Assessing the microbiota of the snail intermediate host of trematodes, Galba truncatula.

BACKGROUND: The microbiome is known to play key roles in health and disease, including host susceptibility to parasite infections. The freshwater snail Galba truncatula is the intermediate host for many trematode species, including the liver and rumen flukes Fasciola hepatica and Calicophoron daubneyi, respectively. The snail-parasite system has previously been investigated. However, the specific interaction between the snail-associated microbiota and intra-snail developmental stages of trematodes has yet to be explored. METHODS: Galba truncatula snails were collected from farms in Northern Ireland and trematode infection was diagnosed using PCR. High-throughput sequencing analysis of the bacterial 16S ribosomal DNA V3-V4 hypervariable regions was subsequently applied to characterise the microbiota of both uninfected and infected snails. RESULTS: We first showed that the snail harboured microbiota that was distinct for its environment. The microbiota of infected snails was found to differ significantly from that of uninfected snails. In particular, the bacterial genera Mycoplasma and Methylotenera were significantly more abundant in infected snails, while genera Sphingomonas and Nocardioides were predominantly associated with uninfected snails. CONCLUSION: These findings pave the way to future studies on the functional roles of bacteria in host-parasite relationships.

Infection Rates of Fasciola Intermediate Host Snail Species and Their Distribution in Africa: A Systematic Review and Meta-Analysis.

This systematic review and meta-analysis aimed to collate the infection rates of Fasciola spp. in intermediate host snails and their distribution in Africa. The overall infectivity prevalences of Galba truncatula, Radix natalensis, and Pseudosuccinea columella are 52%, 8%, and 3%, respectively. The intermediate host snails native to Africa (R. natalensis and G. truncatula) have been examined more than the invasive P. columella. The studies included in the review ranged from 1999 to 2022. North Africa has the highest prevalence of G. truncatula, with an infection rate of 52%. The review reveals that naturally infected intermediate host snails (G. truncatula, R. natalensis, and P. columella) are found in various regions of Africa. G. truncatula accounts for 22% (from three countries) of the studies included in the review and it was only found in the North African region with the highest overall infection rate of 52%. More studies on infection rate and distribution are needed to effectively control and prevent future transmissions.

A proposed ectochory of Galba truncatula snails between wallow sites enhances transmission of Fascioloides magna at gemenc, in Hungary.

Each year temporary puddles are formed on compacted earth roads as a consequence of heavy rains and subsequent flooding in the large floodplain forests of the Danube River, Hungary, Central Europe, Hungary. After the receding of floodwaters, the muddy puddles persist from spring to mid-fall, where they are densely populated by Galba truncatula snails on an annual basis. These snails are the sole intermediate host of Fascioloides magna liver fluke of deer in the forest. According to field observations, G. truncatula is a very rare species on banks of river branches and lakes within the forest but always appears in large aggregations in these semi-natural potholes. Red deer (Cervus elaphus) leave their droppings in puddles while they wallow in the mud, therefore the snails encounter the miracidia of F. magna frequently. Snails are not able to dig themselves into the tamp soil of roads therefore they are destroyed by the wheels of vehicles which drive down the roads from autumn to the end of winter. Therefore, snails colonize these puddles regularly every spring. Since there is no connection between the distant puddles, it is supposed that deer and wild boars repeatedly introduce the snails into the puddles each year carrying snails with the help of mud, which is stuck on their fur while they wallow. This method of transport is supported by the fact that shell remnants of snails can be found on the bark of rubbing trunks situated nearby wallows. It seems that the sequential creation of wallow sites and the repeated introduction of G. truncatula enhance the likelihood for the trematode to infect both hosts.

The giant liver fluke in Europe: A review of Fascioloides magna within cervids and livestock with considerations on an expanding snail-fluke transmission risk.

The giant liver fluke, Fascioloides magna, common in North America and introduced into Europe in the 19th century, is an underappreciated model system for epidemiological studies on biological invasions, interactions with other native parasites and for health impact assessments within both definitive and intermediate snail host populations. Owing to its first contemporary appearance in Europe and then its subsequent spread, fascioloidosis has become a fluke-livestock/wildlife-snail combination of increasing interest for veterinarians, parasitologists and population geneticists. Here, we present a description of its recent epidemiology, biogeography and biology, inclusive of host species lists. Special emphasis is placed upon known definitive hosts of F. magna within Europe and considerations upon this fluke's local intermediate snail hosts. This helps us envisage plausible future epidemiological scenarios for further expansion across Europe, potentially even invasion into the UK. In line with others who draw attention upon needs for better systematic monitoring of putative risk-areas of fluke transmission, we close by highlighting why better surveillance of F. magna across continental Europe, and neighbouring territories, is justified.

Changes in the Populations of Two Lymnaeidae and Their Infection by Fasciola hepatica and/or Calicophoron daubneyi over the Past 30 Years in Central France.

Field investigations were carried out during three periods (from 1976 to 1997, in 2013-2014, and in 2020-2021) on 39 cattle-raising farms on acidic soils to track changes in the populations of two Lymnaeidae (Galba truncatula and Omphiscola glabra) and their infection with Fasciola hepatica and/or Calicophoron daubneyi. Compared to the survey between 1976 and 1997 on these farms, there was a significant decrease in the number of the two lymnaeid populations and the size of the G. truncatula populations in both 2013-2014 and 2020-2021. This decline was significantly faster in the last nine years than it was before 2013. The area of habitats colonized by G. truncatula showed no significant variation over the years, while that of habitats with O. glabra significantly decreased in the period covered by the three surveys. The prevalence of F. hepatica infection in snails significantly decreased over the years, while C. daubneyi infection increased over time in both lymnaeid species. These changes are due to the use of triclabendazole to treat fasciolosis in ruminants since the 1990s, and are probably a consequence of the successive heatwaves that have occurred since 2018 in the region.

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.

Temporal dynamics of trematode intermediate snail host environmental DNA in small water body habitats.

Environmental DNA (eDNA) surveying has potential to become a powerful tool for sustainable parasite control. As trematode parasites require an intermediate snail host that is often aquatic or amphibious to fulfil their lifecycle, water-based eDNA analyses can be used to screen habitats for the presence of snail hosts and identify trematode infection risk areas. The aim of this study was to identify climatic and environmental factors associated with the detection of Galba truncatula eDNA. Fourteen potential G. truncatula habitats on two farms were surveyed over a 9-month period, with eDNA detected using a filter capture, extraction and PCR protocol with data analysed using a generalized estimation equation. The probability of detecting G. truncatula eDNA increased in habitats where snails were visually detected, as temperature increased, and as water pH decreased (P < 0.05). Rainfall was positively associated with eDNA detection in watercourse habitats on farm A, but negatively associated with eDNA detection in watercourse habitats on farm B (P < 0.001), which may be explained by differences in watercourse gradient. This study is the first to identify factors associated with trematode intermediate snail host eDNA detection. These factors should be considered in standardized protocols to evaluate the results of future eDNA surveys.

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.

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.

Endemic occurrence of Fasciola hepatica in an alpine ecosystem, Pyrenees, Northeastern Spain.

Fasciola hepatica is a liver parasite of ruminants whose distribution is determined by its intermediate host, the freshwater snail Galba truncatula. In Europe, F. hepatica is mostly associated with lowlands. Infection from sympatric domestic reservoirs is rarely reported in wild mountain ungulates. This study explores F. hepatica in a multi-host system in a European alpine area. Serum samples (n = 1,209) from Pyrenean chamois (Rupicapra p. pyrenaica), European mouflon (Ovis aries musimon), domestic sheep (Ovis aries) and domestic cattle (Bos taurus) were collected in the National Game Reserve of Freser-Setcases (NGRFS) in Catalonia, Northeastern Spain, from 2008 to 2019, and tested for antibodies against F. hepatica. During the same period, the livers of 214 chamois hunted in the NGRFS were inspected for F. hepatica and associated pathological changes. Finally, 907 freshwater snails were collected in summer 2016 between 1559 and 2,224 metres above sea level (asl) in the NGRFS, and F. hepatica DNA sought by PCR. Antibodies against F. hepatica were detected in all four species, with a higher prevalence in cattle and sheep than in chamois. Fasciola hepatica and hepatic lesions were concurrently observed in 13/214 of the chamois livers inspected (6.1%, CI95 2.9%-9.3%). Fasciola hepatica DNA was detected in one out of the 907 snails (0.1%, Cl95 0.1% - 0.3%; Ct value 33.3) and collected at 2054 m asl. Fasciola hepatica was consistently detected in a high mountain multi-host system, suggesting that its life cycle is completed and that it occurs endemically at the highest elevation reported in Europe. Transhumant livestock are the likely source in this alpine ecosystem, which according to rare occurrence of F. hepatica DNA in G. truncatula is still a suboptimal habitat for F. hepatica life cycle. Studying parasites at their highest distribution range can be useful to monitor climate change in seasonal mountain environments.

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.

Rapid detection of Galba truncatula in water sources on pasture-land using loop-mediated isothermal amplification for control of trematode infections.

BACKGROUND: Fascioliasis caused by the trematodes Fasciola hepatica and F. gigantica, is a global neglected zoonotic disease estimated to cost the livestock industry over €2.5 billion annually. Farm management measures and sustainable use of anthelmintics can, in principle, effectively control trematode infection in livestock and reduce the rate of developing anthelmintic resistance. Previously, we designed an environmental DNA (eDNA) assay to identify a common trematode intermediate host, the freshwater snail Galba truncatula, in water sources to measure specific trematode infection risk areas on pasture-land. To improve this procedure, we now report a loop-mediated isothermal amplification (LAMP) assay to identify G. truncatula eDNA. METHODS: A LAMP assay was designed and optimised (e.g. temperature, time duration and primer concentration) to identify G. truncatula DNA. The ability of the LAMP assay to target G. truncatula DNA was identified, and LAMP assay limit of detection was investigated in comparison to conventional PCR. In the field, 48 water samples were collected from stream, ditch and water pool habitats in four locations at two Aberystwyth University farms over a seven week period to investigate the applicability of the LAMP assay for use on eDNA samples, in comparison to conventional PCR. RESULTS: The LAMP assay delivered detectable results in 30 min at 63 °C. The assay discriminated between G. truncatula DNA and non-target DNA, presenting a level of DNA detection comparable to conventional PCR. No significant difference was found between the ability of the LAMP and PCR assay to identify G. truncatula eDNA in water samples. Kappa coefficient analysis revealed a moderate level of agreement between LAMP and PCR assays. CONCLUSIONS: This study demonstrated that the LAMP assay can detect G. truncatula eDNA in a simple and rapid manner. The LAMP assay may become a valuable tool to determine optimum pasture management for trematode parasite control.

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.

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.

Cryptic intermediate snail host of the liver fluke Fasciola hepatica in Africa.

BACKGROUND: Snails such as Galba truncatula are hosts for trematode flukes causing fascioliasis, a zoonosis that is a major public health problem. Galba truncatula has recently been shown to be a cryptic species complex. African populations of Galba spp. are not yet studied using molecular assessments and is imperative to do so and reconstruct the centre of origin of Galba and to understand when and by what means it may have colonized the highlands of Africa and to what extent humans might have been involved in that process. METHODS: Samples from all known sub-ranges throughout Africa and new samples from Europe and Asia were obtained. We used a combination of two mitochondrial (cox1 and 16S) and one nuclear (ITS2) markers and phylogenetic, divergence time estimates and phylogeographical methods to determine the identity and biogeographical affinities. We also reconstructed the colonization history including the likely mode of dispersal and tested for the presence of cryptic Galba species in Africa. RESULTS: Galba truncatula is restricted to the Palaearctic region of the continent, namely Morocco. All sub-Saharan populations proved to be a distinct species according to the phylogenetic analyses and genetic distance. We propose to use the existing name Galba mweruensis (Connolly, 1929) for this species which is morphologically indistinguishable from the other two species hitherto known to occur in northern Africa, i.e. G. truncatula and G. schirazensis. Sub-tropical Africa has been colonized only once in either the Pliocene and possibly Miocene. Diversification within G. mweruensis is dated to the Plio-Pleistocene and thus human-mediated dispersal can be ruled out for the initial colonization of the isolated mountain ranges. There are potentially even more cryptic species in high altitude areas of Africa as outlined by the distinctness of the population found at the top of Mt. Elgon, Uganda. CONCLUSIONS: From a novel genetic inspection of available African material, a hitherto neglected distinct species, G. mweruensis, now appears a major host of F. hepatica throughout sub-Saharan Africa. A closer examination of trematode parasites hosted by this species is needed in order to understand transmission patterns in highlands throughout eastern and southern Africa. We encourage future studies to inspect other high altitudes areas in Africa in light of parasites of either veterinary or medical importance.

Sub-lethal effects of dimethoate alone and in combination with cadmium on biochemical parameters in freshwater snail, Galba truncatula.

The objective of the present study was to evaluate the influence of the organophosphorus pesticide dimethoate and cadmium on biochemical parameters of a freshwater snail, Galba truncatula, in laboratory conditions. In 14 days, snails were exposed to 0, 100, 200, and 400 µg L-1 of dimethoate and 0.0 and 1000 µg L-1 of cadmium chloride. The results evidenced that dimethoate induces oxidative stress and alters biochemical parameters in freshwater snails. Cadmium also induced significant changes in biochemical parameters. The combination of the dimethoate and cadmium markedly increased the effects on G. truncatula. Dimethoate and cadmium mixture caused a decrease in Acetylcholinesterase (AChE), and Glucose-6-phosphate dehydrogenase (G6PDH) activities, glycogen, and total antioxidant capacity (TAN) levels, and increased aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), glutathione peroxidase (GPx), and catalase (CAT) activities, and malondialdehyde (MDA) level in exposed snails. The increased toxicity and bioaccumulation of cadmium after the exposure to the highest concentration of dimethoate indicates a synergistic effect leading to a reduced performance of the detoxification system in the snail. Dimethoate contributed to the bioconcentration of cadmium in snails and increased its toxic effects evidenced in biomarkers of oxidative stress and cell damage.

Clonal amplification of Fasciola hepatica in Galba truncatula: within and between isolate variation of triclabendazole-susceptible and -resistant clones.

BACKGROUND: Fasciola hepatica is of worldwide significance, impacting on the health, welfare and productivity of livestock and regarded by WHO as a re-emerging zoonosis. Triclabendazole (TCBZ), the drug of choice for controlling acute fasciolosis in livestock, is also the drug used to treat human infections. However TCBZ-resistance is now considered a major threat to the effective control of F. hepatica. It has yet to be demonstrated whether F. hepatica undergoes a genetic clonal expansion in the snail intermediate host, Galba truncatula, and to what extent amplification of genotypes within the snail facilitates accumulation of drug resistant parasites. Little is known about genotypic and phenotypic variation within and between F. hepatica isolates. RESULTS: Six clonal isolates of F. hepatica (3× triclabendazole-resistant, TCBZ-R and 3× triclabendazole-susceptible, TCBZ-S) were generated. Snails infected with one miracidium started to shed cercariae 42-56 days post-infection and shed repeatedly up to a maximum of 11 times. A maximum of 884 cercariae were shed by one clonally-infected snail (FhLivS1) at a single time point, with > 3000 clonal metacercariae shed over its lifetime. Following experimental infection all 12 sheep were FEC positive at the time of TCBZ treatment. Sheep infected with one of three putative TCBZ-S clones and treated with TCBZ had no parasites in the liver at post-mortem, whilst sheep each infected with putative TCBZ-R isolates had 35-165 adult fluke at post-mortem, despite TCBZ treatment. All six untreated control animals had between 15-127 parasites. A single multi-locus genotype was reported for every fluke from each of the six clonal isolates. Adult F. hepatica showed considerable variation in weight, ranging from 20-280 mg, with variation in weight evident within and amongst clonal isolates. CONCLUSIONS: A genetic clonal expansion occurs within G. truncatula, highlighting the potential for amplification of drug resistant genotypes of F. hepatica. Variation in the weight of parasites within and between clonal isolates and when comparing isolates that are either susceptible or resistant to TCBZ represent inherent variation in liver fluke and cannot be attributed to their resistance or susceptibility traits.

Detection of Galba truncatula, Fasciola hepatica and Calicophoron daubneyi environmental DNA within water sources on pasture land, a future tool for fluke control?

BACKGROUND: Increasing trematode prevalence and disease occurrence in livestock is a major concern. With the global spread of anthelmintic resistant trematodes, future control strategies must incorporate approaches focusing on avoidance of infection. The reliance of trematodes on intermediate snail hosts to successfully complete their life-cycle means livestock infections are linked to the availability of respective snail populations. By identifying intermediate snail host habitats, infection risk models may be strengthened whilst farmers may confidently apply pasture management strategies to disrupt the trematode life-cycle. However, accurately identifying and mapping these risk areas is challenging. METHODS: In this study, environmental DNA (eDNA) assays were designed to reveal Galba truncatula, Fasciola hepatica and Calicophoron daubneyi presence within water sources on pasture land. eDNA was captured using a filter-based protocol, with DNA extracted using the DNeasy® PowerSoil® kit and amplified via PCR. In total, 19 potential G. truncatula habitats were analysed on four farms grazed by livestock infected with both F. hepatica and C. daubneyi. RESULTS: Galba truncatula eDNA was identified in 10/10 habitats where the snail was detected by eye. Galba truncatula eDNA was also identified in four further habitats where the snail was not physically detected. Fasciola hepatica and C. daubneyi eDNA was also identified in 5/19 and 8/19 habitats, respectively. CONCLUSIONS: This study demonstrated that eDNA assays have the capabilities of detecting G. truncatula, F. hepatica and C. daubneyi DNA in the environment. Further assay development will be required for a field test capable of identifying and quantifying F. hepatica and C. daubneyi infection risk areas, to support future control strategies. An eDNA test would also be a powerful new tool for epidemiological investigations of parasite infections on farms.

A new method for laboratory rearing of Galba truncatula, the intermediate host of Fasciola hepatica.

In this study a relatively large and open top aquarium was designed, constructed and introduced as a suitable habitat for nutrition, growth and development as well as for egg laying and breeding of Galba truncatula under laboratory conditions. The soil and water used in the aquarium were collected from the locality in which the snails were collected. The aquarium was placed in a laboratory with temperature of 18-32 ºC and relative humidity of 22-37% respectively, according to the season. The artificial light was controlled by a light timer, giving 12 h of light and 12 h of darkness. The snails were fed with dried lettuce leaves, Cyperus alternifolius (aquatic plant), Spirulina (algae), Orthotrichum rupestre (moss) and cuttlebone (a supplementary source of calcium). Approximately five weeks after the start of study, there was evidence of reproduction and success in rearing of G. truncatula by the appearance of eggs and small snails (0.1-0.5 mm) in the aquarium. In conclusion, large scale laboratory rearing of G. truncatula is a feasible task. The method may be improved by balancing the temperature and relative humidity as well as by optimizing the soil type, the water quality and the type of food.

Effectiveness of Chaetogaster limnaei as a controller of Fasciola hepatica in experimental infections of Galba truncatula.

BACKGROUND: Chaetogaster limnaei is an annelid symbiotically associated with lymnaeid snails as Galba truncatula. This association is considered a preventive mechanism against trematode miracidia infection, including Fasciola hepatica. The objective of this study was to determine the effectiveness of Chaetogaster limnaei as a natural controller of Fasciola hepatica in laboratory conditions. PROCEDURES: Fascsola hepatica miracidia were inoculated in parallel into snails carrying Chaetogaster limnaei and snails without the annelid. The degree of infection was measured after 40 days of exposure. Furthermore, the number of annelids per snail was quantified, as well as the ability of Chaetogaster limnaei to devour miracidia at different times of exposure. RESULTS: An effective 70% natural control of Fasciola hepatica in Galba truncatula was observed. The carrying capacity of the snail was established to be of 10.6 ± 1 annelids. Chaetogaster limnaei is a predator of Fasciola hepatica devouring an average of 3.79 ± 0.21 miracidia. The results of these experiments have a potential value as a control measure against fascioliasis in the environment.