WHO : Fascioliasis included it in the list of the great diseases of humanity

Fascioliasis is a parasitic disease caused by two species present in the liver: hepatic fasciola, which is prevalent worldwide, and Fasciola gigantica, which is found in Asia and Africa.

The high pathogenicity of this disease has led the World Health Organisation (WHO) to include it in the list of the great diseases of humanity.

The two Fasciola species cause varied clinical conditions, from cases without symptoms, to severe symptoms that can cause death.

Among the severe cases, there is a broad range of neurological conditions including paralysis of limbs, motor and speech disorders, loss of senses, seizures, epilepsy and coma.

Spain is the country with the second-highest number of cases of diagnosed neurological fascioliasis, after France.

In an article published in the journal Parasitology, María Adela Valero and María Dolores Bargues, professors at the Department of Pharmacy and Pharmacy Technology and Parasitology of the UV, along with professor Santiago Mas-Coma and his collaborators, show that Fasciola excretes and/or secretes a large number of proteins that induce the transformation of a plasma protein called plasminogen into the enzyme plasmin, which has the ability to decompose clots or thrombi.

This transformation of plasminogen into plasmin is part of the fibrinolytic and contact systems, whose end product is a strong proinflammatory peptide called bradykinin, which increases the vasodilation of arteries and veins, as well as vascular permeability.

This is how Fasciola ends up generating the bradykinin capable of opening the hematoencephalic barrier, a small impermeable layer of cells that acts as a protector of the brain.

This barrier is located in the capillaries that irrigate the brain and works as a filter between the blood and nervous systems, blocking a large number of substances from going from the blood to the brain.

The opening of the hematoencephalic barrier by bradykinin makes it possible for different Fasciola secretion/excretion products to access the brain, as well as other toxic substances derived from the pathogenic action of this parasite, with the resulting neurological effects.

Santiago Mas-Coma, WHO expert in tropical diseases and study director, says, “One of the main problems that these patients with neurological symptoms have is that the medical specialists who tend to these patients rarely think of fascioliasis as the source, due to the infrequent nature of these cases.

It has taken us many years of work, because nobody had any idea of the paths that a parasite present in the liver of patients could take to open the hematoencephalic barrier at a distance.

There were many varied hypotheses that different teams had considered, but they had not been confirmed.

Clarifying how the parasite causes these diseases and explaining all the clinical complexity and heterogeneity of these cases has undoubtedly been the most important challenge.”

María Adela says, “There are problems inherent in the necessary experimental part in order to obtain the fresh biological materials to appropriately extract and analyze the excretion/secretion proteins in a parasite of vectoral transmission by way of a freshwater snail, and the subsequent experimental infection of laboratory animals.

They are lengthy experiments where many aspects can go wrong. Fortunately, we had the expected success.”

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Fasciolid transmission

The two species F. hepatica and F. gigantica follow a similar two-host life cycle pattern. It takes about 14–23 weeks and comprises four phases (Mas-Coma and Bargues, 1997):

1. (A) The fluke adult stage infects the large biliary passages and gallbladder of the definitive host, both humans and animals, mainly livestock but also wild herbivores; eggs reach the external milieu by way of bile and intestine; the definitive host is infected by ingestion of metacercariae; metacercariae excyst in the small intestine within 1 h after ingestion, penetrate the host’s intestine wall, and appear in the abdominal cavity by about 2 h after ingestion; most reach the liver within 6 days after excystment; in the liver they migrate for 5–6 weeks, preferentially feeding directly on liver tissue; they eventually penetrate into the bile ducts where they become sexually mature; the prepatent period (from the ingestion of metacercariae to the first appearance of the first eggs in the feces) varies according to the host and also depends on the number of the adult flukes in the liver (Valero et al., 2006b); in man, a period of at least 3–4 months is necessary for the flukes to attain sexual maturity; this period is 1–2 weeks longer in F. gigantica (Valero et al., 2016).
2. (B) The transit between the definitive mammal host and intermediate snail host includes the long resistance phase of the egg and the short active phase of miracidium.
3. (C) At the intermediate host level, the development includes miracidium penetration into the snail, development of sporocyst and redial generations, production of cercariae and shedding of the latter into water.
4. (D) The transit between intermediate snail host and definitive mammal host includes the short swimming phase of cercaria and the long resistance phase of metacercaria until its ingestion by the definitive host; the shedding process takes place independently of light or darkness, between 9 and 26 °C in F. hepatica (at a somewhat higher temperature range in F. gigantica, whose minimum temperature threshold is 16 °C – Afshan et al., 2014); cercariae swim for a short time (1 h) until contacting a solid support, mostly leaves of water plants above or below the water line; they then lose their tails and quickly encyst (Fig. 1A, B), changing into round metacercariae (Fig. 1C) attached to the vegetation (Fig. 1E); floating infective metacercarial cysts (Fig. 1D) are also originated at the level of the water surface line (Fig. 1F) (Vareille-Morel et al., 1993); metacercarial cysts become infective within 24–72 h.Fig. 1. Life cycle stages of Fasciola hepatica involved in the infection of humans: (A) cercarial body beginning the encystment process; (B) cercarial tail after detachment from cercarial body; (C) metacercarial attached cyst; (D) metacercarial floating cyst; (E) metacercariae attached to a green plant leaf; (F) metacercariae floating in water. (Photographs S. Mas-Coma).

The cercarial shedding process seems to follow an infradaily shedding pattern of 7 days in the daily production during the whole emergence and a circadial rhythm with maximum production between midnight and 1.00 h a.m., as seen in the lymnaeid vector Galba truncatula infected by F. hepatica (Audousset et al., 1989). Higher cercarial productions following different shedding chronobiologies have been seen in the same lymnaeid at very high altitude and by other Galba/Fossaria species in the lowlands (Bargues et al., 2017).

Human infection risk

Epidemiological scenarios and transmission patterns

The risk of human infection depends on the fascioliasis transmission rate in the area in question and on its intra- and interannual rate variability linked to climatic factor variations. The marked heterogeneity of human fascioliasis regarding different epidemiological scenarios and transmission patterns throughout the world should be noted in this regard. It may be concluded that well-known situations and patterns of fascioliasis may not always explain the disease characteristics in a given area.

The transmission rate may be inferred from local prevalences and intensities in the inhabitants but also from livestock living in the area. Indeed, different epidemiological situations have been distinguished in human fascioliasis. The classification of epidemiological scenarios proposed by Mas-Coma et al. (1999a, 2009a) still appears to be fully valid and useful. This classification includes: (i) imported cases; (ii) authochthonous, isolated, non-constant cases; (iii) three different human endemic situations, comprising hypoendemic [prevalence of <1%; arithmetic mean intensity <50 eggs per gram (epg) of feces], mesoendemic (prevalence of between 1 and 10%; 5–15-year-old children may present higher prevalences; arithmetic mean intensity in human communities may reach 50 and 300 epg), and hyperendemic (prevalence of more than 10%; 5–15-year-old children usually present higher prevalences; arithmetic mean intensity in human communities may reach values higher than 300 epg); and (iv) two different human epidemic situations (epidemics in animal but non-human endemic areas, and epidemics in human endemic areas).

The intra- and interannual variability of the transmission rate is due to the marked dependence of fasciolid-free larval stages (aforementioned phases B and D) and intramolluscan larval stages (phase C) on the environmental characteristics (Ollerenshaw and Smith, 1969; Ross, 1970; Ollerenshaw, 1971; Fuentes et al., 1999), namely: surface water availability whether from rainfall or from any freshwater body (rivers, streams, lagoons, lakes, subsoil efflorescences, irrigation canals, fountains, etc.) to allow for the presence, development and population dynamics of the lymnaeid vectors (Mas-Coma et al., 1999b) and mainly air and water temperature as main factor. Minimum and maximum larval development temperature thresholds are different for F. hepatica and F. gigantica (Afshan et al., 2014).

Different transmission patterns have been distinguished within the various human endemic areas (Mas-Coma, 2005): (a) a very high altitude pattern related to only F. hepatica in Andean countries, which includes two subpatterns (the altiplanic pattern with transmission throughout the whole year, and the valley pattern, with seasonality and prevalences and intensities related to altitude); (b) a Caribbean insular pattern, with reduced but repeated outbreaks in human hypoendemic areas; (c) a pattern related to Afro-Mediterranean lowlands, including overlapping F. hepatica and F. gigantica, and where seasonality is typical; (d) a pattern occurring in areas surrounding the Caspian, including human hypoendemic areas in which large epidemics occur, occasionally involving up to 10 000 people and with overlapping of F. hepatica and F. gigantica; (e) a pattern recently detected in Vietnam, related to only/mainly F. gigantica, linked to lowland areas and able to give rise to large human epidemics (De et al., 2003; The and Nawa, 2005; Le et al., 2008); and (f) a new pattern very recently found in Argentina corresponding to isolated foci in desertic-arid and semi-arid conditions where transmission factors are concentrated and seasonal transmission depends on the timely overlap of appropriate temperature and river water availability (Bargues et al., 2016).

Seasonality

Seasonality is an important infection risk factor to take into account. Seasonal incidence is pronouncedly determined by climate factors, mainly temperature and rainfall. Human infection has been more frequently observed in the years with heavy rainfall (Ripert et al., 1988), and in, for instance, Western Europe, fascioliasis is referred to as markedly seasonal, with a high percentage (80.9%) of the cases showing the onset of the disease in the autumn months (Garcia-Rodriguez et al., 1985), although the relatively long survival of metacercariae (Valero and Mas-Coma, 2000) explain sporadic individual infections throughout other seasons of the year. Sometimes the seasonality is related to the transmitting plants, most human cases occurring during the watercress season. In Iran, past epidemics of human fascioliasis have been linked to the Ramadan; this period typically driving people to increase the consumption of vegetables.

Almost all fascioliasis endemic areas follow a seasonal transmission of the disease, which is nothing else than the translation of the lymnaeid vector population dynamics in the area in question and which in its turn depends on the local climatic characteristics. Lymnaeids have greatly differing ecological and ethological characteristics depending on the species. Factors such as the type of water collection habitats, lymnaeid population dynamics, different temperature thresholds of the different lymnaeid vector species and their local geographical strains, seasonality or susceptibility regarding liver fluke infection are crucially important for fascioliasis. All this indicates that similarly as known in other vector-borne parasitic diseases, lymnaeids may constitute excellent markers of the disease, useful for differentiating between the various human fascioliasis scenarios and patterns, and consequently also as determinants for the design of appropriate control strategies.

From a global perspective, three main types of transmission seasonality may be distinguished according primarily to latitude and secondarily altitude:

1. (A) Permanent, year-long transmission: this occurs in zones where mean monthly temperatures fluctuate scarcely and are kept within the minimum and maximim fasciolid larval stage development thresholds throughout the year; such a transmission appears in foci where lymnaeid vectors are adapted to permanent water bodies as observed in southern Europe (Valero et al., 1998) and Mediterranean islands (Oviedo et al., 1992); a similar situation appears in Cambodia (Tum et al., 2004, 2007); an effect of the very high altitude of around 4000 m a.s.l. is observed in Andean areas as the Northern Bolivian Altiplano where the high evapotranspiration leads lymnaeids to only inhabit permanent water bodies and daily temperatures counteract the negative effects of the low night temperatures (Fuentes et al., 1999; Mas-Coma et al., 1999b); a higher number of redial generations (up to four) is typical in such places (Mas-Coma et al., 2001).

Where seasonality occurs, temporary transmission is mainly related to lymnaeid vectors able to quickly multiply and colonize temporary water bodies from rainfall and to aestivate and hibernate during the non-appropriate periods.

1. (B) Monoseasonal transmission: this occurs in extreme latitudes, where only an appropriate temperature month window appears throughout the year, whether in usually northern or southern too cold areas as the Patagonia and mountainous areas, or in more tropical, too warm areas. This may be, for instance, the situation of warm areas in south-central Asia where the monsoon period concentrate the rainfall and the absence of irrigation systems does not allow for another yearly transmission period (Afshan et al., 2014).
2. (C) Biseasonal transmission: this is the typical situation in Europe, given areas of the USA and also Australia. This biseasonal model includes low transmission in spring and very high transmission in autumn, with highest animal incidences from August–September expanding even up to December–February (Ross, 1967, 1977; Urquhart et al., 1970; Over and Dijkstra, 1975; Meek and Morris, 1979; Shaka and Nansen, 1979; Craig Hoover et al., 1984; Mage, 1989a, 1989b). This biseasonality inverses in other areas where the highest incidence appears in the first half of the year (Harris and Charleston, 1976; Craig and Bell, 1978; Malone et al., 1984/85; Boyce and Courtney, 1990).

Interestingly, however, a wide global analysis of existing data shows that there is no marked seasonal incidence, human infections occurring nearly throughout the year (Chen and Mott, 1990). This means that metacercarial survival, viability and infectivity during several months if kept under appropriate conditions, mainly sufficient humidity, may qualitatively mask the human infection seasonal distribution due to the higher infectivity of the recent, young metacercariae.

Fascioliasis has been many times reported to be linked to man-made irrigation areas. Everywhere, but more usually in rural areas of developing countries, livestock is freely grazing in irrigated plant cultures and also in their neighbouring irrigation canals where lymnaeids are frequently found. The frequent presence of lymnaeids in rice fields in Northern Africa, Asia and Southern Europe is a good example (Valero et al., 1998). Thus, the different local traditions in the timely artificial floodings of the rice field managements from the irrigation systems modify the fascioliasis transmission seasonality.

In the Punjab, Pakistan, it has been recently proved that human and animal fascioliasis transmission is biseasonal (Qureshi et al., 2016), with one summer peak related to monsoons rainfall and another winter peak related to artificial irrigation (Afshan et al., 2014). In that endemic area, a great amount of water supplied by the Indus basin river system is used for irrigation by means of a very large irrigation system including dams, barrages and a canal network of 60 000 km constructed during British colonial years (Afshan et al., 2014). Such an immense irrigation system is used in great part for the cultivation of crops.

Community, familial and social factors in infection risk

Fascioliasis is predominantly a rural disease because human infection risk is in the field where the disease transmission occurs in freshwater bodies inhabited by the lymnaeid vectors. A thorough epidemiological study in the highest human hyperendemic area known, the Northern Bolivian Altiplano, proved that prevalence and intensity of infection in the communities show a direct correlation with, and are therefore dependent on, the distance of the village from the closest water collection inhabited by lymnaeid snail vectors (Mas-Coma et al., 1999b). In a developed country as France, human infection in 10 000 reports, happened during the 1956–1982 period, correlated well with the zones for cattle and sheep husbandry (Gaillet et al., 1983).

Human infection in urban settlements occurs only sporadically due to consumption, mainly at home but also very rarely at restaurants and hotels, of metacercariae-carrying vegetables acquired in an uncontrolled market to where they were transported from the field. This does however not exclude the possibility of urban inhabitants to become infected in field trips.

The infection distribution by sex appears to be very similar in several areas, as in Europe, although in human hyperendemic areas the females show higher infection rates, whether prevalences as in Egypt (Farag et al., 1979; Esteban et al., 2003), or intensities as in Bolivia (Esteban et al., 1997a, 1997b, 1999). Regarding age relationships, all age groups can be affected, although in human hyperendemic areas children appear to be the most infected (Esteban et al., 1999, 2003; Gonzalez et al., 2011; Zumaquero-Rios et al., 2013).

The incidence of infection is significantly aggregated within family groups because the family shares the same contaminated food and/or water, as it has been observed in for instance Spain (Gallardo et al., 1976; Garcia-Rodriguez et al., 1985; Rodríguez Hernández et al., 1998), Germany (Bechtel et al., 1992), Egypt (Farag et al., 1979) and Peru (Marcos et al., 2005). Familial clustering has been also found in patients from the French island of Corsica (Gil-Benito et al., 1991a) and in community-based surveys in the Northern Bolivian Altiplano (Mas-Coma et al., unpublished data). In a community-based survey in Egypt, among 25 families with at least one infected person, 20% had two members infected and another 20% had three members infected (Farag et al., 1979).

Human infection sources

Studies performed in many countries in the last three decades have demonstrated that there are more sources of human infection than the very few distinguished time ago and traditionally evoked in textbooks (Mas-Coma, 2004). Interestingly, evidence indicates that fascioliasis belongs to the rare diseases which may infect humans by both food and drinking sources. Hence, dietary and drinking habits of the human populations are very important in fascioliasis.

Ingestion of freshwater wild plants

Plant markers of transmission foci

Both field studies and experimental work in the laboratory indicate that fasciolid cercariae do not show preferences for one or other type of aquatic vegetables, plants selected by them to attach and encyst becoming metacercariae depending on the ecology of the lymnaeid vectors in each endemic area. Given that lymnaeids show evident preferences for stagnant or only very slowly running waters, and considering the limited swimming capacity of cercariae, the vegetables selected are those growing in the water body where the lymnaeids are present. There are only two factors which have a clear impact by determining the absence of the lymnaeid vectors, namely the existence of salt and shadow. Lymnaeid vectors do not inhabit brackish waters because they do not tolerate even low salt concentrations, nor water collections (or parts of them) which are under permanent shadow impeding sunshine to allow for the growth of the freshwater algae from which lymnaeids like to feet (Mas-Coma et al., 1999b).

In Europe, although the main lymnaeid vector G. truncatula does not show any direct relationship with the local plant species combination in the water collection, the presence of given plant groups appears to be a good sign of the existence of this lymnaeid (Over, 1962).

Plant combinations having seen as potential markers are Glyceria fluitans and Glyceria plicata (Floating Sweet-grass or water managrass, perennial grass species occurring in wet areas such as ditches, riverbanks and ponds), Alopecurus geniculastus(commonly known as water foxtail or marsh foxtail, a grass species which grows in moist areas) and Ranunculus repens (creeping buttercup or creeping crowfoot, an herbaceous perennial plant very common in damp places, ditches and flooded areas). In other localities, the combination includes Veronica beccabunga (European speedwell, a succulent herb which grows on the margins of brooks and ditches), Glyceria declinata (waxy mannagrass or low glyceria, a small sweet-grass which invades deep vernal pools, swales, ditches and stock ponds), Juncus inflexus (a rush) and the aforementioned R. repens (Over, 1962). Galba truncatula does not inhabit brackisch waters such as those of the Lake Titicaca (Mas-Coma et al., 1999b) and the Caspian Sea (Ashrafi et al., 2015), but may be present in somehow salty environments presenting Juncus gerardii (blackgrass, black needle rush or saltmarsh rush occurring along the shorelines of areas once flooded by the sea), Glaux maritima (Black saltwort growing in humid habitat or water along seashore environments), Carex otrubae (the false fox-sedge) and Festuca arundinacea (a grass commonly known as tall fescue found in damp grasslands, river banks and in coastal seashore locations) (Over, 1962). In grasslands where sheep use to feet, the presence of Ranunculus flammula (lesser spearwort, greater creeping spearwort or banewort, a poisonous species of perennial herbaceous plant) appears to be a good indicator (Over, 1962).

Many other authors have furnished lists of plants which may be used as indicators of the habitats of G. truncatula, such as in Germany (Patzer, 1927; Mehl, 1932) and the UK (de Vries, 1945; Roberts, 1950). In France, rush species as Juncus acutiflorus and J. effusus have already been emphasized (Ghestem et al., 1974; Gaultier et al., 1994; Guy et al., 1996), with J. acutiflorus as the best marker in grasslands and G. fluitans in the banks of streams and ponds (Dreyfuss et al., 1997; Hourdin et al., 2006; Rondelaud et al., 2011). However, in the Northern Bolivian Altiplano hyperendemic area, where the disease transmission is assured by only G. truncatula of European origin, a thorough field study could not establish any positive relationship between freshwater plant combinations and the presence of this lymnaeid vector, probably due to the extreme conditions of the very high altitude (3800–4100 m a.s.l.) of this area (Mas-Coma et al., 1999b).

The lack of direct relationship of the presence of the lymnaeid vector and the vegetation inhabiting the water collection does not mean, however, that outer characteristics of the parts of the plant in contact with the water may offer more or less facilities for cercarial attachment. Thus, an experimental study in Egypt indicated that cercariae of Fasciola spp. prefer to encyst on dark green leaves with the hairy epidermis, followed by leaves with a serrated and mamillated epidermis, whereas plants with a smooth chitinized epidermal surface are those to which the fewest metacercariae attach (WHO, 1995).

Summing up, among the freshwater wild plants carrying metacercariae, their more or less important role in the transmission to humans will mainly depend on the diet and traditions of the humans inhabiting the area in question. Freshwater wild plants are an important human infection source in animal endemic areas and also in given human endemic areas. Among the vegetables incriminated in human infection, freshwater plant species differ according to geographical zones and human dietary habits. Moreover, plant species involved are not necessarily the same in subjects infected ‘at table’ (through vegetables making part of the normal diet) than in subjects ‘infected in the field’ (ingestion, sucking, chewing or stripping with the teeth of vegetables directly taken from the nature and which may not necessarily make part of the usual human diet).

Watercress

Anamnesis in most reports of human infection uses to refer to watercress as the most probable source of the infection of the patient. However, the general term watercress includes different aquatic species such as Nasturtium officinale (common watercress), N. or Roripa silvestris and Roripa amphibia (wild watercress). Wild watercress has been reported as the main source of human infection in areas where fascioliasis in domestic animals is highly endemic.

Watercress is a green leafy vegetable that grows in most temperate and tropical areas of the world. It is the vegetable most involved in patients diagnosed in countries, such as in the USA (Price et al., 1993). In Latin America, wild watercress has been involved in patient infection in many countries, as Mexico (Zumaquero-Rios et al., 2013), Cuba (Diaz et al., 1990; Gonzalez et al., 1987; Brito et al., 1987), Dominican Republic (Noyer et al., 2002), Venezuela (Rodriguez and Gonzalez, 1975; Abdul-Hadi et al., 1996), Peru (Blancas et al., 2004) and Argentina (Mera y Sierra et al., 2011).

In Argentina, several outbreaks appear related to the most common risk factor of ingestion of watercress naturally growing along the river- and stream-beds picked during recreational, weekend or holiday activities. Many of these field excursions are undertaken by a family or as a group activity. A very large number of villages and towns play an important role in these recreational activities. These recreational areas attended by thousands of tourists, campers or weekend visitors present water collections inhabited by lymnaeid vectors and where animals show infection by F. hepatica (Mera y Sierra et al., 2011).

Regarding Europe, watercress consumption appeared linked to liver fluke infection in 69.3% of the fascioliasis patients in Spain (Garcia-Rodriguez et al., 1985) or even in almost all patients (Arjona et al., 1995). In France, wild watercress proved to be the main infection source not only in given areas (Rondelaud, 1978, 1980) but also in the analysis of 10 000 human cases reported between 1956 and 1982 from around the country (Gaillet et al., 1983). This wild freshwater vegetable is repeatedly noted as the infection source in epidemics, whether relatively wide epidemics or smaller familial outbreaks (Fig. 3C, D) already a long time ago (Bouysset et al., 1943) and even in cases of children (Giraud et al., 1955). Watercress appears similarly underlying human infection in other European countries as UK (Hardman et al., 1970) and Irland (LaPook et al., 2000).

In Asia, watercress appears involved in human infection throughout, from Turkey (Gulsen et al., 2006) in the West up to Thailand in the South East (Wong et al., 1985). In Iran, wild watercress is inhabited by G. truncatula in the streams of the Iranian mountains (Fig. 2A, B, C). Similarly occurs in Australia (Wood et al., 1975; Croese et al., 1982). In Africa, human infections with F. gigantica are believed to be caused by ingestion of watercress in Rwanda and Burundi (Janssens et al., 1968).

Fig. 2. Freshwater plants involved in Fasciola transmission: (A) the one-row yellowcress Nasturtium microphyllum in the Zagros mountains close to Yasuj city, Iran; (B) Galba truncatula is frequently associated to this cress; (C) wild vegetables linked to G. truncatula snails in the Talesh mountains of Guilan, Iran; (D) wild watercress in the Northern Bolivian Altiplano; (E) the small rush Juncus ebracteatus is usually sucked and chewed by Altiplano children; (F) edible algae and Nostoc cyanobacteriae are consumed by the Altiplano Aymara inhabitants; (G) the totora Schoenoplectus californicus ssp. tatora inhabiting bank waters of Lake Titicaca, is not associated to lymnaeids due to its noxious secretions; (H) other freshwater plants appear colonized by lymnaeids besides rapid running stream waters in Dominican Republic mountains. (Photographs S. Mas-Coma).

Other freshwater wild plants

Other such plants have been seen to be involved in the transmission of the disease to humans. Aquatic vegetables other than watercress which have been reported as vehicles of human infection are mainly Taraxacum dens leonis or Taraxacum gr. officinale(dandelion leaves) as in France (Garin et al., 1944; Rondelaud, 1980) and Argentina (Mera y Sierra et al., 2011), Valerianella olitoria(lamb’s lettuce) in central France (Rondelaud, 1980) and Mentha viridis (spearmint) (Fig. 3A) (Mas-Coma and Bargues, 1997; Mas-Coma et al., 1999b). Wild common sorrel, Rumex acetosa, also known as spinach dock, collected in a swampy meadow appeared to be the source of infection of two sister girls in France (Mohr et al., 1951). The latter is a slender herbaceous perennial plant that has juicy stems and edible, arrow-shaped leaves and is common in grassland habitats (also cultivated as a garden herb and consumed raw as a salad vegetable).

Fig. 3. Freshwater plant cultures: (A) spearmint Mentha spp. use to live on loamy soils flooded by water where it is colonized by lymnaeid snails, as in Valencia, Spain; (B) plants needing intensive irrigation are consumed in the Nile Delta region; (C) unusual mountainous area in Corsica island, France, where a family outbreak occurred; (D) origin of this outbreak in a garden watercress irrigation canalized from a neighbouring lymnaeid-contaminated spring water; (E) large cultures of water morning glory or water spinach besides Quy Nhon, Vietnam; (F) pond with water caltrop Trapa bispinosainhabited by lymnaeid snail vectors close to urban setting in southern Taiwan; (G) typical south Asian pond besides dwelling presenting floating water lily (Nymphaea sp.); (H) lymnaeid snail vectors attached to a water lily leaf. (Photographs S. Mas-Coma).

In Iran, several species of wild grown aquatic and/or semi-aquatic plants are a main part of the common human diet in many areas, especially in the endemic region of Guilan Province, the most important zone of human fascioliasis in the country. The species Mentha pulegium, Mentha piperita and Eryngium caucasicum are the main species that have been implicated in human fascioliasis transmission in the Guilan endemic province (Asmar et al., 1991; Forghan-Parast et al., 1993; Massoud, 1998). In other Iranian provinces where human fascioliasis has been reported, several species of aquatic plants have been noted to be involved in the disease transmission. Thus, in the southwestern Yasuj district and rural areas of Boyer-Ahmad township the vegetable noted to be involved was the one-row yellowcress Nasturtium microphyllum (locally named ‘bakaloo’ or ‘boolaghuti’) (Fig. 2A, B), Mentha longifolia(known as ‘pooneh’) and spearmint (Sarkari et al., 2012; Hosseini et al., 2015). In the Mazandaran Province at the seashore of the Caspian Sea, reference has been made to Eryngium spp. and Mentha spp. (Moghaddam et al., 2004). In the western Kermanshah Province, Nasturtium spp. and Falcaria vulgaris (locally known as ‘paghaze’) have been mentioned (Emami Al-Agha and Athari, 1995). Interestingly, moreover, a 44% of raw vegetables, including spearmint, were found to be contaminated by eggs of Fasciola sp. in Iran (Abdi et al., 2014).

In the Bolivian Altiplano human hyperendemic area, different freshwater plants have been found carrying metacercariae: 56.3% Compositae; 50.9% Eleocharis sp.; 12.0% Senicio sp.; 10.3% Vallisneria sp.; 3.3% Scirpus sp.; 2.6% Ranunculaceae. In this Andean hyperendemic area, the reports suggest that human infection is related to traditional consumption of uncooked aquatic plants, including (care should be taken with Aymara terms because of usual confusion by Altiplano inhabitants): watercress, berros or ‘okororo’ (Mimulus glabratus and Nasturtium officinale – Scrophulariaceae) (Fig. 2D); matara (Juncus andicola – Juncaceae); totorilla or ‘kosko-oskosko’ (Juncus ebracteatus – Juncaceae) (Fig. 2E); edible algae as cochayuyo or ‘llayta’ (Porphyra purpurea – Chlorophyta) and similar vegetables as Nostoc sp. (Cyanobacteria) (Fig. 2F); and many others (Mas-Coma et al., 1995, 1999b; Esteban et al., 1997a). Regarding the so-called totora or ‘chullu’ (Schoenoplectus californicus ssp. tatora – Cyperaceae) (Fig. 2G), frequently referred to by the Altiplano inhabitants, a negative association between presence of lymnaeids and presence of this plant in the same water collection was observed, most probably because of the noxious secretions of its roots (Mas-Coma et al., 1999b). Among the numerous aquatic and semi-aquatic plant species found in water collections presenting lymnaeids in the Altiplano, mainly J. ebracteatus and M. glabratus and secondarily Nostoc sp. are infection sources for human adults. Concerning the transmission to children, it should be considered that in this endemic very high altitude area, children are malnourished and from an early age many of them help their parents in agricultural activities and in the tending of animals. The many hours spent away from home, in turn, leads them to eat, suck or chew many wild vegetables, which may constitute vehicles that enable the access of metacercariae to their alimentary tract (Esteban et al., 1997a). Among the many other wild freshwater plant species involved in the infection of children, Hydrocotyle ranunculoides, Eleocharis spp. Rorippa spp., other Juncaceae and Scrophulariaceae, Compositae, etc., should be counted (Mas-Coma et al., 1999b).

In Argentina, two patients were mentioned to have chewed blades of grass that grew on a riverbank (Mera y Sierra et al., 2011). This appears to be a problem mainly with children, as they put into mouth all kind of objects and above all wild plants collected in nature during walks, as was the case of a girl frequently sucking wild grass hazardly collected along walks and diagnosed in France (Martin et al., 1944). The high risk of chewing wild grass by children was highlighted in a recent questionnaire survey made in Baños del Inca, Cajamarca, Peru (Rodríguez et al., 2018). Other freshwater plants appeared colonized by lymnaeid snails besides rapid running stream waters in the mountains of the Dominican Republic (Fig. 2H).

In Hawaii, human infection was noted to take place by the accidental ingestion of raw vegetation, including watercress (Alicata and Bonnet, 1956) containing encysted metacercariae, particularly in areas where infected cattle were permitted to roam (Stemmermann, 1953a, 1953b).

In Asia, a human fascioliasis case report in Thailand in which the water morning glory or water spinach (Ipomoea aquatica) was suspected to be the origin of the patient’s infection (Wong et al., 1985) suggested the appropriateness to enlarge the spectrum of freshwater plants involved in the transmission of Fasciola to humans in Asian countries by including the vegetables always considered to underlie human infection by Fasciolopsis buski, another trematode in whose life cycle cercariae behave similarly to those of Fasciola (Mas-Coma et al., 2005). Indeed, this is supported by the coexistence of both Fasciola in ruminants and Fasciolopsisin pigs in the same area of human infection by Fasciola (Manning and Ratanarat, 1969) and by the usual coexistence of the lymnaeid vectors of Fasciola and the planorbid freshwater snails transmitting F. buski in the same water collections. Therefore, the following plant species may be considered: the water caltrop (Trapa natans in China, T. bispinosa in Taiwan and T. bicornis in Bangladesh and Thailand) (Fig. 3F), the water chestnut (Eliocharis tuberosa), the water lotus (Nymphaea lotus), water bamboo (Zizania aquatica) and other freshwater vegetation including eelgrass or tape grass (Valisneria spp.), floating fern, watermoss or water butterfly wings (Salvinia natans), common duckmeat or greater duckweed (Spirodela polyrhiza = Lemna polyrhiza), water hyacinth (Eichhornia crassipes), water lily (Nymphaea sp.), watercress, gankola (Otelia sp.), and the aformentioned water morning glory or water spinach (I. aquatica) (WHO, 1995; Mas-Coma et al., 2005). In Thailand, the species frequently consumed are water caltrop (Trapa hicornis), water hyacinth (E. crassipes), lotus (N. lotus), water mimosa (Neptunia oleraced) and water spinach (I. aquatica) (WHO, 1995). Most of these edible plants grow near the houses, that is, where pollution takes place (Manning and Ratanarat, 1970). Moreover, ‘night soil’ (human excrement collected from latrines) is used to fertilize fish ponds and to feed fish where these plants are present, in that way enhancing disease transmission (Cross, 1984).

In Africa, human infections with F. gigantica may also occur after chewing infested grass or green rice (Janssens et al., 1968). It is believed that one of the reasons why human fascioliasis is rare in southern Africa may be the dietary habits of the Africans in this area (Fig. 3B) where water plants do not seem to be an important source of food or relish and, in any case, are mostly eaten cooked (Gelfand, 1971; Goldsmid, 1975). However, in Malawi, it has been pointed out that some vegetable plants are eaten uncooked, including cabbage, tanaposi and mnadzi, and these may serve as sources of infection in swampy areas. It is also suggested that sugar cane grown in swampy areas may serve as a source of metacercarial ingestion, the cane commonly being stripped by Africans with their teeth (Speckhart, 1969).

Wild plants sold in urban markets

In given reports, information provided by the patient indicated that the infection was from watercress bought in an urban market or bazaar, as seen in Turkey (Kaya et al., 2006) and Australia (Hughes et al., 2003). In France, authorized cresspools were also involved in the infection of patients (Gaillet et al., 1983).

In the Guilan endemic province in Iran, villagers collect the aforementioned aromatic freshwater plants and present them beside the streets and in traditional markets throughout the year (Fig. 4A–D). These vegetables are very popular and may be eaten fresh or used to prepare appetizers (Ashrafi, 2015).

Fig. 4. Uncontrolled markets of vegetables: (A, B, C) edible wild vegetables sold in the market of Rasht city, Iran; note radishes (B) and watercress (C); (D, E) mobile street selling of wild vegetables in Rasht city, Iran (D) and Nile Delta village, Egypt (E); (F) wild vegetable market in Kutaisi city, Georgia; (G, H) wild vegetable market in Quy Nhon, Vietnam; note radishes (G) and carrots (H). (Photographs S. Mas-Coma).

Non-controlled places where wild plants are sold are usually found in city markets of endemic countries. In Egypt, wild vegetables are sold even individually at the street (Fig. 4E) and uncontrolled wild plant selling is also found in city markets of eastern European countries (Fig. 4F). In Uzbekistan, the relatively high human prevalence in the Samarkand region was related to the important percentage (10.5%) of green vegetables sold in the Samarkand market, which presented encysted metacercariae (Sadykov, 1988).

In Vietnam, raw vegetables are an important part of the normal diet and wild freshwater plants are easily available in non-controlled city markets (Fig. 4G, H). Water-plants, particularly water-spinaches that are consumed daily by the whole Vietnamese population, are a huge source of infections. A high percentage of trematode contamination in vegetables has been reported in many south-eastern Asian countries (Abdi et al., 2014; WHO, 2014), especially in Vietnam (Uga et al., 2009). There is a strong correlation between infection and travelling, even if it has been demonstrated that contaminated vegetables reach also the big city markets (Ulukanligil et al., 2001; Fiamma et al., 2015). In the Cambodian capital of Phnom Penh, a study showed contamination in water-spinach samples harvested in a lake located at 5–7 km (Vuong et al., 2007).

Regarding human fascioliasis infection sources through wild vegetables whether directly collected from the field or acquired in urban markets, the recent drive to ‘go green’ as a healthy approach to the modern artificial lifestyle in today developed societies poses evident problems. This recent fashion has shown to underlie an unprecedented increase in the consumption of fresh, raw/green fruit and vegetables (Broglia and Kapel, 2011; Hotez et al., 2014). Unfortunately, this appears to be a real challenge, because this drive appears to be poorly backed by water safety, fertilizer–pesticide use control and waste management. The consumption of poorly monitored, produced and stored fresh green vegetables has contributed to an increased spread of plant/food-borne trematodiases, including fascioliasis among other health problems (Lev and Rager-Zisman, 2014). Indeed, individual private garden cultivation of imported Asian vegetables has been suggested to underlie recent fascioliasis epidemic cases in Switzerland (Gottstein, personal communication).

Ingestion of freshwater cultivated plants

Several metacercariae-carrying species may even be so important in the human diet of a given area, as to be man-produced (at family or even industrial level) and commercially sold in public markets, explaining infection of subjects living far away from the endemic area.

Wild watercress is collected and eaten, but it is also cultivated in small family gardens (Fig. 3C, D) and farms. The plant is also produced commercially on large farms and sold in supermarkets, as in Europe and Australia. A study in France showed that home-grown, wild and commercially grown watercress was the cause in 23, 8 and 2 cases, respectively (Chen and Mott, 1990).

If not controlled, a watercress culture in the garden may become contaminated by lymnaeids and Fasciola eggs shed by livestock moving around close to the garden and which may reach the watercress beds by passive transport through rainwater. In given cases, such familial watercress cultures have allowed understanding human infection presenting familial clustering in places which are non-typical for fascioliasis transmission, such as in mountains lacking wide grass fields (Fig. 3C, D) (Gil-Benito et al., 1991a, 1991b). Gardens provide an efficient and economic means of vegetable production in the periurban areas. If these actívities are poorly managed and untreated human or animal excreta are used as a fertilizer, the potential for transmission should be monitored.

Unexpected problems of contamination of watercress cultures due to disease spread by an introduced sylvatic reservoir animal as the nutria (Menard et al., 2001; Houin et al., 2004) appeared related to the emergence of human fascioliasis in concrete areas of France. It was up to this rodent species Myocastor coypus, originally of South America where it already proved to be a good definitive host for the liver fluke (Gayo et al., 2011), to unexpectedly spread F. hepatica eggs in watercress beds made in the way to avoid contamination from ruminants. The aforementioned fascioliasis emergence was described as the first epidemic due to the ingestion of cultivated watercress (Mailles et al., 2003, 2006).

In Korea, an aquatic plant known as Water dropwort (Oenanthe javanica) is a perennial herb with a distinctive aroma and is cultivated in marshy areas of Asia and Australia. The fresh stems and leaves are widely used as a salad or as a seasoning in soups and stews in Korea. Water dropwort has also been used in Korea as a folk medicine for the treatment of jaundice, hypertension, fever, abdominal pain, leucorrhea, mumps and urinary difficulty. In a survey, the presence of F. hepatica cox1 and ITS-2 DNA markers were detected in two samples among 500 samples assessed, confirming a 0.4% contamination (Choi et al., 2015). The prevalence in this study was lower than that in watercress in France (1.2–2.4% annually) (Dreyfuss et al., 2005).

Throughout Asia, many of the aforementioned edible vegetables involved in trematode metacercariae transmission such as water caltrop, water chestnut, water lotus, water bamboo, water hyacinth, water lily and water morning glory or water spinach are cultivated in several uncontrolled places to respond to the local demand (Mas-Coma et al., 2005). The water morning glory or water spinach, locally known as ‘rau muong’, is widely cultivated in fields neighbouring villages and even cities where human fascioliasis infection appears to be frequent in Vietnam (Fig. 3E). Sources of Fasciola contamination in agricultural products have been noted to include soil, feces, irrigation water, inadequately composted manure, wild and domestic animals, dirty equipment and human handling (Berger et al., 2010). Differences in prevalence may be induced by various factors such as host distribution, locality and environmental conditions (Choi et al., 2015). In southern Taiwan, ponds for the cultivation of the water caltrop Trapa bispinosainhabited by local lymnaeid snail vector species together with the planorbid Segmentina hemisphaerula are typical close to urban settings (Fig. 3F). Cultures of the floating water lily (Nymphaea sp.) in ponds beside dwellings and presenting lymnaeid snails are usual throughout southern Asia (Fig. 3G, H).

Ingestion of terrestrial wild plants

The survival capacity and relative dryness resistance of metacercariae explain the contamination by consumption of wild terrestrial plants collected in dry or moist habitats but which were submerged in water a few weeks or months before, as in places with temporary water bodies in endemic areas of Iran.

In Andean countries, infection of children has been evoked to occur by putting into mouth, sucking, chewing or even eating terrestrial wild plants, mainly with juicy, succulent stems, which grow in places with frequent freshwater on ground, whether because of their presence at the margins of rivers, streams and lagoons or close to such water bodies giving rise to periodic or sporadic floodings of the surrounding areas in periods of water level rise. This typically occurs after rainfall but may also be related to man-made irrigation strategies. The amphibious characteristics of the lymnaeids, very pronounced in the Galba/Fossaria members and also in several species of the Radix group, underlie the possibility of metacercariae to attach to such vegetables during the periods in which the base of their stems is submerged.

It has been shown that cercariae of Fasciola encyst on objects just under the surface of the water (Hodasi, 1972; Ueno and Yoshihara, 1974; Dumag et al., 1976). Hence, this means that metacercariae will be attached to the plant portions which were immersed in water. This explains why the base of the stems but also parts of tubercles protruded over the soil surface, may participate in human and animal infection.

Ingestion of terrestrial cultivated plants

The amphibious characteristics of vector species such as G. truncatula and other Galba/Fossaria species, but also small Asian Radixspecies, explain the fascioliasis transmission foci in plantations of non-aquatic vegetables needing frequent irrigation.

Rice is a good example of a terrestrial plant which needs plenty of irrigation for its cultures. Indeed, rice fields are ideal habitats for lymnaeid vector development, such as G. truncatula in the Albufera rice fields in Spain (Fig. 5A) (Valero et al., 1998), in the wide rice fields inhabited by Radix auricularia in Guilan lowlands, Iran (Fig. 5B), or in those contaminated by R. viridis throughout Vietnam (Fig. 5C). In Egypt, the closeness of rice fields to dwellings of village suburbs is risky for children (Fig. 5D). Rice fields become appropriate for fascioliasis transmission when animal dung or manure (Suhardono et al., 2006a) is used for fertilization or culture fields are visited by livestock (Fig. 10F), both aspects being frequent in Asian countries. In its turn, animals may become infected in these rice fields (Ueno and Yoshihara, 1974) or outside them, as in stall-fed buffaloes by managing the feeding on rice straw (Mahato and Harrison, 2005).

Fig. 5. Rice and other cultivated terrestrial plants needing intense irrigation: (A) the loamy soil after rice irrigation is ideal for lymnaeid snails vectors, as in Valencia, Spain; (B) rice fields with Radix auricularia in Guilan lowlands, Iran; (C) irrigated rice fields with Radix viridis surrounding Hanoi, Vietnam; (D) rice fields close to dwellings of village suburbs in the Nile Delta region, Egypt; (E) terrestrial vegetables irrigated by water canals contaminated by lymnaeid snail vectors in the Nile Delta Region, Egypt; (F) khat bush in Ethiopia; note livestock in the background. (A-E: photographs S. Mas-Coma; F: free online at https://es.dreamstime.com/foto-de-archivo-libre-de-regal%C3%ADas-arbustos-con-las-hojas-del-khat-image39802405, accessed 22.2.2018).

In Egypt, many species of non-aquatic vegetables and weeds are eaten raw as salads. These include the garden rocket Eruca sativa (a salad vegetable locally known as ‘El gargeer’), the lettuce Lactuca sativa (known as ‘EI khas’, a leaf vegetable most often used for salads) and the leek Allium porrum (‘EI korrat’). These plants are cultivated along the banks of water channels and need frequent irrigation (Fig. 5E). On collection, they are washed in the nearby water body during their preparation for marketing to have a beautiful green colour. Irrigation and washing expose them to become a carrier of the encysted metacercariae of Fasciola species, thus conveying infection to man (Motawea et al., 2001a).

Other such vegetables on which attached metacercariae have been found in Egypt are the parsley Petroselinum sativum whose edible aromatic leaves are used as a seasoning or garnish and also used in cooking, and the common purslane Portulaca oleracea, an annual succulent that may be eaten as a leaf vegetable used raw in salads (El-Sayed et al., 1997; Motawea et al., 2001a).

Thanks to transport of vegetables, both aquatic and terrestrial, from rural endemic zones to cities, plants carrying metacercariae can be sold in non-controlled city markets giving rise to urban infection, as in Europe, Egypt (Fig. 4E), Bolivia (Mas-Coma et al., 1999b) or Vietnam (Fig. 4G, H). Metacercariae of F. hepatica were found in 1% of lettuces of a local market in the Mantaro valley, Peru (Bendezu, 1969). In the Peruvian capital of Lima, among 277 patients infected by F. hepatica, 31.6% mentioned having eaten lettuce, 10.5% alfalfa and 5.3% spinach (Blancas et al., 2004).

In the recent decades, a slow-growing shrub or tree native to the Horn of Africa and the Arabian Peninsula has repeatedly been involved in human fascioliasis infection, namely Catha edulis (Celastraceae), known as khat, qat, gat or Arabian tea (Fig. 5F) (Doherty et al., 1995; Cats et al., 2000; De Bree et al., 2013). This plant grows especially well in moist conditions. Therefore, a heavy irrigation of khat cultures starts around a month before they are harvested to make the leaves and stems soft and moist. The fresh leaves or the soft part of the stem are chewed with either chewing gum or fried peanuts to make it easier to chew, in the way to achieve a state of euphoria and stimulation. They are also, but less frequently, dried and consumed as a tea.

Khat chewing is a social tradition since thousands of years among people of countries of the aforementioned region. WHO classified it in 1980 as a drug of abuse that can produce mild to moderate psychological dependence (Nutt et al., 2007). Its production, sale and consumption are legal in countries such as Djibouti, Ethiopia, Somalia and Yemen (Chevalier, 1949). Khat leaves have been chewed by generations in countries of the Horn of Africa, mainly Yemen, for their stimulant properties. Its young fresh leaves are especially valued for their potency. For khat transport, freshly picked leaves are, therefore, usually kept damp and wrapped in banana leaves. In Yemen, khat is so popular that its cultivation requires much of the agricultural resources of the country. Around 40% of the water supply of the country is destinated to its irrigation. Additionally, khat is important in Yemen because it provides a high income for farmers. All this defines a worrying scenario of khat-underlying human fascioliasis in these countries.

Khat is nevertheless a controlled substance in other countries as the USA, Canada and Germany. Traditionally, the use of khat has been confined to regions where it is grown, given that only the fresh leaves offer the desired stimulating effects. However, improved roads, off-road motor vehicles, and air transportation have increased its global distribution in recent years, therefore allowing to understand reports of this plant in the UK, Italy, The Netherlands, Israel, Canada, USA, Australia and New Zealand.

The diagnosis of patients infected in European countries due to the consumption of imported non-controlled khat demonstrates that contaminated cultivated plants in one country may be exported to other countries bearing still viable metacercariae. Indeed, a 45-year-old woman originally from Yemen have not travelled outside the UK was supposed to acquire fascioliasis through chewing khat leaves in London (Doherty et al., 1995). In The Netherlands, a 36-year-old Somalian man with fascioliasis reported that he had never eaten liver or watercress or other wild water plants but chewed leaves of khat imported from Kenya where khat shrubs were cultivated in areas housing sheep and were irrigated with local water. The survival of the encysted metacercariae was deduced to have been prolonged because the freshly plucked khat leaves were kept damp and wrapped in banana leaves during transport (Cats et al., 2000). Also in The Netherlands, another 24-year-old Somalian man infected by Fasciola admitted chewing freshly imported khat leaves, which was most likely the source of infection (De Bree et al., 2013). It becomes evident that laws for the control of fascioliasis risks posed by khat exportation are needed.

Ingestion of traditional local dishes made with contaminated sylvatic plants

In the fascioliasis endemic Iranian province of Guilan, there are several very popular kinds of wild aromatic plants, such as species of Eryngium and Mentha, which are (i) eaten raw, (ii) ground and mixed with walnuts, various spices, garlic and fresh olives for the preparation of an appetizer called ‘zeitoon-parvardeh’ (Fig. 6A, B) or (iii) used in the preparation of a paste called ‘delar’ along with a great quantity of salt as a condiment (Fig. 6C, D). The very high quantity of salt used for ‘delar’ preparation is at the base for the local name ‘green salt’ also given to this specialty. This paste may be stored for consumption over several months.

Fig. 6. Traditional culinary specialities made from popular aromatic wild plants, involved in human fascioliasis in the endemic province of Gilan, Iran: (A, B) “Zeitoon-Parvardeh”, a speciality which is made by mixing the grounded local wild plants with other ingredients and fresh olives (A) and used as an appetizer dish (B); (C, D) ”Delar”, a speciality which may be stored for consumption over several months (C) and is served as a traditional herbal paste (D). (A, D: photographs K. Ashrafi; B, C: photographs S. Mas-Coma).

The aromatic vegetables used for these two traditional home-made foods are usually sold throughout the year mainly in the streets of all endemic areas in Guilan Province (Fig. 6A–D). The consumption of these two traditional local foods has been shown to be the main source of human infections in that area. They are also believed to have played an important role in the large outbreak involving around 10 000 people in the Bandar-Anzali and Rasht districts in 1989 and in the subsequent outbreak affecting around 5000 people of the same area of the Guilan province occurred some 10 years later (Ashrafi et al., 2006a, 2006b). Additionally, these foods represent a risk for fascioliasis spread when Guilan inhabitants give these appetizer and condiment as a present to other family members living in other Iranian provinces.

Ingestion of raw liver

Given experimental results suggested that humans consuming raw liver dishes prepared from fresh livers infected with immature flukes may also become infected, because early migrating flukes present in the ingested infected liver may keep the capacity to re-start the intraorganic migration. In a first experiment, twenty-four mice were inoculated orally, each with a mean number of 68 freshly recovered immature flukes. The livers of 7 of the 24 recipient mice showed migratory lesions of capsular and subcapsular granulomatous infiltration and two of those mice also had haemorrhagic lesions typical of those caused by active migration of early immature flukes (Taira et al., 1997). In a second experiment, 10 piglets were given fresh livers of mice harbouring approximately 2000 live immature flukes aged 3–7 days. Granulomatous lesions were found in all pigs at necropsy, except in those that were given livers containing flukes aged 7 days. From the 10 pigs given livers, 65 live flukes were recovered, 0.29% of the estimated number of immature flukes given (Taira et al., 1997).

Such an infection source may give rise to confusion with the so-called spurious fascioliasis throughout (Mas-Coma et al., 2014a; 2014b) and also with spurious paramphistomiasis in Africa.

People ingesting infected domestic animal livers (mainly cattle, sheep, goat and pig) a short time before may reflect ‘false’ fascioliasis when the fluke eggs are found in their stools (Stork et al., 1973; Ragab and Farag, 1978; Campo et al., 1980). Such spurious infection may give rise to false positives. Although an expert microscopist may differentiate the somewhat degenerated aspect of ‘eggs in transit’ from ‘normal eggs’, to avoid confusion in such cases diagnosis requires placing the patient on a liver-free diet and performing repeated follow-up stool examinations.

It should also be considered that paramphistomid flukes are frequently infecting livestock throughout and that their eggs are very similar to those of fasciolids, both in form and size, and may therefore be easily confused when diagnosing animals by coprological analyses. Fortunately, however, paramphistomid flukes do not develop in humans, but this does not exclude the possibility to find ‘paramphistomid eggs in transit’ in human stools shortly after having consumed an animal meat (mainly rumen) infected by paramphistomid flukes, mainly in several parts of Africa.

Six main features allow for the ascription of eggs to Fasciola and differentiate them from the also oval, operculate and non-embryonated eggs from Paramphistomidae:

1. (a) fasciolid eggs are brownish-yellowish, whereas paramphistomid eggs are clear, transparent or silver-grey;
2. (b) fasciolid eggs are oval with a common trend to become slender at both ends, whereas paramphistomid eggs are oval with a rectangular trend, i.e. with wide ends;
3. (c) as a consequence of the aforementioned characteristic, the operculum width/maximum egg width ratio is smaller in fasciolid eggs than in paramphistomid eggs;
4. (d) in fasciolid eggs the inner contents shows brownish granules smaller in size than the clear cells inside paramphistomid eggs;
5. (e) at the abopercular end of the shell surface of Fasciola eggs there is often a typical roughened or irregular, more intense brownish dark area which may sometimes appear laterally displaced (Valero et al., 2009), whereas this is absent in paramphistomid eggs.

Moreover, it has been noted that the eggs of these trematodes may be differentiated by contrast stain, eggs of Fasciola showing yellowish colour while eggs of paramphistomids staining when adding a drop of methylene blue or methyl green solution to the sample sediment (Hansen and Perry, 1994).

In fact, the possibility of acquiring a Fasciola infection by eating an infected animal liver has been suggested since long ago, although not regarding the capacity to give rise to a hepatic infection by the flukes but to a clinical syndrome known as ‘Halzoun’ (i.e., ‘suffocation’), that manifests as an acute allergic edematous reaction involving the upper respiratory tract and nasopharyngeal mucosa. Such a syndrome is known to follow the consumption of raw sheep or goat liver, a food presentation in some countries such as Lebanon, Syria and Iran. Halzoun consists in the temporary attachment to the pharyngeal mucosa of adult worms, which have been ingested along with raw livers of goats and sheep, used for sacrificial purposes and later eaten in religious festivals (Siavashi et al., 2002). This localized infection produces an edematous congestion of the soft palate, pharynx, larynx, nasal fossae and Eustachian tubes, accompanied by dyspnea, dysphagia, deafness, and, in a few cases, resulting in asphyxiation (Faust, 1949).

Different causal agents have been involved in that syndrome (Khalil et al., 2013). Fasciola hepatica was the first to be described as an agent causing Halzoun. The immature worms of F. hepatica were considered to be the causative agent at that time (Khouri, 1905). Much time later, a similar syndrome referred to as ‘buccopharyngeal distomatosis’ was attributed to F. hepatica or another unknown trematode (Brumpt, 1936). Since then, similar clinical syndromes, considered to be synonymous to Halzoun syndrome, were described in the literature and were attributed to different pathogens and other parasites with buccopharyngeal tropism (Khalil et al., 2013). On the basis of clinical cases, Halzoun was attributed to the leech Limnatis nilotica, without ruling out the existence of another form linked to the trematode Clinostomum complanatum (Witenberg, 1944). Again on the basis of clinical cases, two forms of Halzoun were described, one caused by F. hepatica with the other by L. nilotica (Watson and Kerim, 1956). When comparing Lebanese Halzoun and the nasopharyngeal linguatuliasis known as Marrara syndrome in Sudan, the larva of the pentastomid Linguatula serrata was also identified as the parasite of Halzoun (Schacher et al., 1969; Yagi et al., 1996). More recent publications have attributed Halzoun to accidental infestation by the larvae of L. serrata, including also Greece (Papadakis and Hourmouziadis, 1958) and Egypt (Morsy et al., 1999) Despite their striking resemblance in clinical presentation, few differences exist between these food-borne diseases, i.e. expectoration of worms is rarely observed in the Lebanese Halzoun patients whereas it is a common event in Marrara syndrome (Khalil et al., 2013). The most recent study on Halzoun, performed in Lebanon, has added a new causal agent, the trematode Dicrocoelium dendriticum (Khalil et al., 2013). It has moreover suggested that the immature forms of F. hepatica described by A. Khoury as an agent of Halzoun in 1905, may, in fact, have been D. dendriticum, since both trematode species are frequently found together in the liver of ruminants. Hence, this finding indicates the need to verify whether Fasciola worms are really able to cause pharyngeal fascioliasis. Indeed, experimental work confirmed that F. hepatica adults are unable to attach themselves to the mucosa of the pharynx and can be swallowed without symptoms of pharyngeal irritation arising (Watson and Kerim, 1956), and the experimental use of young F. hepatica failed in the attempt to infect dogs (Brumpt, 1936). However, the fact that the disease was successfully reproduced in rabbits (Khouri, 1905) keeps the question open about the possibility of Fasciola also being involved in Halzoun after ingestion of raw meat from sheep and goats.

Drinking of contaminated water

The possibility of being infected by means of drinking water carrying floating metacercariae originated from natural water collections inhabited by lymnaeid snail vectors in the field in an endemic area cannot be neglected (Bargues et al., 1996).

Indeed, natural freshwater drinking was known to be a potential human infection source according to the anamnesis of many patients in the past (Bürgi, 1936). Contaminated water has even been mentioned to have been the infection source in collective infections (Sedallian et al., 1949). However, the infection of the definitive host, whether humans or animals, was traditionally considered secondary or even rare when compared with infection rates by metacercariae attached to vegetables, both in developed countries (Arjona et al., 1995) and in developing countries (Mas-Coma et al., 1995).

In Europe, in an only one patient of the 75 cases reviewed was the antecedent ingestion of watercress not mentioned, and in a series of 18 patients, only four of them reported frequent ingestion of water from unsanitized sources (Arjona et al., 1995). In the Northern Bolivian Altiplano, the hyperendemic area with the highest prevalences and intensities known, only in one community was reference made to the potential role of water as a human infection source. In this community, all children surveyed consumed previously untreated water from a well and a canal shared with animals. Both well and canal were noted to be places where humans and animals eliminated biological excreta, thus causing their contamination. Food, mainly local vegetables, were washed with contaminated water, increasing the risk of infection. In this survey, it was concluded that fascioliasis may be acquired by drinking contaminated water instead of eating watercress and other aquatic plants, since the inhabitants mentioned to not usually eat these plants (Mas-Coma et al., 1995).

The rarity of human infection by water drinking when compared with infection by ingestion of contaminated vegetables had been explained by two facts:

1. (i) Non-attached metacercariae were proved to sink down to the bottom of the water body quite shortly after cercarial shedding by the snail, due to the progressive impregnation of the metacercarial floaters with different natural water materials giving rise to the increase of the weight of the metacercariae. This was observed in both laboratory (Vareille-Morel et al., 1993) and natural conditions (Rondelaud et al., 2004).
2. (ii) Other results suggested that humans and animals might be infected with Fasciola sp. in endemic areas by drinking the water of small streams or banks contaminated with the metacercariae appearing in the feces (45% have lost their rough gelatinous outer cyst coat, appearing white or cream coloured) shed by lymnaeid snails having previously ingested normal encysted metacercariae (Taylor and Parfitt, 1957; Yoshihara and Ueno, 2004), although here also the faecal metacercariae, chained inside faecal cylinders, fell to the bottom. These metacercariae shed in snail feces experimentally proved to be similarly infective than normal metacercariae (Yoshihara and Ueno, 2004).

However, the importance of fascioliasis transmission through water is under reconsideration today after many more recent indirect results suggesting a higher role of water as human infection source (Mas-Coma, 2004).

In the French Mediterranean island of Corsica where several human infection cases were reported (Gil Benito et al., 1991b), water fountains for humans together or beside watering troughs for livestock constructed on the borders of the roads are inhabited by lymnaeid snails and visited by free moving livestock for water drinking, thus representing an evident human infection risk place (Fig. 7A; Gil Benito et al., 1991a). Similar fountains to be shared by humans and livestock are found in Eastern Europe, as for instance along roads in Georgia (Fig. 7B).

Fig. 7. Drinking water as a human fascioliasis infection source: (A) lymnaeid contaminated on-road fountain including bilateral watering troughs for free moving livestock in Corsica, France; (B) lymnaeid contaminated fountain and watering trough in endemic area of Georgia; (C) collecting water for home in the overflowed water from a broken artificial fountain in the Northern Altiplano of Bolivia; (D) child collecting water for home in canal in the Nile Delta region, Egypt; (E) man-made irrigation system canal on soil inhabited by lymnaeids for the water supply of dwellings in the Peruvian Altipllano; (F) bathing/washing the buffalo in large lateral canal of the Nile river, Egypt. (A, B, D, E, F: photographs S. Mas-Coma; C: photograph J.G. Esteban).

In the Northern Bolivian Altiplano human hyperendemic area, 13% of the metacercariae of all isolates proved experimentally to be floating (Bargues et al., 1996). This is worth mentioning owing to the very high number of cercariae-shedding lymnaeids which may be found in that hyperendemic area, e.g. 31.6% prevalence in lymnaeids from the locality of Tambillo. Up to seven metacercariae in only half a liter of water from the small river crossing Tambillo were found (Mas-Coma, 2004; Bargues and Mas-Coma, unpublished). In the Bolivian Altiplano, moreover, people collect water to home from overflowed waters from broken man-made fountains, these overflowed waters presenting lymnaeid snail vectors (Fig. 7C).

Similarly appears to happen in high altitude Andean valleys of Peru, where for instance a high prevalence of 20.5% was detected among 462 lymnaeids collected in front of the school of Santa Rosa de Chaquil, Cajamarca province (Bargues et al., 2012), a locality in which 47.7% of the children were infected (Gonzalez et al., 2011). Also in Peru, in a series of 277 patients with fascioliasis diagnosed in Lima, a 10.5% mentioned having drunk water from ‘puquiales’ (natural water from small streams) (Blancas et al., 2004).

Human infection by water drinking is also suggested by the statistically significant positive associations between liver fluke infection and infection by protozoans and other helminths which are waterborne. Thus, F. hepatica infection appears associated to Giardia intestinalis infection in the Northern Altiplano of Bolivia (Esteban et al., 1997a) and Peru (Esteban et al., 2002), to Entamoeba hartmanni in Cajamarca, Peru, where G. intestinalis also shows high prevalences paralleling those very high fascioliasis prevalences (Gonzalez et al., 2011), and to Entamoeba coli, Chilomastix mesnili and Schistosoma mansoni in the Nile Delta, Egypt (Fig. 7D; Esteban et al., 2003).

In many human hyperendemic areas of the Americas, people do not have a history of eating watercress (Hillyer and Apt, 1997). Of particular interest in that sense are the reports on human infection in artificial irrigation areas, even giving rise to human fascioliasis endemic situations. In the Peruvian Altiplano, the human endemic area of Asillo is a man-made irrigation area where human inhabitants do not have the tradition to consume freshwater plants but to collect water for drinking from the irrigation canals inhabited by the snail vector G. truncatula (Fig. 7E; Esteban et al., 2002).

In Argentina, several outbreaks appear related to visits of recreational field areas by thousands of tourists and campers during weekends. These areas include natural water collections inhabited by lymnaeid vectors and frequented by Fasciola-infected livestock (Mera y Sierra et al., 2011). The increasing infection risk posed by recreational waters due to the impact of climate change is undoubtedly a future challenge (de Roda Husman and Schets, 2010).

In the Nile Delta region, persons living in houses where piped water is present showed to have a higher infection risk (Curtale et al., 2003). In Egypt, children have a risk to become infected when washing the livestock in the waters of the large irrigation canals and also when drinking water from the smaller irrigation canals bordering the plantations surrounding the outer village suburbs, the lymnaeid vectors having colonized all such canals (Bargues et al., unpublished).

The importance of water as human infection source is also proved by the high prevalence reduction effectivity obtained in an assay performed by the Egyptian Ministry of Health and Population, the Italian Cooperation and the WHO Collaborating Centre of Valencia in an endemic village of the Nile Delta human hyperendemic area with the construction of the so-called ‘washing units’ (Curtale et al., unpublished). In these ‘washing units’, water from a big canal was pipped and filtered by means of a swimming pool filtration equipment before flowing up to the taps of each one of several laundries. Women of this village were a successfully convinced to daily use such installation as a source for (i) water drinking, (ii) collecting water for food preparation and washing and (iii) water for washing of kitchen utensils and dresses (Fig. 8A–D). The drastic human prevalence reduction obtained, from a previous 18% (Esteban et al., 2003) to a subsequent 2% after the construction and use of these ‘washing units’ (Curtale et al., unpublished), suggested human infection in this village to be mainly related to water drinking (Mas-Coma, 2004).

Fig. 8. The “Washing Units” control pilot intervention implemented in the hyperendemic village of Tiba, Delengat, Nile Delta, Egypt: (A) building with several laundries where filtered water flows through the taps; (B, C) women of this village were successfully convinced to daily use such installation as a source for water drinking and collecting water to home for food preparation (B), as well as for washing of kitchen utensils (B) and dresses (C); (D) water from a big canal was pipped and filtered by means of a swimming pool filtration equipment. (Photographs S. Mas-Coma).

Anyway, this result indicating the importance of water as human infection source in a village does not mean that the sources are the same in other villages, not even in the same endemic area. The epidemiological heterogeneity of human fascioliasis is also evident in that aspect. In the Northern Altiplano, vegetables play a more important role than water drinking in the Bolivian part eastward from Lake Titicaca (Mas-Coma et al., 1995), whereas water drinking appears to be the almost only human infection source on the Peruvian side of the Lake (Esteban et al., 2002). Opposite to that, vegetables and mainly watercress underlie human infection in Europe (Arjona et al., 1995).

Cercariae of F. gigantica can also encyst on very small floating particles or on the water surface, in which case they may be swallowed in contaminated drinking water (Mas-Coma and Bargues, 1997). Thus, in enzootic areas of F. gigantica as in Africa, contraction of the infection by the animals and their contamination of the area with eggs of the parasite in the feces take place when the animals go to drink (Fig. 7F), rather than when they are grazing in the pasture. Accordingly, avoiding the watering of the animals from swampy banks of rivers and from bodies of water rich in vegetation would considerably reduce the chances of infection.

Drinking of beverages and juices made from local plants

Local beverages and juices have been linked to human infection by Fasciola according to the results of the patient’s anamnesis or surveys about risky foods and drinks. Contamination of such beverages and juices may be whether from the plants or the natural water used for their washing and beverage production.

In Andean countries, beverages and juices are produced from leaves of plants and traditionally consumed in mainly Peru, but also Bolivia, Ecuador and Colombia. In Peru, warm beverages called ‘emolientes’ (emollients) were introduced during the colonization period and became so popular as to be sold in every street corner of the capital Lima. These emollients are aqueous drinks made from various medicinal plants, mainly alfalfa and watercress, and are supposed to be good for liver diseases among other illnesses.

These emollients are believed to also play a role in rural areas of Peru, according to their regular appearance among the risk factors deduced from results of questionnaire surveys in endemic areas (Blancas et al., 2004; Raymundo et al., 2004; Valencia et al., 2005; Marcos et al., 2006). In a hospital in Lima, a 5.3% among a total of 277 Fasciola-infected patients mentioned to be consumers of emollients (Blancas et al., 2004).

When surveying children in the endemic rural areas of Peru, drinking alfalfa juice appeared as the main exposure factor for F. hepatica infection (Raymundo et al., 2004; Marcos et al., 2006). A similar result indicating the importance of alfalfa juice in fascioliasis infection was obtained in a survey of schoolchildren in Puebla, Mexico (Zumaquero-Rios et al., 2013). Alfalfa (Medicago sativa) is a perennial plant which is cultivated as livestock fodder since the antiquity. Alfalfa has an extremely high nutritive value and it is used in traditional medicine due to its high content in protein, calcium, vitamins, enzymes and amino acids (Amraie et al., 2015). This is why its sprouts are a common ingredient in dishes made in South Indian cuisine. The correlation of alfalfa juice with fascioliasis in children led to suggest that human fascioliasis in Peru should be suspected in patients who have a history of consumption of alfalfa juice when they come from livestock-rearing areas and present with jaundice and eosinophilia (Marcos et al., 2006).

The Cape Verde Republic is an Atlantic archipelago in which human fascioliasis has repeatedly been reported. Although most of the patients refer to having consumed watercress (see e.g. Nozais et al., 1998), the possibility for the traditional beverages to be also involved in the transmission to humans should not be neglected (Mas-Coma, 2004). One of the most important aspects of Cape Verdean culture is beverage production, for which sugar cane is used on the islands of Santiago and Santo Antão. Indeed, sugar cane grown in swampy areas has already been emphasized as a potential source of metacercarial ingestion in Africa (Speckhart, 1969).

Ingestion of dishes and soups made with contaminated water

Water containing metacercariae may also contaminate food. Liver fluke infection by ingestion of salads contaminated with metacercariae-carrying water has been reported in Basse-Normandie, France (Cadel et al., 1996).

In the human hyperendemic area of the Northern Altiplano of Bolivia, edible algae as cochayuyo or ‘llayta’ (P. purpurea – Chlorophyta) and similar vegetables as gelatinous species of Nostoc (Cyanobacteria) (Mas-Coma et al., 1995) present in water collections show sometimes lymnaeid snail specimens moving on their surface, indicating the risk of presenting attached metacercariae. These vegetables are locally used to make soups and frequently appear in questionnaire surveys correlating with infection in children.

Washing of vegetables, fruits, tubercles, kitchen utensils or other objects with contaminated water

Natural water may be an indirect source of fortuitous infection when containing infective metacercariae and contaminating vegetable foods, kitchen utensils and other objects by two ways: whether by washing or when water is used to soak vegetables, fruits or tubercles and utensils. This is an additional way to understand the contamination of terrestrial vegetables (Fig. 9A, B) and may be the only to tell how aerial fruits and subterraneous tubercles may become contaminated by fasciolid metacercariae. Several studies have confirmed that vegetables are good vectors for the transmission of some parasitic diseases in different countries due to the use of contaminated water to irrigate or more often to clean the vegetables (de Oliviera and Germano, 1992; Eraky et al., 2014). Indeed, fecal contamination of vegetables and fruits during cultivation and processing for the market is well known since long ago (Geldreich and Bordner, 1971).

Fig. 9. Washing or soaking with contaminated waters: (A) lymnaeid snail vectors on loamy soil surrounding lettuce leaves in Hong Kong; (B) terrestrial wild vegetables collected from wet habitats and washed before consumption in Talesh mountains, Guilan, Iran; (C) housewives and children washing in water canal inhabited by lymnaeid snails and frequented by livestock for drinking, in Atlixco, Puebla State, Mexico; (D) carrots freshen and soaked in stream waters for subsequent marketing in Apartaderos, Merida State, Venezuela; (E, F) housewives and children in traditional washing at river presenting lymnaeid snails and frequented by livestock for drinking, in Mantaro valley, Peru; (G) women washing dresses and utensils in large canals in the Nile Delta region, Egypt; (H) small stream (marked by shrubs and trees linearly arranged from left to right) inhabited by Galba truncatula vectors separating tourist camping (in the background) and football field used by livestock for grazing, in the Mediterranean Corsica island, France. (Photographs S. Mas-Coma).

A curious case was that of a girl who became infected with Fasciola when frequently munching apples without previously peeling them (Ehlers and Knüttgen, 1949). This girl collected these apples after falling down into the stagnant water of a canal just beside where she was playing and a canal which was later verified to be inhabited by G. truncatula (Minning and Vogel, 1950). This case speaks about the opportunistic way of transmission Fasciola may take advantage of.

Of particular interest is the possibility for tubercles to play a role as human fascioliasis infection source. An association between child fascioliasis and the habit of eating raw vegetables was identified in Mexico. The link of fascioliasis risk with consumption of raw vegetables other than watercress should be highlighted, as it suggests contamination when washing terrestrial vegetables with untreated water and/or in plant cultures using natural water for irrigation (Zumaquero-Rios et al., 2013). Radish appeared with an 82% among the questionnaires fulfilled by parents of children infected with F. hepatica in a survey of schoolchildren in Puebla, Mexico. In the same survey, terrestrial plants as lettuce, broccoli and spinach appeared however with pronouncedly lower percentages (34, 12 and 8%, respectively) (Zumaquero-Rios et al., 2013). In this human endemic area of Puebla, there is the tradition for women and children to wash at small rivers where lymnaeid vectors are present and livestock move around (Fig. 9C). The consumption of radishes has also appeared highlighted as an important risk factor for child infection in a questionnaire survey recently made in Baños del Inca, Cajamarca, Peru (Rodriguez et al., 2018).

In Venezuela, in Apartaderos, in the endemic high altitude area of Merida, carrots are soaked or freshen in waters of natural streams (Fig. 9D) (Mas-Coma et al., unpublished). In Peru, a similar behaviour of women and children washing in rivers with lymnaeids and livestock is observed in the human hyperendemic area of the Mantaro valley (Fig. 9E, F).

In the hyperendemic area of Cajamarca, Peru, it is a common practice for young children to help their parents in agricultural activities and in the tending of animals. Thus adults and children, whilst performing these activities, stay away from home for many hours. This leads them to drink freshwater and to eat vegetables such as watercress and ‘chocho’ or ‘tarwi’ (Lupinus mutabilis). The latter is an annual plant, a species of lupin grown in the Andes mainly for its edible bean. Lupin seeds are soaked with waters of streams and small rivers of the endemic area and that may therefore present lymnaeid vectors and consequently become contaminated with metacercariae of F. hepatica (Ortiz et al., 2000). ‘Chocho’ is also sold in the market of Cajamarca city, thus allowing for urban infection. When asking mothers of the rural endemic area of Cajamarca about risky activities at home, 74% mentioned to consume ‘chocho’ and 39% to drink emollients (Rivera-Jacinto et al., 2010).

In the city of La Paz, just bordering the human fascioliasis hyperendemic area of the Northern Bolivian Altiplano, a high contamination by protozoan and helminth enteroparasites was found in vegetables sold in the city markets, including F. hepaticacontaminating a 3.6% of carrots and 17% of watercress (Muñoz Ortiz and Laura, 2008).

In the human fascioliasis hyperendemic area of the Nile Delta region, Egypt, a possible reason for the high prevalence is thought to be the habit of farmers of picking vegetables and then leaving them immersed in the canals to keep them fresh while they continue picking (Hotez et al., 2012). Additionally, in the marketplace as well as in the home, contaminated water containing metacercariae may be used to freshen vegetables, particularly leafy vegetables.

In the same hyperendemic region of Egypt, the presence of farm animals and their sheds inside the houses of their farmers’ owners has been highlighted to be a risk factor for them (Hussein et al., 2000). These Egyptian farmers living indoors with animals sheds are at high risk of fascioliasis infection (Motawea et al., 2001a). Most of these farmers, their housewives and children wash the animals in canals together with the vegetables, dresses and utensils (Fig. 9G). Afterward, they eat such contaminated vegetables and drink water in such contaminated utensils. During animal washing, their children swim in water and may involuntarily swallow such contaminated water. In such circumstances, the risk of infection with Fasciola species has been observed to be higher in situations of low housing score, little or very low social score, and large family size (Hussein et al., 2000; Motawea et al., 2001b). Anyway, another study reported that there was no significant association between the social class of the family and the increased risk of infection by Fasciola (El-Sahn et al., 1995).

Also in the Nile Delta of Egypt, the most exhaustive study on the relationships of the dietary habits and household characteristics with human infection by Fasciola so far made in a human fascioliasis hyperendemic area was carried out (Curtale et al., 2003). The habit to consume daily raw seeds was confirmed as an important risk factor (OR = 8.6), followed by the presence of piped water in the latrine (OR = 6.9), the habit to bring the animals to the canal for drinking and/or bathing them (OR = 3.2), and the use to cultivate the vegetables eaten in the household (OR = 3.1) (Curtale et al., 2003).

Among the factors investigated, only the presence of cows, buffaloes and/or goats in the household, and the habit to bring those animals to the canal for bathing was significantly associated with the infection. The possibility that people use the irrigation canal to wash dishes, clothes, vegetables and even themselves, close to where domestic ruminants are taken to bath and where also the lymnaeid snail host is present, represents an obvious link between the animal and human infections implying the importance of controlling the animal reservoir to the benefit of humans (Curtale et al., 2003). In the French Corsica island, human infection was reported to occur in touristic campings where washing kitchen and table-eating utensils were made in lateral stream inhabited by G. truncatula snails and frequented by cattle for drinking (Fig. 9H).

Two more risk factors, apparently not significant in the univariate analysis, emerged as closely associated with the infection in the logistic regression analysis, namely the direct production of the vegetables eaten in the household and the presence of piped water. The habit of eating raw seeds emerged from the analyses as significantly more frequent among cases than controls. This finding should be interpreted taking into account that those seeds are usually washed several times in the canal before being dried. The washing process makes it possible that metacercaria encyst on the skin of the seeds, and then resist to the drying process, being ingested by the consumer breaking the skins with his teeth (Curtale et al., 2003).

However, while it is easy to assume that water used for irrigation is derived from the same canal where animals are bathing, it is more difficult to interpret the association between piped water in the household and infection. The presence of piped water in the household has always been considered an essential element for the control of intestinal parasites. However, the possibility that water sources may be polluted with encysted metacercaria cannot be excluded and should be verified in further studies (Curtale et al., 2003).

In the same sense indicates the finding of the lymnaeid vector G. truncatula living on a somewhat muddy sheet over the water border of a geosynthetic waterproof ground of the large artificial water reservoir at the origin of the sewage network for the water supply of a city in Corsica Island (Oviedo et al., 1992).

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More information: J. González-Miguel et al. Numerous Fasciola plasminogen-binding proteins may underlie blood-brain barrier leakage and explain neurological disorder complexity and heterogeneity in the acute and chronic phases of human fascioliasis, Parasitology (2018). DOI: 10.1017/S0031182018001464

ournal information: Parasitology
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