Evidence of population structuring following population genetic analyses of Fasciola hepatica from Argentina.

Fasciola hepatica, the liver fluke, is a trematode parasite that causes disease of economic importance in livestock. As a zoonosis this parasite also poses a risk to human health in areas where it is endemic. Population genetic studies can reveal the mechanisms responsible for genetic structuring (non-panmixia) within parasite populations and provide valuable insights into population dynamics, which in turn enables theoretical predictions of evolutionary dynamics such as the evolution of drug resistance. Here we genotyped 320 F. hepatica collected from 14 definitive hosts from four provinces in Argentina. STRUCTURE analysis indicated three population clusters, and principal coordinate analysis confirmed this, showing population clustering across provinces. Similarly, pairwise FST values amongst all four provinces were significant, with standardised pairwise FST (F'ST) ranging from 0.0754 to 0.6327. Therefore, population genetic structure was evident across these four provinces in Argentina. However, there was no evidence of deviation from Hardy-Weinberg equilibrium, so it appears that within these sub-populations there is largely random mating. We identified 263 unique genotypes, which gave a clonal diversity of 82%. Parasites with identical genotypes, clones, accounted for 26.6% of the parasites studied and were found in 12 of the 14 hosts studied, suggesting some clonemate transmission.

Use of the PCR in a Combined Methodological Approach for the Study of Human Fascioliasis in an Endemic Area.

PURPOSE: Fascioliasis is a worldwide distributed trematodiasis considered a neglected disease. Diagnosis in humans has been traditionally based on parasitological and immunological techniques. Recently we reported the use of the PCR in stool samples for the individual diagnosis. The purpose of this study was to evaluate human fascioliasis by a combination of diagnostic methods in an area where the disease is highly endemic in animals. METHODS: We studied all the inhabitants (N = 240) of Tatón village, Argentina, by Fasciola hepatica rproCL1-ELISA. Among them, we continued the study with 13 cases that had at least two positive serological tests, who performed a questionnaire, physical examination, abdominal ultrasonography, and collection of blood and faeces. Blood/serum samples were used for Fh rproCL1-ELISA and liver function tests. Faeces were used for parasitological analysis and PCR of a repetitive fragment of Fasciola sp. RESULTS: Among the 13 patients, 9 presented symptoms of biliary colic. All patients repeated positive serology. F. hepatica eggs were not detected. PCR was positive in 11 cases. CONCLUSION: This is the first report employing an approach based on the combination of methods for the evaluation of human fascioliasis in an endemic area, which includes molecular tools with a high value in detecting low infections.

First genetic characterization of Fasciola hepatica in Argentina by nuclear and mitochondrial gene markers.

Fasciola hepatica is a trematode showing genetic variation among isolates from different regions of the world. The objective of this work was to characterize for the first time F. hepatica isolates circulating in different regions of Argentina. Twenty-two adult flukes were collected from naturally infected bovine livers in different areas from Argentina and used for DNA extraction. We carried out PCR amplification and sequence analysis of the ribosomal internal transcribed spacer 1 (ITS1), mitochondrial nicotinamide adenine dinucleotide dehydrogenase subunits 4 and 5 (nad4 and nad5) and mitochondrial cytochrome c oxidase subunit I (cox1) genes as genetic markers. Phylogenies were reconstructed using maximum parsimony algorithm. A total of 6 haplotypes were found for cox1, 4 haplotypes for nad4 and 3 haplotypes for nad5. The sequenced ITS1 fragment was identical in all samples. The analyzed cox1 gene fragment is the most variable marker and is recommended for future analyses. No geographic association was found in the Argentinean samples.

Fasciola hepatica infection in humans: overcoming problems for the diagnosis.

Fascioliasis is a zoonosis actually considered as a foodborne trematode disease priority by the World Health Organization. Our study presents three cases of F. hepatica infection diagnosed by direct, indirect and/or imaging diagnostic techniques, showing the need of the combined use of them. In order to overcome some difficulties of the presently available methods we show for the first time the application of molecular tools to improve human fascioliasis diagnosis by employing a PCR protocol based on a repetitive element as target sequence. In conclusion, diagnosis of human fascioliasis has to be carried out by the combination of diagnostic techniques that allow the detection of infection in different disease phases, different epidemiological situations and known/new transmission patterns in the actual scenario.

Human fascioliasis endemic areas in Argentina: multigene characterisation of the lymnaeid vectors and climatic-environmental assessment of the transmission pattern.

BACKGROUND: In South America, fascioliasis stands out due to the human endemic areas in many countries. In Argentina, human endemic areas have recently been detected. Lymnaeid vectors were studied in two human endemic localities of Catamarca province: Locality A beside Taton and Rio Grande villages; Locality B close to Recreo town. METHODS: Lymnaeids were characterised by the complete sequences of rDNA ITS-2 and ITS-1 and fragments of the mtDNA 16S and cox1. Shell morphometry was studied with the aid of a computer image analysis system. Climate analyses were made by nearest neighbour interpolation from FAO data. Koeppen & Budyko climate classifications were used. De Martonne aridity index and Gorczynski continentality index were obtained. Lymnaeid distribution was assessed in environmental studies. RESULTS: DNA sequences demonstrated the presence of Lymnaea neotropica and L. viator in Locality A and of L. neotropica in Locality B. Two and four new haplotypes were found in L. neotropica and L. viator, respectively. For interspecific differentiation, ITS-1 and 16S showed the highest and lowest resolution, respectively. For intraspecific analyses, cox1 was the best marker and ITS-1 the worst. Shell intraspecific variability overlapped in both species, except maximum length which was greater in L. viator. The desertic-arid conditions surrounding Locality A, the semiaridity-aridity surrounding Locality B, and the very low yearly precipitation in both localities, are very different from the typical fascioliasis transmission foci. Lymnaeids are confined to lateral river side floodings and small man-made irrigation systems. Water availability only depends on the rivers flowing from neighbouring mountains. All disease transmission factors are concentrated in small areas where humans and animals go for water supply, vegetable cultures and livestock farming. CONCLUSIONS: The unusually high number of DNA haplotypes and the extreme climate unsuitable for F. hepatica and lymnaeid development, demonstrate that the transmission foci are isolated. Seasonal transmission may depend on the timely overlap of appropriate temperature and river water availability. Lymnaeids and F. hepatica have probably reached these localities by livestock introduction. DNA differences regarding other populations of L. neotropica and L. viator in Argentina suggest an introduction independent from the spreading movements which allowed these two lymnaeids to expand throughout the country.

Estudio epidemiológico de fasciolosis en el valle de Fiambalá, Catamarca

Con el objetivo de contribuir al conocimiento de la epidemiología de la fasciolosis en el Valle de Fiambalá, Catamarca, Argentina, se realizó un estudio descriptivo, prospectivo, con diseño no experimental transeccional, durante el período 008-2012. En 305 individuos con seguimiento de la cohorte en humanos se estudió la presencia de anticuerpos anti-rproCL1 de fasciola hepática (fh), eosinofilia, proteínas totales, ácidos billares, enzimas hepáticas y bilirrubina sérica. Se analizó la presencia de fasciola hepática en la materia fecal y utilizó el diagnóstico por imagen en los individuos estudiados. La interpretación de las manifestaciones clínicas (n=174), la detección de anticuerpos antifasciola por ensayo de inmunoadsorción ligado a enzimas (ELISA), las imágenes (n=57) y la aplicación de métodos de diagnóstico etiológico por microscopía en materiales de heces (n=96); mediante su utilización sistemática e integrada, permitieron realizar el diagnóstico de fasciolosis.

SUMMARY: In order to contriburte to the knowledge of fascioliasis epidemiology in the Valley of Fiambalá, Catamarca, Argentina, a prospective descriptive study with no experimental cross-sectional design was done during the period 2008-2012. Within 305 individuals with cohort tracing in human, the presence of antibodies anti-rproCL1 of fasciola hepatica, eosinophilia, total proteins, bile acids, live enzymes and serum blirubin was studies. The presence of faciola hepatica (fh) in the stool was analyzed and diagnostic imaging was used in individuals studied. The interpretation of clinical manifestations (n=174), the use of serological diagnosis techniques by immunosorbent assay for enzyme-linked (ELISA), images (n=57) and the application of methods of etiological diagnosis by microscopy in materials feces (n=96); through its systematic and integrated use, allowed the fascioliasis diagnosis.

Molecular diagnosis of natural fasciolosis by DNA detection in sheep faeces.

Fasciolosis is an important parasitic zoonosis considered the most important helminth infection of ruminants in tropical countries. The aim of this study was to develop a PCR assay for the sensitive and specific detection of F. hepatica in formalin preserved sheep faeces. A 405-bp fragment of the cytochrome c oxidase subunit 1 gene of F. hepatica was amplified from stool samples of infected sheep. The PCR assay showed a detection limit of 20 pg of F. hepatica DNA. No cross-reactions were observed with samples containing coccidian oocysts or gastrointestinal nematodes eggs. Our PCR technique showed to be effective for specific detection of F. hepatica infections in sheep.

Direct, immunological and molecular techniques for a fasciolosis survey in a rural area of San Luis, Argentina.

Fasciolosis is a zoonosis caused by the trematode Fasciola hepatica, prevalent in cattle, that is actually emerging as a cause of disease in humans. The goal of this work was to describe the characteristics of fasciolosis in arroyo El Juncal region, La Toma, San Luis province, Argentina. In order to get this objective, a transversal, quantitative study was carried out by a fieldwork that allowed the collection of data, human, animal, and environmental samples. The materials were processed by direct, immunological and/or molecular diagnostic techniques. According to the geographical characteristics and in presence of all the definitive and intermediate hosts, reservoirs, and sources of infection, it was possible to describe the persistence of fasciolosis in the area. The prevalence was 11.90 % in humans (by serology), 5.26 % in cattle (by coprological analysis) and 61.76 % in snails (by PCR). The situation that was found for this area indicates that any measure of intervention for the control of this zoonosis should be adopted by multidisciplinary teams.