Wide variation of heterozygotic genotypes of recent fasciolid hybrids from livestock in Bangladesh assessed by rDNA internal transcribed spacer region sequencing and cloning.

Fascioliasis causes high economic losses in livestock and underlies public health problems in rural areas, mainly of low-income countries. The increasing animal infection rates in Bangladesh were assessed, by focusing on host species, different parts of the country, and rDNA sequences. Fasciolid flukes were collected from buffaloes, cattle, goats and sheep from many localities to assess prevalences and intensities of infection. The nuclear rDNA internal transcribed spacer (ITS) region including ITS-1 and ITS-2 spacers was analyzed by direct sequencing and cloning, given the detection of intermediate phenotypic forms in Bangladesh. The 35.4% prevalence in goats and 55.5% in buffaloes are the highest recorded in these animals in Bangladesh. In cattle (29.3%) and sheep (26.8%) prevalences are also high for these species. These prevalences are very high when compared to lowlands at similar latitudes in neighboring India. The high prevalences and intensities appear in western Bangladesh where cross-border importation of animals from India occur. The combined haplotype CH3A of Fasciola gigantica widely found in all livestock species throughout Bangladesh fits its historical connections with the western Grand Trunk Road and the eastern Tea-Horse Road. The "pure" F. hepatica sequences only in clones from specimens showing heterozygotic positions indicate recent hybridization events with local "pure" F. gigantica, since concerted evolution did not yet have sufficient time to homogenize the rDNA operon. The detection of up to six different sequences coexisting in the cloned specimens evidences crossbreeding between hybrid parents, indicating repeated, superimposed and rapidly evolving hybridization events. The high proportion of hybrids highlights an increasing animal infection trend and human infection risk, and the need for control measures, mainly concerning goats in household farming management. ITS-1 and ITS-2 markers prove to be useful for detecting recent hybrid fasciolids. The introduction of a Fasciola species with imported livestock into a highly prevalent area of the other Fasciola species may lead to a high nucleotide variation in the species-differing positions in the extremely conserved fasciolid spacers. Results suggest that, in ancient times, frequent crossbreeding inside the same Fasciola species gave rise to the very peculiar characteristics of the present-day nuclear genome of both fasciolids.

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.

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.

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.

First morphogenetic analysis of parasite eggs from Schistosomiasis haematobium infected sub-Saharan migrants in Spain and proposal for a new standardised study methodology.

Schistosomiasis is a Neglected Tropical Disease caused by trematode species of the genus Schistosoma. Both, autochthonous and imported cases of urogenital schistosomiasis have been described in Europe. The present study focuses on eggs, considered pure S. haematobium by genetic characterisation (intergenic ITS region of the rDNA and cox1 mtDNA). A phenotypic characterisation of S. haematobium eggs was made by morphometric comparison with experimental populations of S. bovis and S. mansoni, to help in the diagnosis of S. haematobium populations infecting sub-Saharan migrants in Spain. Analyses were made by Computer Image Analysis System (CIAS) applied on the basis of new standardised measurements and geometric morphometric tools. The principal component analysis (PCA), including seventeen non-redundant measurements, showed three phenotypic patterns in eggs of S. haematobium, S. bovis and S. mansoni. PCA showed that the S. bovis population presented a large egg size range with a pronouncedly larger maximum size. Similarly, S. bovis shows bigger spine values than S. haematobium. Mahalanobis distances between each pair of groups were calculated for each discriminant analysis performed. In general, S. mansoni and S. bovis present larger distances between them than with S. haematobium, i.e. they present the greatest differences. Regarding the spine, S. haematobium and S. mansoni are the most distant species. Results show the usefulness of this methodology for the phenotypic differentiation between eggs from these Schistosoma species, capable of discerning morphologically close eggs, as is the case of the haematobium group. Schistosoma egg phenotyping approaches may be applied to assess not only hybrid forms but also potential influences of a variety of other factors.