Human fascioliasis emergence in southern Asia: Complete nuclear rDNA spacer and mtDNA gene sequences prove Indian patient infection related to fluke hybridization in northeastern India and Bangladesh.

Fascioliasis is a snail-borne zoonotic disease with impact on the development of human subjects and communities. It is caused by two liver-infecting fasciolid trematode species, the globally-distributed Fasciola hepatica and the Africa/Asia-restricted but more pathogenic, larger F. gigantica. Fasciola gigantica is the cause of endemicity in livestock throughout the warm lowlands from Pakistan to southeastern Asia since old times. Human fascioliasis is emerging in this region at present, with an increase of patient reports. Complete sequences of rDNA ITS-1 and ITS-2 spacers and mtDNA nad1 and cox1 genes were obtained from fasciolid eggs found in the endoscopic bile aspirate from a patient of Arunachal Pradesh, northeastern India. Egg measurements, pronounced ITS heterozygosity, and pure F. gigantica mtDNA haplotypes demonstrate an infection by a recent F. gigantica-like hybrid. Sequence identities and similarities with the same DNA markers found in livestock from Bangladesh prove the human-infecting fasciolid to present identical ITSs and nad1 haplotypes and only one silent transversion in cox1 when compared to a widely-spread combined haplotype in animals. In northeastern India and Bangladesh, human fascioliasis emergence appears linked to increasing livestock prevalences due to: ruminant importation from other countries because of the increasing demand of rapidly growing human populations; numerous livestock movements, including transborder corridors, due to the uncontrolled small-scale household farming practices; and man-made introduction of F. hepatica with imported livestock into an area originally endemic for F. gigantica leading to frequent hybridization. Sequences, phylogenetic trees, and networks indicate that the origins of intermediate/hybrid fasciolids and factors underlying human infection risk differ in eastern and western South Asia. The emergence scenario in southern China and Vietnam resembles the aforementioned of northeastern India and Bangladesh, whereas in Pakistan it is linked to increasing monsoon rainfall within climate change combined with an impact of an extensive irrigation system. Past human-guided movements of pack animals along the western Grand Trunk Road and the eastern Tea-Horse Road explain the F. gigantica mtDNA results obtained. Physicians should be aware about these emerging scenarios, clinical pictures, diagnostic techniques and treatment. Government authorities must appropriately warn health professionals, ensure drug availability and improve livestock control.

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.

One Health for fascioliasis control in human endemic areas.

Fasciola hepatica and F. gigantica are liver flukes causing fascioliasis, a worldwide zoonotic, complex disease. Human infection/reinfection occurs in endemic areas where preventive chemotherapy is applied, because of fasciolid transmission ensured by livestock and lymnaeid snail vectors. A One Health control action is the best complement to decrease infection risk. The multidisciplinary framework needs to focus on freshwater transmission foci and their environment, lymnaeids, mammal reservoirs, and inhabitant infection, ethnography and housing. Local epidemiological and transmission knowledge furnished by previous field and experimental research offers the baseline for control design. A One Health intervention should be adapted to the endemic area characteristics. Long-term control sustainability may be achieved by prioritizing measures according to impact depending on available funds.

Human and Animal Fascioliasis: Origins and Worldwide Evolving Scenario.

Fascioliasis is a plant- and waterborne zoonotic parasitic disease caused by two trematode species: (i) Fasciola hepatica in Europe, Asia, Africa, the Americas, and Oceania and (ii) F. gigantica, which is restricted to Africa and Asia. Fasciolid liver flukes infect mainly herbivores as ruminants, equids, and camelids but also omnivore mammals as humans and swine and are transmitted by freshwater Lymnaeidae snail vectors. Two phases may be distinguished in fasciolid evolution. The long predomestication period includes the F. gigantica origin in east-southern Africa around the mid-Miocene, the F. hepatica origin in the Near-Middle East of Asia around the latest Miocene to Early Pliocene, and their subsequent local spread. The short postdomestication period includes the worldwide spread by human-guided movements of animals in the last 12,000 years and the more recent transoceanic anthropogenic introductions of F. hepatica into the Americas and Oceania and of F. gigantica into several large islands of the Pacific with ships transporting livestock in the last 500 years. The routes and chronology of the spreading waves followed by both fasciolids into the five continents are redefined on the basis of recently generated knowledge of human-guided movements of domesticated hosts. No local, zonal, or regional situation showing disagreement with historical records was found, although in a few world zones the available knowledge is still insufficient. The anthropogenically accelerated evolution of fasciolids allows us to call them "peridomestic endoparasites." The multidisciplinary implications for crucial aspects of the disease should therefore lead the present baseline update to be taken into account in future research studies.

Variability of Calodium hepaticum eggs from sigmodontine host species through geometric morphometric analysis.

Calodium hepaticum is a zoonotic nematode with a worldwide distribution. Although the host range of C. hepaticum includes a wide spectrum of mammals (including humans), this parasite is predominantly associated with the families Muridae and Cricetidae. Several Sigmodontinae species from Argentina were found to be infected by C. hepaticum, with a high prevalence in Akodon azarae. The present study focuses on C. hepaticum eggs from natural infection of three species of sigmodontine rodents from Argentina. Eggs were genetically characterized (intergenic 18S rRNA region). The objectives of this work are: (i) to propose a new analytical methodology; and (ii) to morphologically characterize C. hepaticum eggs, from three Sigmodontinae species (A. azarae, Calomys callidus and Oligoryzomys flavescens). Analyses were made by the Computer Image Analysis System based on the new standardized measurements and geometric morphometric tools. The resulting factor maps clearly illustrate global size differences in the parasite eggs from the three Sigmodontinae species analysed. The degree of similarity between egg populations was assessed through pairwise Mahalanobis distances, showing that the largest distances were detected between parasite eggs from C. callidus and O. flavescens. Herein, the phenotypical plasticity of C. hepaticum eggs is shown. Significant positive correlations were obtained between each egg parasite principal component 1 and rodent corporal characteristics: weight; liver weight; rodent length; and rodent body condition. The usefulness of the geometric morphometric analysis in studies of the relationship between C. hepaticum and its host must be highlighted. The high prevalence observed in A. azarae, associated with the wide size range of the parasite eggs evidenced by principal component analysis, suggests A. azarae to be the Sigmodontinae host species that plays the most important role as reservoir host for C. hepaticum in the New World.

One Health Action against Human Fascioliasis in the Bolivian Altiplano: Food, Water, Housing, Behavioural Traditions, Social Aspects, and Livestock Management Linked to Disease Transmission and Infection Sources.

The Northern Bolivian Altiplano is the fascioliasis endemic area with the reported highest human prevalence and intensities. A multidisciplinary One Health initiative was implemented to decrease infection/reinfection rates detected by periodic monitoring between the ongoing yearly preventive chemotherapy campaigns. Within a One Health axis, the information obtained throughout 35 years of field work on transmission foci and affected rural schools and communities/villages is analysed. Aspects linked to human infection risk are quantified, including: (1) geographical extent of the endemic area, its dynamics, municipalities affected, and its high strategic importance; (2) human population at risk, community development and mortality rates, with emphasis on problems in infancy and gender; (3) characteristics of the freshwater collections inhabited by lymnaeid snail vectors and constituting transmission foci; (4) food infection sources, including population surveys with questionnaire and reference to the most risky edible plant species; (5) water infection sources; (6) household characteristics; (7) knowledge of the inhabitants on Fasciola hepatica and the disease; (8) behavioural, traditional, social, and religious aspects; (9) livestock management. This is the widest and deepest study of this kind ever performed. Results highlight prevention and control difficulties where inhabitants follow century-old behaviours, traditions, and beliefs. Intervention priorities are proposed and discussed.

Very High Fascioliasis Intensities in Schoolchildren from Nile Delta Governorates, Egypt: The Old World Highest Burdens Found in Lowlands.

Quantitative coprological analyses of children were performed in Alexandria and Behera governorates, Egypt, to ascertain whether individual intensities in the Nile Delta lowlands reach high levels as those known in hyperendemic highland areas of Latin America. Analyses focused on subjects presenting intensities higher than 400 eggs per gram of faeces (epg), the high burden cut-off according to WHO classification. A total of 96 children were found to shed between 408 and 2304 epg, with arithmetic and geometric means of 699.5 and 629.07 epg, respectively. Intensities found are the highest hitherto recorded in Egypt, and also in the whole Old World. A total of 38 (39.6%) were males and 58 (60.4%) were females, with high intensities according to gender following a negative binomial distribution. The high burden distribution shows a peak in the 7-10 year-old children group, more precocious in females than males. Results showed high burdens in winter to be remarkably higher than those known in summer. The fascioliasis scenario in Egyptian lowlands shows similarities to highlands of Bolivia and Peru. Diagnostic methods, pathogenicity and morbidity in high burdens should be considered. The need for an appropriate quantitative assessment of heavy infected children to avoid post-treatment colic episodes is highlighted.

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.

Spread of the fascioliasis endemic area assessed by seasonal follow-up of rDNA ITS-2 sequenced lymnaeid populations in Cajamarca, Peru.

Fascioliasis is a worldwide emerging snail-borne zoonotic trematodiasis with a great spreading capacity linked to animal and human movements, climate change, and anthropogenic modifications of freshwater environments. South America is the continent with more human endemic areas caused by Fasciola hepatica, mainly in high altitude areas of Andean regions. The Peruvian Cajamarca area presents the highest human prevalences reported, only lower than those in the Bolivian Altiplano. Sequencing of the complete rDNA ITS-2 allowed for the specific and haplotype classification of lymnaeid snails collected in seasonal field surveys along a transect including 2007-3473 m altitudes. The species Galba truncatula (one haplotype preferentially in higher altitudes) and Pseudosuccinea columella (one haplotype in an isolated population), and the non-transmitting species Lymnaea schirazensis (two haplotypes mainly in lower altitudes) were found. Climatic seasonality proved to influence G. truncatula populations in temporarily dried habitats, whereas L. schirazensis appeared to be more climatologically independent due to its extreme amphibious ecology. Along the southeastern transect from Cajamarca city, G. truncatula and L. schirazensis shared the same site in 7 localities (46.7% of the water collections studied). The detection of G. truncatula in 11 new foci (73.3%), predominantly in northern localities closer to the city, demonstrate that the Cajamarca transmission risk area is markedly wider than previously considered. Lymnaea schirazensis progressively increases its presence when moving away from the city. Results highlight the usefulness of lymnaeid surveys to assess borders of the endemic area and inner distribution of transmission foci. Similar lymnaeid surveys are still in need to be performed in the wide northern and western zones of the Cajamarca city. The coexistence of more than one lymnaeid transmitting species, together with a morphologically indistinguishable non-transmitting species and livestock movements inside the area, conform a complex scenario which poses difficulties for the needed One Health control intervention.

Domestic pig prioritized in one health action against fascioliasis in human endemic areas: Experimental assessment of transmission capacity and epidemiological evaluation of reservoir role.

The Northern Bolivian Altiplano is the human fascioliasis hyperendemic area where the highest prevalences and intensities in humans have been reported. Preventive chemotherapy was implemented in the last ten years. Surveillance showed high human infection and re-infection rates in between the annual triclabendazole monodose treatments. A complementary One Health control action was launched to decrease the infection risk. Among the multidisciplinary axes, there is the need to establish animal reservoir species priorities for a more efficient control. Laboratory and field studies were performed for the first time to assess the Fasciola hepatica transmission capacity of the pig and its potential reservoir role. The experimental follow-up of altiplanic pig isolates through altiplanic Galba truncatula snail vector isolates were performed at different miracidial doses and different day/night temperatures. Experiments included egg embryonation, miracidial infectivity, lymnaeid snail infection, intramolluscan larval development, cercarial production, chronobiology of the cercarial shedding, vector survival to infection, metacercarial infectivity of mammal host, and adult stage development. Surveys included the assessment of prevalence, intensity, egg measurements and egg shedding rates in nature. Pig contribution was evaluated by comparing with the main altiplanic reservoirs sheep and cattle. Results demonstrated that the pig assures the whole F. hepatica life cycle and participates in its transmission in this area. The fast egg embryonation, high cercarial production, long multi-wave shedding chronobiological pattern in monomiracidial infections at permanent 20 °C temperature, and the high daily egg outputs per pig are worth mentioning. The high infection risk suggests early infection of freely running piglets and evolutionary long-term adaptation of the liver fluke to this omnivorous mammal, despite its previously evoked resistance or non-suitability. Genetic, physiological and immune similarities with humans may also underlie the parasite adaptation to humans in this area. The pig should be accordingly included for appropriate control measures within a One Health action against human fascioliasis. The pig should henceforth be considered in epidemiological studies and control initiatives not only in fascioliasis endemic areas with human infection risk on other Andean countries, but also in rural areas of Latin America, Africa and Asia where domestic pigs are allowed to run freely.

Under pressure: phenotypic divergence and convergence associated with microhabitat adaptations in Triatominae.

BACKGROUND: Triatomine bugs, the vectors of Chagas disease, associate with vertebrate hosts in highly diverse ecotopes. It has been proposed that occupation of new microhabitats may trigger selection for distinct phenotypic variants in these blood-sucking bugs. Although understanding phenotypic variation is key to the study of adaptive evolution and central to phenotype-based taxonomy, the drivers of phenotypic change and diversity in triatomines remain poorly understood. METHODS/RESULTS: We combined a detailed phenotypic appraisal (including morphology and morphometrics) with mitochondrial cytb and nuclear ITS2 DNA sequence analyses to study Rhodnius ecuadoriensis populations from across the species' range. We found three major, naked-eye phenotypic variants. Southern-Andean bugs primarily from vertebrate-nest microhabitats (Ecuador/Peru) are typical, light-colored, small bugs with short heads/wings. Northern-Andean bugs from wet-forest palms (Ecuador) are dark, large bugs with long heads/wings. Finally, northern-lowland bugs primarily from dry-forest palms (Ecuador) are light-colored and medium-sized. Wing and (size-free) head shapes are similar across Ecuadorian populations, regardless of habitat or phenotype, but distinct in Peruvian bugs. Bayesian phylogenetic and multispecies-coalescent DNA sequence analyses strongly suggest that Ecuadorian and Peruvian populations are two independently evolving lineages, with little within-lineage phylogeographic structuring or differentiation. CONCLUSIONS: We report sharp naked-eye phenotypic divergence of genetically similar Ecuadorian R. ecuadoriensis (nest-dwelling southern-Andean vs palm-dwelling northern bugs; and palm-dwelling Andean vs lowland), and sharp naked-eye phenotypic similarity of typical, yet genetically distinct, southern-Andean bugs primarily from vertebrate-nest (but not palm) microhabitats. This remarkable phenotypic diversity within a single nominal species likely stems from microhabitat adaptations possibly involving predator-driven selection (yielding substrate-matching camouflage coloration) and a shift from palm-crown to vertebrate-nest microhabitats (yielding smaller bodies and shorter and stouter heads). These findings shed new light on the origins of phenotypic diversity in triatomines, warn against excess reliance on phenotype-based triatomine-bug taxonomy, and confirm the Triatominae as an informative model system for the study of phenotypic change under ecological pressure .

Donkey Fascioliasis Within a One Health Control Action: Transmission Capacity, Field Epidemiology, and Reservoir Role in a Human Hyperendemic Area.

A One Health initiative has been implemented for fascioliasis control in a human hyperendemic area for the first time. The area selected for this multidisciplinary approach is the Northern Bolivian Altiplano, where the highest prevalences and intensities in humans have been reported. Within the strategic intervention axis of control activities concerning animal reservoirs, complete experimental studies, and field surveys have been performed to assess the fascioliasis transmission capacity and epidemiological role of the donkey for the first time. Laboratory studies with altiplanic donkey-infecting Fasciola hepatica and altiplanic Galba truncatula snail vector isolates demonstrate that the donkey assures the viability of the whole fasciolid life cycle. Several aspects indicate, however, that F. hepatica does not reach, in the donkey, the level of adaptation it shows in sheep and cattle in this high altitude hyperendemic area. This is illustrated by a few-day delay in egg embryonation, longer prepatent period despite similar miracidial infectivity and shorter patent period in the intramolluscan development, lower cercarial production per snail, different cercarial chronobiology, shorter snail survival after shedding end, shorter longevity of shedding snails, and lower metacercarial infectivity in Wistar rats. Thus, the role of the donkey in the disease transmission should be considered secondary. Field survey results proved that liver fluke prevalence and intensity in donkeys are similar to those of the main reservoirs sheep and cattle in this area. Fasciolid egg shedding by a donkey individual contributes to the environment contamination at a rate similar to sheep and cattle. In this endemic area, the pronounced lower number of donkeys when compared to sheep and cattle indicates that the epidemiological reservoir role of the donkey is also secondary. However, the donkey plays an important epidemiological role in the disease spread because of its use by Aymara inhabitants for good transport, movements, and travel from one locality/zone to another, a repercussion to be considered in the present geographical spread of fascioliasis in the Altiplano due to climate change. Donkey transport of parasite and vector, including movements inside the zone under control and potential introduction from outside that zone, poses a problem for the One Health initiative.

Sheep and Cattle Reservoirs in the Highest Human Fascioliasis Hyperendemic Area: Experimental Transmission Capacity, Field Epidemiology, and Control Within a One Health Initiative in Bolivia.

The Northern Bolivian Altiplano is the human fascioliasis hyperendemic area where the highest prevalences and intensities of infection by Fasciola hepatica in humans have been reported. Four animal species are the reservoir species for F. hepatica in this area, namely, sheep, cattle, pigs, and donkeys. Livestock for the Aymara inhabitants is crucial because vegetable cultures are not viable due to the inhospitality of the very high altitude of 3,820-4,100 m. A One Health initiative has been implemented in this area in recent years, as the first such control action in a human endemic area ever. Among the different control axes included, special focus is devoted to the two main reservoirs sheep and cattle. Egg embryonation, miracidial infectivity, intramolluscan development, cercarial production, infected snail survival, and metacercarial infectivity were experimentally studied in altiplanic sheep and cattle isolates. These laboratory studies were performed using altiplanic isolates of the lymnaeid species Galba truncatula, the only vector present in the hyperendemic area. Experiments were made at constant 12 h day/12 h night and varying 20/20°C and 22/5°C photoperiods. Infections were implemented using mono-, bi-, and trimiracidial doses. Results demonstrate that sheep and cattle have the capacity to assure F. hepatica transmission in this very high-altitude area. Field surveys included prevalence studies by coprology on fecal samples from 1,202 sheep and 2,690 cattle collected from different zones of the Northern Bolivian Altiplano. Prevalences were pronouncedly higher and more homogeneous in sheep (63.1%; range: 38.9-68.5%) than in cattle (20.6%; range: 8.2-43.3%) in each one of the different zones. Although similarities between the prevalences in sheep and cattle appeared in the zones of the highest and lowest infection rates, this disappeared in the other zones due to cattle treatments. Comparison with past surveys demonstrates that this hyperendemic area is stable from the disease transmission point of view. Therefore, the control design should prioritize sheep and cattle within the One Health action. Studies performed in the Bolivian Altiplano furnish a baseline for future initiatives to assess the transmission and epidemiological characteristics of fascioliasis in the way for its control in other high altitude Andean endemic areas.

Equines as reservoirs of human fascioliasis: transmission capacity, epidemiology and pathogenicity in Fasciola hepatica-infected mules.

Fascioliasis is a zoonotic disease caused by liver flukes transmitted by freshwater lymnaeid snails. Donkey and horse reservoir roles have been highlighted in human endemic areas. Liver fluke infection in mules has received very limited research. Their role in disease transmission, epidemiological importance and Fasciola hepatica pathogenicity are studied for the first time. Prevalence was 39.5% in 81 mules from Aconcagua, and 24.4% in 127 from Uspallata, in high-altitude areas of Mendoza province, Argentina. A mean amount of 101,242 eggs/mule/day is estimated. Lymnaeids from Uspallata proved to belong to ribosomal DNA internal transcribed spacer (ITS) markers ITS-1 and ITS-2 combined haplotype 3C of Galba truncatula. These lymnaeids were experimentally susceptible to infection by egg miracidia from mules. Infectivity, number of cercariae/snail and shedding period fit the enhanced F. hepatica/G. truncatula transmission pattern at very high altitude. This indicates that the mule is able to maintain the F. hepatica cycle independently. Individual burdens of 20 and 97 flukes were found. Mule infection susceptibility is intermediate between donkey and horse, although closer to the latter. Anatomo-pathology and histopathology indicate that massive infection may cause mule death. Haematological value decreases of red blood cells, haemoglobin, leucocytes and lymphocytes indicate anaemia and strong immunosuppression. Strongly increased biochemical marker values indicate liver function alterations. The mule probably played a role in the past exchanges with Chile and Bolivia through Mendoza province. Evidence suggests that mules could contribute to the spread of both F. hepatica and G. truncatula to human fascioliasis-endemic areas in these countries.

Impact of fascioliasis reinfection on Fasciola hepatica egg shedding: relationship with the immune-regulatory response.

Fascioliasis is a disease caused by liver flukes. In human fascioliasis hyperendemic areas, reinfection and chronicity are the norm. Control strategies in humans require the use of egg count techniques to calculate the appropriate treatment dose for colic risk prevention. The present study investigates how fascioliasis reinfection affects liver fluke egg shedding and its relationship with the immune-regulatory response. The experimental design reproduced the usual reinfection/chronicity conditions in human fascioliasis endemic areas and included Fasciola hepatica primo-infected Wistar rats (PI) and rats reinfected at 4 weeks (R4), 8 weeks (R8), 12 weeks (R12), and negative control rats. In a longitudinal study (0-20 weeks post-infection, p.i.), serical IgG1 levels and eggs per gram of faeces (epg) were analyzed. 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. In R8 and R12, transiently higher averages of epg and epg/worm in reinfected groups vs PI group were detected at least in the weeks following reinfection. The kinetics of IgG1 levels shows that reinfected groups followed a pattern similar to the one in the PI group, but transiently higher averages of IgG1 levels in reinfected groups vs the PI group were detected in the weeks following reinfection. Epg correlated with IgG1 levels and also with systemic Il10 and thymic Ifng, and Il10 expression levels. These results suggest that epg depends on the Th1 and Treg phenotype and that the determination of the fluke burden by epg is likely to be an overestimation in cases of recent reinfection in low burden situations. A strategy to facilitate the implementation of epg count techniques and the subsequent decision on the appropriate treatment dose for each patient to prevent colic risk is required.

Genetic uniformity, geographical spread and anthropogenic habitat modifications of lymnaeid vectors found in a One Health initiative in the highest human fascioliasis hyperendemic of the Bolivian Altiplano.

BACKGROUND: Fascioliasis is a snail-borne zoonotic trematodiasis emerging due to climate changes, anthropogenic environment modifications, and livestock movements. Many areas where Fasciola hepatica is endemic in humans have been described in Latin America altitude areas. Highest prevalences and intensities were reported from four provinces of the northern Bolivian Altiplano, where preventive chemotherapy is ongoing. New strategies are now incorporated to decrease infection/re-infection risk, assessment of human infection sources to enable efficient prevention measures, and additionally a One Health initiative in a selected zone. Subsequent extension of these pilot interventions to the remaining Altiplano is key. METHODS: To verify reproducibility throughout, 133 specimens from 25 lymnaeid populations representative of the whole Altiplano, and 11 used for population dynamics studies, were analyzed by rDNA ITS2 and ITS1 and mtDNA cox1 and 16S sequencing to assess their classification, variability and geographical spread. RESULTS: Lymnaeid populations proved to belong to a monomorphic group, Galba truncatula. Only a single cox1 mutation was found in a local population. Two cox1 haplotypes were new. Comparisons of transmission foci data from the 1990's with those of 2018 demonstrated an endemic area expansion. Altitudinal, northward and southward expansions suggest movements of livestock transporting G. truncatula snails, with increasing temperatures transforming previously unsuitable habitats into suitable transmission areas. Transmission foci appear to be stable when compared to past field observations, except for those modified by human activities, including construction of new roads or control measures undertaken in relation to fascioliasis. CONCLUSIONS: For a One Health initiative, the control of only one Fasciola species and snail vector species simplifies efforts because of the lower transmission complexity. Vector monomorphism suggests uniformity of vector population responses after control measure implementation. Hyperendemic area outer boundary instability suggests a climate change impact. All populations outside previously known boundaries were close to villages, human dwellings and/or schools, and should therefore be considered during disease control planning. The remarkable southward expansion implies that a fifth province, Aroma, should now be included within preventive chemotherapy programmes. This study highlights the need for lymnaeid molecular identification, transmission foci stability monitoring, and potential vector spread assessment.

Lymnaeid Snail Vectors of Fascioliasis, Including the First Finding of Lymnaea neotropica in Ecuador, Assessed by Ribosomal DNA Sequencing in the Southern Zone Close to the Peru Border.

PURPOSE: Fascioliasis is a freshwater snail-borne zoonotic trematodiasis of high pathogenicity and wide veterinary repercussions. In South America, moreover, it causes serious public health problems, with high human infection rates in Andean countries. Ecuador offers a worrying risky scenario due to its physiography, including many human infection reports and animal endemicity throughout its Andean highlands. METHODS: Endemic areas with increasing animal fascioliasis were surveyed for lymnaeid snails in the province of Loja, southern Ecuador, close to the border of Peru, the country known to present the widest human fascioliasis endemic zone. The altitude of the sampling sites ranged between 150 and 1770 m a.s.l., and their location was close to human villages. Biotopes surveyed were characterized according to fascioliasis transmission needs. RESULTS: The species Lymnaea schirazensis and L. neotropica were identified by rDNA ITS-2 and ITS-1 sequencing. The non-transmitting L. schirazensis combined haplotype agreed with populations of this species previously reported from northern Ecuador. The finding of the efficient vector L. neotropica is reported for the first time in Ecuador and suggests a passive introduction from neighbouring Peru by uncontrolled livestock transport. CONCLUSIONS: Rice irrigation system implementation, lymnaeid finding on Taraxacum (dandelion) plants which are consumed fresh in salads by people, and Saccharum (sugarcane), whose bark is peeled off with the teeth, represent potential infection sources for humans. The closeness to the Cajamarca human hyperendemic area in northern Peru, where the same two lymnaeids have been also found and triclabendazole resistance reported, is an additional risk to be considered regarding the livestock transborder exchange.