Experimental Assays

For the experimental study of the embryonation of the eggs of F. hepatica, fecal samples from three naturally infected sheep individuals from the locality of Batallas and from three naturally infected cattle from Kallutaca were used (Figure 1). Eggs isolated by filtration (filter pore size of 40 μm) were conserved in natural water under complete darkness at 4°C at very high altitude until starting of the embryogenesis follow-up study.

Map showing the Northern Bolivian Altiplano human fascioliasis hyperendemic area, at 3,820–4,100 m altitude, including zones where sheep and cattle were surveyed and localities where lymnaeid snail specimens of Galba truncatula were collected.

Given the irresolvable difficulties in performing the necessary experimental studies at the very high altitude in Bolivia due to the unavailability of the appropriate infrastructures (equipments, devices) and logistics (permanent functioning provisions, energy supplies, and security systems), an appropriate experimental approach was designed. Experiments were performed in a laboratory at low altitude. To minimize the influence of oxygen pressure (low at the very high altitude when compared to low altitude), experimental studies were started immediately after each field mission and arrival to the laboratory, i.e., altiplanic F. hepatica eggs were kept at low altitude only hours or maximum 1 and 2 days before beginning of egg embryonation, and only the first laboratory generation of altiplanic lymnaeids were used. Moreover, all life-cycle phases experimentally studied occur in freshwater, in which the difference of oxygen pressure is lower than in air.

For the experimental infection of lymnaeid snails, F. hepatica eggs were similarly obtained from sheep from Batallas and from cattle from Kallutaca and Batallas (Figure 1). Eggs were similarly isolated by filtration and conserved until used for snail infection in the laboratory. No distinction was made between isolates, which may be considered representative of the whole endemic area, as previous molecular studies have demonstrated the uniformity of parasite and snail involved in the disease throughout the whole Northern Bolivian Altiplano (38).

For the experimental infection of Wistar rats, F. hepatica metacercariae were obtained from the aforementioned experimentally infected lymnaeid snails. These metacercariae were stored in natural water in total darkness 4°C until required (42).

Embryogenesis was microscopically followed at constant 20°C, with analytical observations every 4 days. This study was made in the climatic chambers of the Parasitology Department of the University of Valencia, Spain. Egg development was made by differentiation of (i) eggs in the phase of morula, showing vitelline granules and/or spheroidal cells (EM), (ii) eggs in the phase of outlined miracidium, in which a miracidial form begins to be observed (EOM), and (iii) eggs in the phase of developed miracidium, in which a fully developed miracidium is observed inside (EDM). For each 4-day study, a total of 33 eggs from each host individual were analyzed. Counting included not only EM, EOM, and EDM but also (i) degenerated eggs, (ii) empty eggs, and (iii) broken eggs. Egg counts were noted in percentages independently for sheep or cattle per observational day.

Developed miracidia were forced to hatch by putting fully embryonated eggs under light and used for the experimental infection of snails (43, 44). Only laboratory-reared specimens were used. Lymnaeid snails of a size of 4–5 mm were used to assess infection susceptibility, by exposing each snail to miracidia for 4 h in a small Petri dish containing 2 ml of freshwater. Mono-, bi-, and trimiracidial infections were carried out. The disappearance of the miracidia was taken as verification of its successful penetration into the snail.

The sheep F. hepatica isolate was used for several infection assays under different experimental conditions concerning (i) miracidial dose and (ii) day/night temperature according to a photoperiod of 12-h light/12-h darkness in high-accuracy climatic chambers (HPS-1500, VB-0714, and HPS-500 models of Heraeus-Vötsch) (45). A similar procedure was followed with the cattle F. hepatica isolate. The characteristics, conditions, and number of snails in all these infection experiments are detailed in Table 1. After the infection, snails were returned to 2,000-ml containers, at 90% relative humidity (r.h.), 12/12 h light/darkness, and dry lettuce ad libitum, until day 30 post-infection. Afterward, they were again isolated in Petri dishes to allow daily monitoring of cercarial shedding by individual snails. Lettuce was provided ad libitum to each snail in a Petri dish during both shedding and post-shedding periods until death of the snail. The cercarial shedding was followed by daily counting of metacercariae in each Petri dish.

Experimental infections of altiplanic lymnaeid snails with altiplanic sheep and cattle isolates of Fasciola hepatica.

dpi, days post-infection.

The presence of only G. truncatula as only lymnaeid species inhabiting the Northern Bolivian Altiplano hyperendemic area has recently been confirmed by the sequencing of complete nuclear ribosomal DNA and mitochondrial DNA markers (38). This species is of European origin and differs from the Neotropical species of the Galba/Fossaria group of lymnaeids which also act as vectors of fascioliasis in South America (46). Live G. truncatula snails were collected in the localities of Huacullani, Batallas, Tambillo, and Viacha (Figure 1) and transported under isothermal conditions for their laboratory adaptation in Valencia to standardized controlled conditions of 20°C, 90% relative humidity, and a 12/12-h light/darkness photoperiod in the aforementioned precision climatic chambers. The possible natural infection by fasciolids was always individually verified prior to the launch of laboratory cultures. This was performed by keeping each lymnaeid snail isolated in a Petri dish containing a small amount of natural water. After 24 h, the presence or absence of motionless metacercarial cysts or moving cercariae was verified in each Petri dish. Non-infected lymnaeids were arranged in standard breeding boxes containing 2,000 ml freshwater, to assure locality-pure cultures. The water was changed weekly and lettuce added ad libitum.

A total of 45 male Wistar rats (Iffa Credo, Barcelona, Spain) aged 4–5 weeks were used throughout. A balanced commercial rodent diet (Panlab Chow A04) and water were provided ad libitum, according to standards previously described (47).

Wistar rats were infected according to methods previously described (48). Metacercariae were inoculated orally by means of a gastric tube. A dose of 20 F. hepatica metacercariae per rat was used (Table 2). Animal care, animal health, body condition, and well-being were assessed on a weekly basis by means of checking their body weight and the appearance of the fur. Infected animals presented a lower body weight than negative controls at the end of the experiment. No mortality occurred. The number of Wistar rats in each infection experiment is noted in Table 2. Finally, animals were humanely euthanized with an overdose of an anesthetic (IsoFlo; Dr Esteve SA, Barcelona, Spain), and F. hepatica worms were collected under a dissecting microscope, according to methods already outlined before (49). Infection prevalence and intensity (number of worms successfully developed in each rat) were established by necropsy 12 weeks after infection. Initially, the bile duct was examined for the presence of worms, though the rest of the organs were also evaluated. Afterward, the thoracic and abdominal viscera and cavities were examined and thoroughly rinsed with water to assure the recovery of all worms. After the analysis of the definitive host infectivity of the sheep isolate in a sufficient number of experimentally infected rats, and considering present knowledge indicating the absence of infectivity differences between sheep and cattle isolates, in agreement with present ethical recommendations, a minimum number of rats were used to assess this aspect in the cattle isolate.

Experimental infections of Wistar rats with experimentally obtained Fasciola hepatica metacercariae from sheep and cattle isolates from the Northern Bolivian Altiplano human hyperendemic area.