years ago when the New World phlebotomines were organized into two tribes Hertigiini and Phlebotomini. The latter was structured into four subtribes (Brumptomyiina, Sergentomyiina, Lutzomyiina, and Psychodopygina) and 20 genera. The subtribe Psychodopygina, including most of the American vectors of tegumentary Leishmania comprises seven genera from which no molecular work has been produced to support this classification. Here, we carried out a molecular phylogeny based on combined sequences (1,334 bp) of two genes: partial 28S rDNA and mtDNA cytochrome b from 47 taxa belonging to the Psychodopygina. The Bayesian phylogenetic reconstruction agreed with the classification based on morphological characters, supporting the monophyly of the genera Psychodopygus and Psathyromyia, whereas Nyssomyia and Trichophoromyia seemed to be paraphyletic. The paraphylies of the two latter groups were exclusively caused by the doubtful position of the species Ny. richardwardi. Our molecular analysis provides additional support to adopt the morphologic classification of Psychodopygina.

Title: La phylogénie moléculaire des Psychodopygina (Diptera, Psychodidae) soutient la systématique morphologique de ce groupe de vecteurs de leishmaniose cutanée du Nouveau Monde. Abstract: Les phlébotomes du nouveau monde sont des vecteurs de leishmaniose, de bartonellose et de certains arbovirus. Une classification basée sur 88 caractères morphologiques a été proposée il y a 27 ans lorsque les phlébotomes du nouveau monde ont été organisés en deux tribus Hertigiini et Phlebotomini. Cette dernière était structurée en quatre sous-tribus (Brumptomyiina, Sergentomyiina, Lutzomyiina et Psychodopygina) et 20 genres. La sous-tribu des Psychodopygina, qui inclut la plupart des vecteurs américains de la leishmaniose cutanée, comprend sept genres mais aucun travail moléculaire n'a été produit pour soutenir cette classification. Dans cet article, nous avons réalisé une phylogénie moléculaire basée sur des séquences combinées (1334 pb) de deux gènes : ADNr 28S partiel et cytochrome b (ADNmt) chez 47 taxons appartenant aux Psychodopygina. La reconstruction phylogénique bayésienne est en accord avec la classification basée sur les caractères morphologiques, soutenant la monophylie des genres Psychodopygus et Psathyromyia, alors que Nyssomyia et Trichophoromyia semblent être paraphylétiques. Les paraphylies des deux derniers groupes sont exclusivement causées par la position douteuse de la seule espèce Ny. richardwardi. Notre analyse moléculaire fournit une raison supplémentaire pour adopter la classification morphologique des Psychodopygina.

Ecological Niche Modelling Approaches: Challenges and Applications in Vector-Borne Diseases.

Vector-borne diseases (VBDs) pose a major threat to human and animal health, with more than 80% of the global population being at risk of acquiring at least one major VBD. Being profoundly affected by the ongoing climate change and anthropogenic disturbances, modelling approaches become an essential tool to assess and compare multiple scenarios (past, present and future), and further the geographic risk of transmission of VBDs. Ecological niche modelling (ENM) is rapidly becoming the gold-standard method for this task. The purpose of this overview is to provide an insight of the use of ENM to assess the geographic risk of transmission of VBDs. We have summarised some fundamental concepts and common approaches to ENM of VBDS, and then focused with a critical view on a number of crucial issues which are often disregarded when modelling the niches of VBDs. Furthermore, we have briefly presented what we consider the most relevant uses of ENM when dealing with VBDs. Niche modelling of VBDs is far from being simple, and there is still a long way to improve. Therefore, this overview is expected to be a useful benchmark for niche modelling of VBDs in future research.

Fasciola hepatica juveniles interact with the host fibrinolytic system as a potential early-stage invasion mechanism.

BACKGROUND: The trematode Fasciola hepatica is the most widespread causative agent of fasciolosis, a parasitic disease that mainly affects humans and ruminants worldwide. During F. hepatica infection, newly excysted juveniles (FhNEJ) emerge in the duodenum of the mammalian host and migrate towards their definitive location, the intra-hepatic biliary ducts. Understanding how F. hepatica traverses the intestinal wall and migrates towards the liver is pivotal for the development of more successful strategies against fasciolosis. The central enzyme of the mammalian fibrinolytic system is plasmin, a serine protease whose functions are exploited by a number of parasite species owing to its broad spectrum of substrates, including components of tissue extracellular matrices. The aim of the present work is to understand whether FhNEJ co-opt the functions of their host fibrinolytic system as a mechanism to facilitate trans-intestinal migration. METHODOLOGY/PRINCIPAL FINDINGS: A tegument-enriched antigenic extract of FhNEJ (FhNEJ-Teg) was obtained in vitro, and its capability to bind the zymogen plasminogen (PLG) and enhance its conversion to the active protease, plasmin, were analyzed by a combination of enzyme-linked immunosorbent, chromogenic and immunofluorescence assays. Additionally, PLG-binding proteins in FhNEJ-Teg were identified by bidimensional electrophoresis coupled to mass spectrometry analysis, and the interactions were validated using FhNEJ recombinant proteins. CONCLUSIONS/SIGNIFICANCE: Our results show that FhNEJ-Teg contains proteins that bind PLG and stimulate its activation to plasmin, which could facilitate the traversal of the intestinal wall by FhNEJ and contribute to the successful establishment of the parasite within its mammalian host. Altogether, our findings contribute to a better understanding of host-parasite relationships during early fasciolosis and may be exploited from a pharmacological and/or immunological perspective for the development of treatment and control strategies against this global disease.

Geographical Influence on Morphometric Variability of Genetically "Pure" Schistosoma haematobium Eggs from Sub-Saharan Migrants in Spain.

Schistosome eggs play a key role in schistosomiasis diagnosis and research. The aim of this work is to morphogenetically study the eggs of Schistosoma haematobium found in sub-Saharan migrants present in Spain, analyzing their morphometric variation in relation to the geographical origin of the parasite (Mali, Mauritania and Senegal). Only eggs considered "pure" S. haematobium by genetic characterization (rDNA ITS-2 and mtDNA cox1) have been used. A total of 162 eggs obtained from 20 migrants from Mali, Mauritania and Senegal were included in the study. Analyses were made by the Computer Image Analysis System (CIAS). Following a previously standardized methodology, seventeen measurements were carried out on each egg. The morphometric analysis of the three morphotypes detected (round, elongated and spindle) and the biometric variations in relation to the country of origin of the parasite on the egg phenotype were carried out by canonical variate analysis. Mahalanobis distances, when all egg measurements were analyzed, showed differences between: (i) Mali-Mauritania, Mali-Senegal and Mauritania-Senegal in the round morphotype; (ii) Mali-Mauritania and Mauritania-Senegal in the elongated morphotype; and (iii) Mauritania-Senegal in the spindle morphotype. Mahalanobis distances, when spine variables were analyzed, showed differences between Mali-Senegal in the round morphotype. In conclusion, this is the first phenotypic study performed on individually genotyped "pure" S. haematobium eggs, allowing the assessment of the intraspecific morphological variations associated with the geographical origin of the schistosome eggs.

Paleobiogeographical origins of Fasciola hepatica and F. gigantica in light of new DNA sequence characteristics of F. nyanzae from hippopotamus.

Fascioliasis is a highly pathogenic disease affecting humans and livestock worldwide. It is caused by the liver flukes Fasciola hepatica transmitted by Galba/Fossaria lymnaeid snails in Europe, Asia, Africa, the Americas and Oceania, and F. gigantica transmitted by Radix lymnaeids in Africa and Asia. An evident founder effect appears in genetic studies as the consequence of their spread by human-guided movements of domestic ruminants, equines and Old World camelids in the post-domestication period from the beginning of the Neolithic. Establishing the geographical origins of fasciolid expansion is multidisciplinary crucial for disease assessment. Sequencing of selected nuclear ribosomal and mitochondrial DNA markers of F. nyanzae infecting hippopotamuses (Hippopotamus amphibius) in South Africa and their comparative analyses with F. hepatica and F. gigantica, and the two Fascioloides species, Fs. jacksoni from Asian elephants and Fs. magna from Holarctic cervids, allow to draw a tuned-up evolutionary scenario during the pre-domestication period. Close sequence similarities indicate a direct derivation of F. hepatica and F. gigantica from F. nyanzae by speciation after host capture phenomena. Phylogenetic reconstruction, genetic distances and divergence estimates fully fit fossil knowledge, past interconnecting bridges between continents, present fasciolid infection in the wild fauna, and lymnaeid distribution. The paleobiogeographical analyses suggest an origin for F. gigantica by transfer from primitive hippopotamuses to grazing bovid ancestors of Reduncinae, Bovinae and Alcelaphinae, by keeping the same vector Radix natalensis in warm lowlands of southeastern Africa in the mid-Miocene, around 13.5 mya. The origin of F. hepatica should have occurred after capture from primitive, less amphibious Hexaprotodon hippopotamuses to mid-sized ovicaprines as the wild bezoar Capra aegagrus and the wild mouflon Ovis gmelini, and from R. natalensis to Galba truncatula in cooler areas and mountainous foothills of Asian Near East in the latest Miocene to Early Pliocene, around 6.0 to 4.0 mya and perhaps shortly afterwards.

Research on Schistosomiasis in the Era of the COVID-19 Pandemic: A Bibliometric Analysis.

The objectives of this work are to check whether the COVID-19 pandemic affected the research on schistosomiasis, to provide an insight into the most productive countries and journals and the most cited publications, and to analyse any association between the total publications of countries and a set of socio-economic and demographic factors. Based on PRISMA methodology, we used the Scopus database to search for articles published between 1 January 2020 and 26 March 2022. VOSviewer was used to generate the co-authorship and the co-occurrence networks, and Spearman's rank correlation was applied to study associations. A total of 1988 articles were included in the study. Although we found that the year-wise distribution of publications suggests no impact on schistosomiasis research, many resources have been devoted to research on COVID-19, and the Global Schistosomiasis Alliance revealed the main activities for eradication of schistosomiasis had been affected. The most productive country was the United States of America. The articles were mainly published in PLoS Neglected Tropical Diseases. The most prolific funding institution was the National Natural Science Foundation of China. The total publications per country were significantly correlated with population, GERD, and researchers per million inhabitants, but not with GDP per capita and MPM.

Mating Interactions between Schistosoma bovis and S. mansoni and Compatibility of Their F1 Progeny with Biomphalaria glabrata and Bulinus truncatus.

Contrary to the majority of other Trematoda, Schistosoma species are gonochoric. Consequently, in endemic areas where several schistosome species overlap and can co-infect the same definitive host, there may be frequent opportunities for interspecific pairing. Our experimental study provides novel insight on the pairing behavior between Schistosoma bovis and S. mansoni in mixed infections in mice. We used six mate choice experiments to assess mating interactions between the two schistosome species. We show that mating between the two Schistosoma species is not random and that S. mansoni exhibits greater mate recognition compared to S. bovis. We also performed reciprocal crosses (male S. mansoni × female S. bovis) and (female S. mansoni × male S. bovis) that produce active swimming miracidia. These miracidia were genotyped by ITS2 sequencing and proposed for mollusc infection. Molecular analyses show that all the miracidia are parthenogenetically produced (i.e., their harbor the mother ITS2 genotype) and as a consequence can only infect the mollusc of the maternal species. Offspring produced by male S. mansoni × female S. bovis pairing can only infect Bulinus truncatus whereas offspring produced by female S. mansoni × male S. bovis can only infect Biomphalaria glabrata snails. Evolutionary and epidemiological consequences are discussed.

West Nile virus in Spain: Forecasting the geographical distribution of risky areas with an ecological niche modelling approach.

West Nile virus (WNV), a well-known emerging vector-borne arbovirus with a zoonotic life cycle, represents a threat to both public and animal health. Transmitted by ornithophilic mosquitoes, its transmission is difficult to predict and even more difficult to prevent. The massive and unprecedented number of human cases and equid outbreaks in Spain during 2020 interpellates for new approaches. For the first time, we present an integrate analysis from a niche perspective to provide an insight to the situation of West Nile disease (WND) in Spain. Our modelling approach benefits from the combined use of global occurrence records of outbreaks of WND in equids and of its two alleged main vectors in Spain, Culex pipiens and Cx. perexiguus. Maps of the climatic suitability for the presence of the two vectors species and for the circulation of WNV are provided. The main outcome of our study is a map delineating the areas under certain climatic risk of transmission. Our analyses indicate that the climatic risk of transmission of WND is medium in areas nearby the south Atlantic coastal area of the Cadiz Gulf and the Mediterranean coast, and high in southwestern Spain. The higher risk of transmission in the basins of the rivers Guadiana and Guadalquivir cannot be attributed exclusively to the local abundance of Cx. pipiens, but could be ascribed to the presence and abundance of Cx. perexiguus. Furthermore, this integrated analysis suggests that the WNV presents an ecological niche of its own, not fully overlapping the ones of its hosts or vector, and thus requiring particular environmental conditions to succeed in its infection cycle.

DNA Multi-Marker Genotyping and CIAS Morphometric Phenotyping of Fasciola gigantica-Sized Flukes from Ecuador, with an Analysis of the Radix Absence in the New World and the Evolutionary Lymnaeid Snail Vector Filter.

Fascioliasis is a disease caused by Fasciola hepatica worldwide transmitted by lymnaeid snails mainly of the Galba/Fossaria group and F. gigantica restricted to parts of Africa and Asia and transmitted by Radix lymnaeids. Concern has recently risen regarding the high pathogenicity and human infection capacity of F. gigantica. Abnormally big-sized fasciolids were found infecting sheep in Ecuador, the only South American country where F. gigantica has been reported. Their phenotypic comparison with F. hepatica infecting sheep from Peru, Bolivia and Spain, and F. gigantica from Egypt and Vietnam demonstrated the Ecuadorian fasciolids to have size-linked parameters of F. gigantica. Genotyping of these big-sized fasciolids by rDNA ITS-2 and ITS-1 and mtDNA cox1 and nad1 and their comparison with other countries proved the big-sized fasciolids to belong to F. hepatica. Neither heterozygotic ITS position differentiated the two species, and no introgressed fragments and heteroplasmic positions in mtDNA were found. The haplotype diversity indicates introductions mainly from other South American countries, Europe and North America. Big-sized fasciolids from Ecuador and USA are considered to be consequences of F.gigantica introductions by past livestock importations. The vector specificity filter due to Radix absence should act as driving force in the evolution in such lineages.

Fascioliasis in Llama, Lama glama, in Andean Endemic Areas: Experimental Transmission Capacity by the High Altitude Snail Vector Galba truncatula and Epidemiological Analysis of Its Reservoir Role.

South American camelids are definitive hosts of Fasciola hepatica. However, their capacity to participate in the transmission and epidemiology of fascioliasis has never been appropriately studied. Therefore, an F. hepatica isolate from Argentine llama is for the first time analyzed using Galba truncatula lymnaeids from Bolivia. Experimental follow-up studies included egg embryogenesis, miracidial infection of lymnaeid snails, intramolluscan larval development, cercarial production, chronobiology of cercarial shedding, vector survival to infection, and metacercarial infectivity of mammal host. Shorter prepatent and patent periods were leading to markedly lower cercarial production, shorter cercarial shedding, and a higher negative impact on snail survival. The usually low liver fluke prevalences and intensities and low daily fecal outputs indicate that llamas do not substantially contribute to fascioliasis transmission. The defecating behavior in dung piles far from freshwater collections prevents lymnaeid infection by eggs shed by this camelid. All results suggest the reservoir role of the llama to be negligible and, therefore, no priority within control measures in endemic areas. However, llamas may play a disease-spreading role if used as pack animals in rural areas. In the Northern Bolivian Altiplano human hyperendemic area, neither llamas nor alpacas should be considered for control measures within a One Health action.

Evidence of autochthonous transmission of urinary schistosomiasis in Almeria (southeast Spain): An outbreak analysis.

BACKGROUND: Schistosomiasis is endemic in 78 countries belonging to tropical and subtropical areas. However, autochthonous transmission of urogenital schistosomiasis was reported in Corsica (France) in 2013. We present evidence of autochthonous transmission of urogenital schistosomiasis in Almería (Spain) in 2003. METHODS: Description of the outbreak in farmers and subsequent epidemiological studies aimed at searching for Bulinus snails and their genotypic characteristics. RESULTS: The outbreak affected 4 farmers out of a group of 5 people who repeatedly bathed that summer in an irrigation pool in the area. Two of them presented macroscopic hematuria with bilharziomas, showing the presence of Schistosoma eggs in bladder biopsies. Two others were asymptomatic but the serology for schistosomiasis was positive. In 2015, the presence of the vector Bulinus truncatus was demonstrated in Almería in water collections of appropriate characteristics. DNA sequencing proving that local B. truncatus species were base-to-base identical to B. truncatus from Senegal. CONCLUSIONS: We present a new outbreak of autochthonous transmission of urogenital schistosomiasis in Europe. Although no new cases of autochthonous transmission have been reported, some other cases may have occurred at that time or later on and be unnoticed as many cases of schistosomiasis are asymptomatic or present mild and unspecific symptoms.

One Health initiative in the Bolivian Altiplano human fascioliasis hyperendemic area: Lymnaeid biology, population dynamics, microecology and climatic factor influences.

Fascioliasis is a freshwater snail-borne zoonotic disease. The Northern Bolivian Altiplano is a very high altitude endemic area where the highest human prevalences and intensities have been reported. Preventive chemotherapy by treatment campaigns is yearly applied. However, liver fluke infection of cattle, sheep, pigs and donkeys assures endemicity and consequent human infection and re-infection risks. A One Health action has therefore been implemented. Activity concerns lymnaeid vectors and environment diversity. Studies included growth, egg-laying and life span in laboratory-reared lymnaeids. Different habitat types and influencing factors were assessed. All populations proved to belong to Galba truncatula by rDNA sequencing. Analyses comprised physico-chemical characteristics and monthly follow-up of water temperature, pH and quantity, and lymnaeid abundance and density. Population dynamics in the transmission foci differed. Mean environmental temperature was lower than fluke development minimum temperature threshold, but water temperature was higher, except during winter. A two generations/year pattern appeared in permanent water habitats, and one generation/year pattern in habitats drying out for months. The multidisciplinary control measures can be extended from one part of the endemic area to another. These studies, made for the first time at very high altitude, constitute a baseline useful for fascioliasis control in other countries.

Aedes albopictus diversity and relationships in south-western Europe and Brazil by rDNA/mtDNA and phenotypic analyses: ITS-2, a useful marker for spread studies.

BACKGROUND: Aedes albopictus is a very invasive mosquito, which has recently colonized tropical and temperate regions worldwide. Of concern is its role in the spread of emerging or re-emerging mosquito-borne diseases. Ae. albopictus from south-western Europe and Brazil were studied to infer genetic and phenetic diversity at intra-individual, intra-population and inter-population levels, and to analyse its spread. METHODS: Genotyping was made by rDNA 5.8S-ITS-2 and mtDNA cox1 sequencing to assess haplotype and nucleotide diversity, genetic distances and phylogenetic networks. Male and female phenotyping included combined landmark-and outlined-based geometric morphometrics of wing size and shape. RESULTS: Specimens from seven populations from Spain, France and Brazil provided 12 cox1 and 162 5.8S-ITS-2 haplotypes, with great genetic variability difference between both markers (0.9% vs 31.2%). Five cox1 haplotypes were shared with other countries, mainly Italy, USA and China, but none was shared between Europe and Brazil. The 5.8S-ITS-2 showed 2-7 intra-individual (mean 4.7) and 16-34 intra-/inter-population haplotypes (24.7), including haplotypes shared between Spain, France and Brazil. A 4.3% of ITS-2 haplotypes were shared, mainly with Italy, USA and Thailand, evidencing worldwide spread and introductions from areas where recent outbreaks of Ae. albopictus-transmitted pathogens occurred. Wing size showed sex differences. Wing shape distinguished between Brazilian and European specimens. Both genetic and morphometric markers showed differences between insular Spain and continental Spain, France and Brazil. CONCLUSIONS: ITS-2 proves to be a useful marker to assess Ae. albopictus spread, providing pronouncedly more information than cox1, including intra-individual, intra-population and inter-population levels, furnishing a complete overview of the evolutionary exchanges followed by this mosquito. Wing morphometry proves to be a useful phenotyping marker, allowing to distinguish different populations at the level of both male and female specimens. Results indicate the need for periodic surveillance monitorings to verify that no Ae. albopictus with high virus transmission capacity is introduced into Europe.

Buffalo Infection by Fasciola gigantica Transmitted by Radix acuminata in Uttar Pradesh, India: A Molecular Tool to Improve Snail Vector Epidemiology Assessments and Control Surveillance.

PURPOSE: Fascioliasis is caused by Fasciola species transmitted by freshwater Lymnaeidae snails and infecting herbivorous mammals and humans worldwide. In southern Asia, fascioliasis is a problem in livestock from the Near East to Bangladesh, where recent human infection reports are worrying. In this region, Fasciola gigantica is transmitted by species of the Radix auricularia superspecies group. In the densely populated northern Indian state of Uttar Pradesh, livestock appears infected throughout. The economic importance of buffaloes highlights the need to control their very high infection rates. METHODS: In the Gorakhpur area, a molecular method based on the two specific primer sets of genomic DNA was applied to fasciolids from buffaloes slaughtered in local abattoirs and cercariae from R. acuminata snails from freshwater collections. RESULTS: PCR products and sequences demonstrated that the cercariae belonged to F. gigantica and that R. acuminata acts as vector for its transmission to buffaloes. The 72.0% rate found in one transmission focus appears to be the highest worldwide record of fasciolid infection in a lymnaeid population. Lymnaeid prevalences and burdens found close to human communities indicate a very high infection risk. CONCLUSION: This method is simple, fast and cheap because there is no need for sequencing, it differentiates between fasciolid species and between fasciolids and other trematodes infecting R. acuminata, facilitates epidemiological surveys, and is useful for surveillance to evaluate the efficiency of control measures. Within climate change predictions, future increases of rain events and floods suggest the need for control and surveillance efforts in this endemic area.

One Health initiative in the Bolivian Altiplano human fascioliasis hyperendemic area: Lymnaeid biology, population dynamics, microecology and climatic factor influences / Aplicação do conceito One Health na área hiperendêmica de fasciolíase humana do Altiplano Boliviano: biologia dos limneídeos, dinâmica populacional, microecologia e influência de fatores climáticos

Abstract Fascioliasis is a freshwater snail-borne zoonotic disease. The Northern Bolivian Altiplano is a very high altitude endemic area where the highest human prevalences and intensities have been reported. Preventive chemotherapy by treatment campaigns is yearly applied. However, liver fluke infection of cattle, sheep, pigs and donkeys assures endemicity and consequent human infection and re-infection risks. A One Health action has therefore been implemented. Activity concerns lymnaeid vectors and environment diversity. Studies included growth, egg-laying and life span in laboratory-reared lymnaeids. Different habitat types and influencing factors were assessed. All populations proved to belong to Galba truncatula by rDNA sequencing. Analyses comprised physico-chemical characteristics and monthly follow-up of water temperature, pH and quantity, and lymnaeid abundance and density. Population dynamics in the transmission foci differed. Mean environmental temperature was lower than fluke development minimum temperature threshold, but water temperature was higher, except during winter. A two generations/year pattern appeared in permanent water habitats, and one generation/year pattern in habitats drying out for months. The multidisciplinary control measures can be extended from one part of the endemic area to another. These studies, made for the first time at very high altitude, constitute a baseline useful for fascioliasis control in other countries.

Resumo A fasciolíase é uma doença zoonótica transmitida para os humanos por formas evolucionárias de Fasciola hepatica oriundas de limneídeos infectados. O Altiplano Boliviano Norte é uma área endêmica de altitude muito alta, onde foram relatadas as maiores prevalências e intensidades em infecções humanas. A quimioterapia preventiva por campanhas de tratamento é aplicada anualmente. No entanto, infecção por Fasciola hepatica em bovinos, ovelhas, suínos e asininos garante endemicidade e consequentes riscos de infecção e reinfecção humana. Portanto, ações norteadas no conceito "One Health" foram implementadas. As atividades envolveram os limneídeos e a avaliação da diversidade de ambientes. Os estudos incluíram o crescimento, postura de ovos e expectativa de vida de limneídeos criados em laboratório, bem como a avaliação da influência dos diferentes hábitats. Todas as populações foram identificadas como Galba truncatula por meio do sequenciamento de rDNA. As análises incluíram características físico-químicas e acompanhamento mensal da temperatura, pH, quantidade da água, abundância e densidade de limneídeos. A dinâmica populacional nos focos de transmissão diferiu. A temperatura ambiente média foi mais baixa do que o limite mínimo de temperatura do desenvolvimento do helminto, mas a temperatura da água foi mais alta, exceto durante o inverno. Um padrão de duas gerações/ano apareceu em hábitats com água permanente, enquanto um padrão de uma geração/ano foi observado em habitats que ficam secos durante meses. Os resultados permitem concluir que as medidas multidisciplinares de controle podem ser estendidas de uma parte da área endêmica para outra. Esses estudos, realizados pela primeira vez em altitudes muito elevadas, constituem uma base útil e extrapolável para o controle da fasciolíase.

Angiogenic response in an in vitro model of dog microvascular endothelial cells stimulated with antigenic extracts from Dirofilaria immitis adult worms.

BACKGROUND: Angiogenesis can occur under pathological conditions when stimuli such as inflammation, vascular obstruction or hypoxia exist. These stimuli are present in cardiopulmonary dirofilariosis (Dirofilaria immitis). The aim of this study was to analyze the capacity of D. immitis antigens to modify the expression of angiogenic factors and trigger the formation of pseudocapillaries (tube-like structures) in an in vitro model of endothelial cells. METHODS: The expression of VEGF-A, sFlt, mEndoglin and sEndoglin in cultures of canine microvascular endothelial cells stimulated with extract of adult worms of D. immitis obtained from an untreated dog (DiSA) and from a dog treated for 15 days with doxycycline (tDiSA), was determined by using commercial kits. The capacity of pseudocapillary formation was evaluated analyzing cell connections and cell groups in Matrigel cell cultures stimulated with DiSA and tDiSA. In both cases non-stimulated cultures were used as controls. RESULTS: First, we demonstrated that worms obtained from the dog treated with doxycycline showed a significantly lower amount of Wolbachia (less than 60%) than worms removed from the untreated dog. Only DiSA was able to significantly increase the expression of the proangiogenic factor VEGF-A in the endotelial cells cultures. None of the D. immitis extracts modified the expression of sFlt. tDiSA extract was able to modify the expression of the endoglins, significantly decreasing the expression of the pro-angiogenic mEndoglin and increasing the anti-angiogenic sEndoglin. The formation of pseudocapillaries was negatively influenced by tDiSA, which reduced the organization and number of cellular connections. CONCLUSIONS: The ability of antigens from adult D. immitis worms to modify the expression of pro and anti-angiogenic factors in endotelial cell cultures was demonstrated, as well as the trend to form pseudocapillaries in vitro. The capacity of stimulation may be linked to the amount of Wolbachia present in the antigenic extracts.